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Recent Articles in Microbiology and Molecular Biology Reviews

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Umeno D, Tobias AV, Arnold FH
Diversifying carotenoid biosynthetic pathways by directed evolution.
Microbiol Mol Biol Rev. 2005 Mar;69(1):51-78.
Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products--those that could be made biosynthetically--remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these "evolved" pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed. [Abstract/Link to Full Text]

Wolfe AJ
The acetate switch.
Microbiol Mol Biol Rev. 2005 Mar;69(1):12-50.
To succeed, many cells must alternate between life-styles that permit rapid growth in the presence of abundant nutrients and ones that enhance survival in the absence of those nutrients. One such change in life-style, the "acetate switch," occurs as cells deplete their environment of acetate-producing carbon sources and begin to rely on their ability to scavenge for acetate. This review explains why, when, and how cells excrete or dissimilate acetate. The central components of the "switch" (phosphotransacetylase [PTA], acetate kinase [ACK], and AMP-forming acetyl coenzyme A synthetase [AMP-ACS]) and the behavior of cells that lack these components are introduced. Acetyl phosphate (acetyl approximately P), the high-energy intermediate of acetate dissimilation, is discussed, and conditions that influence its intracellular concentration are described. Evidence is provided that acetyl approximately P influences cellular processes from organelle biogenesis to cell cycle regulation and from biofilm development to pathogenesis. The merits of each mechanism proposed to explain the interaction of acetyl approximately P with two-component signal transduction pathways are addressed. A short list of enzymes that generate acetyl approximately P by PTA-ACKA-independent mechanisms is introduced and discussed briefly. Attention is then directed to the mechanisms used by cells to "flip the switch," the induction and activation of the acetate-scavenging AMP-ACS. First, evidence is presented that nucleoid proteins orchestrate a progression of distinct nucleoprotein complexes to ensure proper transcription of its gene. Next, the way in which cells regulate AMP-ACS activity through reversible acetylation is described. Finally, the "acetate switch" as it exists in selected eubacteria, archaea, and eukaryotes, including humans, is described. [Abstract/Link to Full Text]

Gottesman ME, Weisberg RA
Little lambda, who made thee?
Microbiol Mol Biol Rev. 2004 Dec;68(4):796-813.
The study of the bacteriophage lambda has been critical to the discipline of molecular biology. It was the source of key discoveries in the mechanisms of, among other processes, gene regulation, recombination, and transcription initiation and termination. We trace here the events surrounding these findings and draw on the recollections of the participants. We show how a particular atmosphere of interactions among creative scientists yielded spectacular insights into how living things work. [Abstract/Link to Full Text]

Ghosh P
Process of protein transport by the type III secretion system.
Microbiol Mol Biol Rev. 2004 Dec;68(4):771-95.
The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry. [Abstract/Link to Full Text]

Zientz E, Dandekar T, Gross R
Metabolic interdependence of obligate intracellular bacteria and their insect hosts.
Microbiol Mol Biol Rev. 2004 Dec;68(4):745-70.
Mutualistic associations of obligate intracellular bacteria and insects have attracted much interest in the past few years due to the evolutionary consequences for their genome structure. However, much less attention has been paid to the metabolic ramifications for these endosymbiotic microorganisms, which have to compete with but also to adapt to another metabolism--that of the host cell. This review attempts to provide insights into the complex physiological interactions and the evolution of metabolic pathways of several mutualistic bacteria of aphids, ants, and tsetse flies and their insect hosts. [Abstract/Link to Full Text]

Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala'Aldeen D
Type V protein secretion pathway: the autotransporter story.
Microbiol Mol Biol Rev. 2004 Dec;68(4):692-744.
Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function. [Abstract/Link to Full Text]

Schloss PD, Handelsman J
Status of the microbial census.
Microbiol Mol Biol Rev. 2004 Dec;68(4):686-91.
Over the past 20 years, more than 78,000 16S rRNA gene sequences have been deposited in GenBank and the Ribosomal Database Project, making the 16S rRNA gene the most widely studied gene for reconstructing bacterial phylogeny. While there is a general appreciation that these sequences are largely unique and derived from diverse species of bacteria, there has not been a quantitative attempt to describe the extent of sequencing efforts to date. We constructed rarefaction curves for each bacterial phylum and for the entire bacterial domain to assess the current state of sampling and the relative taxonomic richness of each phylum. This analysis quantifies the general sense among microbiologists that we are a long way from a complete census of the bacteria on Earth. Moreover, the analysis indicates that current sampling strategies might not be the most effective ones to describe novel diversity because there remain numerous phyla that are globally distributed yet poorly sampled. Based on the current level of sampling, it is not possible to estimate the total number of bacterial species on Earth, but the minimum species richness is 35,498. Considering previous global species richness estimates of 10(7) to 10(9), we are certain that this estimate will increase with additional sequencing efforts. The data support previous calls for extensive surveys of multiple chemically disparate environments and of specific phylogenetic groups to advance the census most rapidly. [Abstract/Link to Full Text]

Handelsman J
Metagenomics: application of genomics to uncultured microorganisms.
Microbiol Mol Biol Rev. 2004 Dec;68(4):669-85.
Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na(+)(Li(+))/H(+) antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies. [Abstract/Link to Full Text]

Dennis PP, Ehrenberg M, Bremer H
Control of rRNA synthesis in Escherichia coli: a systems biology approach.
Microbiol Mol Biol Rev. 2004 Dec;68(4):639-68.
The first part of this review contains an overview of the various contributions and models relating to the control of rRNA synthesis reported over the last 45 years. The second part describes a systems biology approach to identify the factors and effectors that control the interactions between RNA polymerase and rRNA (rrn) promoters of Escherichia coli bacteria during exponential growth in different media. This analysis is based on measurements of absolute rrn promoter activities as transcripts per minute per promoter in bacterial strains either deficient or proficient in the synthesis of the factor Fis and/or the effector ppGpp. These absolute promoter activities are evaluated in terms of rrn promoter strength (V(max)/K(m)) and free RNA polymerase concentrations. Three major conclusions emerge from this evaluation. First, the rrn promoters are not saturated with RNA polymerase. As a consequence, changes in the concentration of free RNA polymerase contribute to changes in rrn promoter activities. Second, rrn P2 promoter strength is not specifically regulated during exponential growth at different rates; its activity changes only when the concentration of free RNA polymerase changes. Third, the effector ppGpp reduces the strength of the rrn P1 promoter both directly and indirectly by reducing synthesis of the stimulating factor Fis. This control of rrn P1 promoter strength forms part of a larger feedback loop that adjusts the synthesis of ribosomes to the availability of amino acids via amino acid-dependent control of ppGpp accumulation. [Abstract/Link to Full Text]

García-Fernández JM, de Marsac NT, Diez J
Streamlined regulation and gene loss as adaptive mechanisms in Prochlorococcus for optimized nitrogen utilization in oligotrophic environments.
Microbiol Mol Biol Rev. 2004 Dec;68(4):630-8.
Prochlorococcus is one of the dominant cyanobacteria and a key primary producer in oligotrophic intertropical oceans. Here we present an overview of the pathways of nitrogen assimilation in Prochlorococcus, which have been significantly modified in these microorganisms for adaptation to the natural limitations of their habitats, leading to the appearance of different ecotypes lacking key enzymes, such as nitrate reductase, nitrite reductase, or urease, and to the simplification of the metabolic regulation systems. The only nitrogen source utilizable by all studied isolates is ammonia, which is incorporated into glutamate by glutamine synthetase. However, this enzyme shows unusual regulatory features, although its structural and kinetic features are unchanged. Similarly, urease activities remain fairly constant under different conditions. The signal transduction protein P(II) is apparently not phosphorylated in Prochlorococcus, despite its conserved amino acid sequence. The genes amt1 and ntcA (coding for an ammonium transporter and a global nitrogen regulator, respectively) show noncorrelated expression in Prochlorococcus under nitrogen stress; furthermore, high rates of organic nitrogen uptake have been observed. All of these unusual features could provide a physiological basis for the predominance of Prochlorococcus over Synechococcus in oligotrophic oceans. [Abstract/Link to Full Text]

Phipps AJ, Premanandan C, Barnewall RE, Lairmore MD
Rabbit and nonhuman primate models of toxin-targeting human anthrax vaccines.
Microbiol Mol Biol Rev. 2004 Dec;68(4):617-29.
The intentional use of Bacillus anthracis, the etiological agent of anthrax, as a bioterrorist weapon in late 2001 made our society acutely aware of the importance of developing, testing, and stockpiling adequate countermeasures against biological attacks. Biodefense vaccines are an important component of our arsenal to be used during a biological attack. However, most of the agents considered significant threats either have been eradicated or rarely infect humans alive today. As such, vaccine efficacy cannot be determined in human clinical trials but must be extrapolated from experimental animal models. This article reviews the efficacy and immunogenicity of human anthrax vaccines in well-defined animal models and the progress toward developing a rugged immunologic correlate of protection. The ongoing evaluation of human anthrax vaccines will be dependent on animal efficacy data in the absence of human efficacy data for licensure by the U.S. Food and Drug Administration. [Abstract/Link to Full Text]

Melo AM, Bandeiras TM, Teixeira M
New insights into type II NAD(P)H:quinone oxidoreductases.
Microbiol Mol Biol Rev. 2004 Dec;68(4):603-16.
Type II NAD(P)H:quinone oxidoreductases (NDH-2) catalyze the two-electron transfer from NAD(P)H to quinones, without any energy-transducing site. NDH-2 accomplish the turnover of NAD(P)H, regenerating the NAD(P)(+) pool, and may contribute to the generation of a membrane potential through complexes III and IV. These enzymes are usually constituted by a nontransmembrane polypeptide chain of approximately 50 kDa, containing a flavin moiety. There are a few compounds that can prevent their activity, but so far no general specific inhibitor has been assigned to these enzymes. However, they have the common feature of being resistant to the complex I classical inhibitors rotenone, capsaicin, and piericidin A. NDH-2 have particular relevance in yeasts like Saccharomyces cerevisiae and in several prokaryotes, whose respiratory chains are devoid of complex I, in which NDH-2 keep the balance and are the main entry point of electrons into the respiratory chains. Our knowledge of these proteins has expanded in the past decade, as a result of contributions at the biochemical level and the sequencing of the genomes from several organisms. The latter showed that most organisms contain genes that potentially encode NDH-2. An overview of this development is presented, with special emphasis on microbial enzymes and on the identification of three subfamilies of NDH-2. [Abstract/Link to Full Text]

Brüssow H, Canchaya C, Hardt WD
Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion.
Microbiol Mol Biol Rev. 2004 Sep;68(3):560-602.
Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like "swarms" of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework. [Abstract/Link to Full Text]

Brehm-Stecher BF, Johnson EA
Single-cell microbiology: tools, technologies, and applications.
Microbiol Mol Biol Rev. 2004 Sep;68(3):538-59.
The field of microbiology has traditionally been concerned with and focused on studies at the population level. Information on how cells respond to their environment, interact with each other, or undergo complex processes such as cellular differentiation or gene expression has been obtained mostly by inference from population-level data. Individual microorganisms, even those in supposedly "clonal" populations, may differ widely from each other in terms of their genetic composition, physiology, biochemistry, or behavior. This genetic and phenotypic heterogeneity has important practical consequences for a number of human interests, including antibiotic or biocide resistance, the productivity and stability of industrial fermentations, the efficacy of food preservatives, and the potential of pathogens to cause disease. New appreciation of the importance of cellular heterogeneity, coupled with recent advances in technology, has driven the development of new tools and techniques for the study of individual microbial cells. Because observations made at the single-cell level are not subject to the "averaging" effects characteristic of bulk-phase, population-level methods, they offer the unique capacity to observe discrete microbiological phenomena unavailable using traditional approaches. As a result, scientists have been able to characterize microorganisms, their activities, and their interactions at unprecedented levels of detail. [Abstract/Link to Full Text]

Gil R, Silva FJ, Peretó J, Moya A
Determination of the core of a minimal bacterial gene set.
Microbiol Mol Biol Rev. 2004 Sep;68(3):518-37.
The availability of a large number of complete genome sequences raises the question of how many genes are essential for cellular life. Trying to reconstruct the core of the protein-coding gene set for a hypothetical minimal bacterial cell, we have performed a computational comparative analysis of eight bacterial genomes. Six of the analyzed genomes are very small due to a dramatic genome size reduction process, while the other two, corresponding to free-living relatives, are larger. The available data from several systematic experimental approaches to define all the essential genes in some completely sequenced bacterial genomes were also considered, and a reconstruction of a minimal metabolic machinery necessary to sustain life was carried out. The proposed minimal genome contains 206 protein-coding genes with all the genetic information necessary for self-maintenance and reproduction in the presence of a full complement of essential nutrients and in the absence of environmental stress. The main features of such a minimal gene set, as well as the metabolic functions that must be present in the hypothetical minimal cell, are discussed. [Abstract/Link to Full Text]

Schweizer E, Hofmann J
Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems.
Microbiol Mol Biol Rev. 2004 Sep;68(3):501-17.
The present review focuses on microbial type I fatty acid synthases (FASs), demonstrating their structural and functional diversity. Depending on their origin and biochemical function, multifunctional type I FAS proteins form dimers or hexamers with characteristic organization of their catalytic domains. A single polypeptide may contain one or more sets of the eight FAS component functions. Alternatively, these functions may split up into two different and mutually complementing subunits. Targeted inactivation of the individual yeast FAS acylation sites allowed us to define their roles during the overall catalytic process. In particular, their pronounced negative cooperativity is presumed to coordinate the FAS initiation and chain elongation reactions. Expression of the unlinked genes, FAS1 and FAS2, is in part constitutive and in part subject to repression by the phospholipid precursors inositol and choline. The interplay of the involved regulatory proteins, Rap1, Reb1, Abf1, Ino2/Ino4, Opi1, Sin3 and TFIIB, has been elucidated in considerable detail. Balanced levels of subunits alpha and beta are ensured by an autoregulatory effect of FAS1 on FAS2 expression and by posttranslational degradation of excess FAS subunits. The functional specificity of type I FAS multienzymes usually requires the presence of multiple FAS systems within the same cell. De novo synthesis of long-chain fatty acids, mitochondrial fatty acid synthesis, acylation of certain secondary metabolites and coenzymes, fatty acid elongation, and the vast diversity of mycobacterial lipids each result from specific FAS activities. The microcompartmentalization of FAS activities in type I multienzymes may thus allow for both the controlled and concerted action of multiple FAS systems within the same cell. [Abstract/Link to Full Text]

Tropel D, van der Meer JR
Bacterial transcriptional regulators for degradation pathways of aromatic compounds.
Microbiol Mol Biol Rev. 2004 Sep;68(3):474-500.
Human activities have resulted in the release and introduction into the environment of a plethora of aromatic chemicals. The interest in discovering how bacteria are dealing with hazardous environmental pollutants has driven a large research community and has resulted in important biochemical, genetic, and physiological knowledge about the degradation capacities of microorganisms and their application in bioremediation, green chemistry, or production of pharmacy synthons. In addition, regulation of catabolic pathway expression has attracted the interest of numerous different groups, and several catabolic pathway regulators have been exemplary for understanding transcription control mechanisms. More recently, information about regulatory systems has been used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment. The topic of biodegradation is relatively coherent, and this review presents a coherent overview of the regulatory systems involved in the transcriptional control of catabolic pathways. This review summarizes the different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to other known protein families. Specific attention has been paid to describing the genetic organization of the regulatory genes, promoters, and target operon(s) and to discussing present knowledge about signaling molecules, DNA binding properties, and operator characteristics, and evidence from regulatory mutants. For each regulator family, this information is combined with recently obtained protein structural information to arrive at a possible mechanism of transcription activation. This demonstrates the diversity of control mechanisms existing in catabolic pathways. [Abstract/Link to Full Text]

Roberts GP, Youn H, Kerby RL
CO-sensing mechanisms.
Microbiol Mol Biol Rev. 2004 Sep;68(3):453-73.
Carbon monoxide (CO) has long been known to have dramatic physiological effects on organisms ranging from bacteria to humans, but recently there have a number of suggestions that organisms might have specific sensors for CO. This article reviews the current evidence for a variety of proteins with demonstrated or potential CO-sensing ability. Particular emphasis is placed on the molecular description of CooA, a heme-containing CO sensor from Rhodospirillum rubrum, since its biological role as a CO sensor is clear and we have substantial insight into the basis of its sensing ability. [Abstract/Link to Full Text]

Wang Q, Carmichael GG
Effects of length and location on the cellular response to double-stranded RNA.
Microbiol Mol Biol Rev. 2004 Sep;68(3):432-52.
Since double-stranded RNA (dsRNA) has not until recently generally been thought to be deliberately expressed in cells, it has commonly been assumed that the major source of cellular dsRNA is viral infections. In this view, the cellular responses to dsRNA would be natural and perhaps ancient antiviral responses. While the cell may certainly react to some dsRNAs as an antiviral response, this does not represent the only response or even, perhaps, the major one. A number of recent observations have pointed to the possibility that dsRNA molecules are not seen only as evidence of viral infection or recognized for degradation because they cannot be translated. In some instances they may also play important roles in normal cell growth and function. The purpose of this review is to outline our current understanding of the fate of dsRNA in cells, with a focus on the apparent fact that their fates and functions appear to depend critically not only on where in the cell dsRNA molecules are found, but also on how long they are and perhaps on how abundant they are. [Abstract/Link to Full Text]

Thompson FL, Iida T, Swings J
Biodiversity of vibrios.
Microbiol Mol Biol Rev. 2004 Sep;68(3):403-31, table of contents.
Vibrios are ubiquitous and abundant in the aquatic environment. A high abundance of vibrios is also detected in tissues and/or organs of various marine algae and animals, e.g., abalones, bivalves, corals, fish, shrimp, sponges, squid, and zooplankton. Vibrios harbour a wealth of diverse genomes as revealed by different genomic techniques including amplified fragment length polymorphism, multilocus sequence typing, repetetive extragenic palindrome PCR, ribotyping, and whole-genome sequencing. The 74 species of this group are distributed among four different families, i.e., Enterovibrionaceae, Photobacteriaceae, Salinivibrionaceae, and Vibrionaceae. Two new genera, i.e., Enterovibrio norvegicus and Grimontia hollisae, and 20 novel species, i.e., Enterovibrio coralii, Photobacterium eurosenbergii, V. brasiliensis, V. chagasii, V. coralliillyticus, V. crassostreae, V. fortis, V. gallicus, V. hepatarius, V. hispanicus, V. kanaloaei, V. neonatus, V. neptunius, V. pomeroyi, V. pacinii, V. rotiferianus, V. superstes, V. tasmaniensis, V. ezurae, and V. xuii, have been described in the last few years. Comparative genome analyses have already revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by decay or deletion, and gene acquisitions through duplication or horizontal transfer (e.g., in the acquisition of bacteriophages, pathogenicity islands, and super-integrons), that are probably important driving forces in the evolution and speciation of vibrios. Whole-genome sequencing and comparative genomics through the application of, e.g., microarrays will facilitate the investigation of the gene repertoire at the species level. Based on such new genomic information, the taxonomy and the species concept for vibrios will be reviewed in the next years. [Abstract/Link to Full Text]

Barth H, Aktories K, Popoff MR, Stiles BG
Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins.
Microbiol Mol Biol Rev. 2004 Sep;68(3):373-402, table of contents.
Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic "A-B" paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The "B" components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated "B" components form homoheptameric rings that subsequently dock with an "A" component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, "A" molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria. [Abstract/Link to Full Text]

Longworth MS, Laimins LA
Pathogenesis of human papillomaviruses in differentiating epithelia.
Microbiol Mol Biol Rev. 2004 Jun;68(2):362-72.
Human papillomaviruses (HPV) are the etiological agents of cervical and other anogenital malignancies. Over 100 different types of HPVs have been identified to date, and all target epithelial tissues for infection. One-third of HPV types specifically infect the genital tract, and a subset of these are the causative agents of anogenital cancers. Other HPV types that infect the genital tract induce benign hyperproliferative lesions or genital warts. The productive life cycle of HPVs is linked to epithelial differentiation. Papillomaviruses are thought to infect cells in the basal layer of stratified epithelia and establish their genomes as multicopy nuclear episomes. In these cells, viral DNA is replicated along with cellular chromosomes. Following cell division, one of the daughter cells migrates away from the basal layer and undergoes differentiation. In highly differentiated suprabasal cells, vegetative viral replication and late-gene expression are activated, resulting in the generation of progeny virions. Since virion production is restricted to differentiated cells, infected basal cells can persist for up to several decades or until the immune system clears the infection. The E6 and E7 genes encode viral oncoproteins that target Rb and p53, respectively. During the viral life cycle, these proteins facilitate stable maintenance of episomes and stimulate differentiated cells to reenter the S phase. The E1 and E2 proteins act as origin recognition factors as well as regulators of early viral transcription. The functions of the E5 and E1--E4 proteins are still largely unknown, but these proteins have been implicated in modulating late viral functions. The L1 and L2 proteins form icosahedral capsids for progeny virion generation. The characterization of the cellular targets of these viral proteins and the mechanisms regulating the differentiation-dependent viral life cycle remain active areas for the study of these important human pathogens. [Abstract/Link to Full Text]

Nickerson CA, Ott CM, Wilson JW, Ramamurthy R, Pierson DL
Microbial responses to microgravity and other low-shear environments.
Microbiol Mol Biol Rev. 2004 Jun;68(2):345-61.
Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters. [Abstract/Link to Full Text]

Roux PP, Blenis J
ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions.
Microbiol Mol Biol Rev. 2004 Jun;68(2):320-44.
Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries. [Abstract/Link to Full Text]

Szurmant H, Ordal GW
Diversity in chemotaxis mechanisms among the bacteria and archaea.
Microbiol Mol Biol Rev. 2004 Jun;68(2):301-19.
The study of chemotaxis describes the cellular processes that control the movement of organisms toward favorable environments. In bacteria and archaea, motility is controlled by a two-component system involving a histidine kinase that senses the environment and a response regulator, a very common type of signal transduction in prokaryotes. Most insights into the processes involved have come from studies of Escherichia coli over the last three decades. However, in the last 10 years, with the sequencing of many prokaryotic genomes, it has become clear that E. coli represents a streamlined example of bacterial chemotaxis. While general features of excitation remain conserved among bacteria and archaea, specific features, such as adaptational processes and hydrolysis of the intracellular signal CheY-P, are quite diverse. The Bacillus subtilis chemotaxis system is considerably more complex and appears to be similar to the one that existed when the bacteria and archaea separated during evolution, so that understanding this mechanism should provide insight into the variety of mechanisms used today by the broad sweep of chemotactic bacteria and archaea. However, processes even beyond those used in E. coli and B. subtilis have been discovered in other organisms. This review emphasizes those used by B. subtilis and these other organisms but also gives an account of the mechanism in E. coli. [Abstract/Link to Full Text]

Gage DJ
Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes.
Microbiol Mol Biol Rev. 2004 Jun;68(2):280-300.
Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads. [Abstract/Link to Full Text]

Elsen S, Swem LR, Swem DL, Bauer CE
RegB/RegA, a highly conserved redox-responding global two-component regulatory system.
Microbiol Mol Biol Rev. 2004 Jun;68(2):263-79.
The Reg regulon from Rhodobacter capsulatus and Rhodobacter sphaeroides encodes proteins involved in numerous energy-generating and energy-utilizing processes such as photosynthesis, carbon fixation, nitrogen fixation, hydrogen utilization, aerobic and anaerobic respiration, denitrification, electron transport, and aerotaxis. The redox signal that is detected by the membrane-bound sensor kinase, RegB, appears to originate from the aerobic respiratory chain, given that mutations in cytochrome c oxidase result in constitutive RegB autophosphorylation. Regulation of RegB autophosphorylation also involves a redox-active cysteine that is present in the cytosolic region of RegB. Both phosphorylated and unphosphorylated forms of the cognate response regulator RegA are capable of activating or repressing a variety of genes in the regulon. Highly conserved homologues of RegB and RegA have been found in a wide number of photosynthetic and nonphotosynthetic bacteria, with evidence suggesting that RegB/RegA plays a fundamental role in the transcription of redox-regulated genes in many bacterial species. [Abstract/Link to Full Text]

Hilbert DW, Piggot PJ
Compartmentalization of gene expression during Bacillus subtilis spore formation.
Microbiol Mol Biol Rev. 2004 Jun;68(2):234-62.
Gene expression in members of the family Bacillaceae becomes compartmentalized after the distinctive, asymmetrically located sporulation division. It involves complete compartmentalization of the activities of sporulation-specific sigma factors, sigma(F) in the prespore and then sigma(E) in the mother cell, and then later, following engulfment, sigma(G) in the prespore and then sigma(K) in the mother cell. The coupling of the activation of sigma(F) to septation and sigma(G) to engulfment is clear; the mechanisms are not. The sigma factors provide the bare framework of compartment-specific gene expression. Within each sigma regulon are several temporal classes of genes, and for key regulators, timing is critical. There are also complex intercompartmental regulatory signals. The determinants for sigma(F) regulation are assembled before septation, but activation follows septation. Reversal of the anti-sigma(F) activity of SpoIIAB is critical. Only the origin-proximal 30% of a chromosome is present in the prespore when first formed; it takes approximately 15 min for the rest to be transferred. This transient genetic asymmetry is important for prespore-specific sigma(F) activation. Activation of sigma(E) requires sigma(F) activity and occurs by cleavage of a prosequence. It must occur rapidly to prevent the formation of a second septum. sigma(G) is formed only in the prespore. SpoIIAB can block sigma(G) activity, but SpoIIAB control does not explain why sigma(G) is activated only after engulfment. There is mother cell-specific excision of an insertion element in sigK and sigma(E)-directed transcription of sigK, which encodes pro-sigma(K). Activation requires removal of the prosequence following a sigma(G)-directed signal from the prespore. [Abstract/Link to Full Text]

Tjalsma H, Antelmann H, Jongbloed JD, Braun PG, Darmon E, Dorenbos R, Dubois JY, Westers H, Zanen G, Quax WJ, Kuipers OP, Bron S, Hecker M, van Dijl JM
Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome.
Microbiol Mol Biol Rev. 2004 Jun;68(2):207-33.
Secretory proteins perform a variety of important "remote-control" functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which approximately 90 extracellular proteins were identified. Analysis of these proteins disclosed various "secrets of the secretome," such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only approximately 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance. [Abstract/Link to Full Text]

Gray JV, Petsko GA, Johnston GC, Ringe D, Singer RA, Werner-Washburne M
"Sleeping beauty": quiescence in Saccharomyces cerevisiae.
Microbiol Mol Biol Rev. 2004 Jun;68(2):187-206.
The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one "model" quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available. [Abstract/Link to Full Text]

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Recent Articles in Applied and Environmental Microbiology

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Coutard F, Lozach S, Pommepuy M, Hervio-Heath D
Real-time reverse transcription-PCR for transcriptional expression analysis of virulence and housekeeping genes in viable but nonculturable Vibrio parahaemolyticus after recovery of culturability.
Appl Environ Microbiol. 2007 Aug;73(16):5183-9.
A real-time reverse transcription-PCR method was developed to determine whether the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus induced the expression of virulence genes coding for the thermostable direct hemolysin and for type III secretion system 2 (TTSS2). The culturability of clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4 degrees C was monitored, and the VBNC state was obtained. One day after entry into the VBNC state, temperature upshifts to 20 and 37 degrees C allowed the recovery of culturability. Standard curves for the relative quantification of expression of the housekeeping genes rpoS, pvsA, fur, and pvuA; the tdh2 gene; and the TTSS2 genes (VPA1354, VPA1346, and VPA1342) were established. The levels of expression of the pvsA and pvuA genes were stable and were used to normalize the levels of expression of the other genes. No transcriptional induction of the selected virulence genes under the temperature conditions used to recover the culturability of the VBNC bacteria was observed. The study results demonstrate that the recovery of culturability of VBNC cells of pathogenic V. parahaemolyticus is restricted to regrowth, without correlation with the induction of virulence gene expression. Disease induction would depend mainly on host-pathogen interactions that allow the expression of the virulence genes. This is the first time that the use of mRNA to detect viable cells was evaluated by computing the half-lives of multiple mRNA species under conditions inducing the VBNC state. [Abstract/Link to Full Text]

Harrison JJ, Ceri H, Yerly J, Rabiei M, Hu Y, Martinuzzi R, Turner RJ
Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms.
Appl Environ Microbiol. 2007 Aug;73(15):4940-9.
Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO(4)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), AsO(2)(-), and SeO(3)(2-)) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated "domed," "layer cake," "flat," and "mycelial." This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation. [Abstract/Link to Full Text]

de María N, Guevara A, Serra MT, García-Luque I, González-Sama A, García de Lacoba M, de Felipe MR, Fernández-Pascual M
Putative porin of Bradyrhizobium sp. (Lupinus) bacteroids induced by glyphosate.
Appl Environ Microbiol. 2007 Aug;73(16):5075-82.
Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed. [Abstract/Link to Full Text]

Spain AM, Peacock AD, Istok JD, Elshahed MS, Najar FZ, Roe BA, White DC, Krumholz LR
Identification and isolation of a Castellaniella species important during biostimulation of an acidic nitrate- and uranium-contaminated aquifer.
Appl Environ Microbiol. 2007 Aug;73(15):4892-904.
Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site. [Abstract/Link to Full Text]

Kamgang-Youbi G, Herry JM, Bellon-Fontaine MN, Brisset JL, Doubla A, Naïtali M
Evidence of temporal postdischarge decontamination of bacteria by gliding electric discharges: application to Hafnia alvei.
Appl Environ Microbiol. 2007 Aug;73(15):4791-6.
This study aimed to characterize the bacterium-destroying properties of a gliding arc plasma device during electric discharges and also under temporal postdischarge conditions (i.e., when the discharge was switched off). This phenomenon was reported for the first time in the literature in the case of the plasma destruction of microorganisms. When cells of a model bacterium, Hafnia alvei, were exposed to electric discharges, followed or not followed by temporal postdischarges, the survival curves exhibited a shoulder and then log-linear decay. These destruction kinetics were modeled using GinaFiT, a freeware tool to assess microbial survival curves, and adjustment parameters were determined. The efficiency of postdischarge treatments was clearly affected by the discharge time (t*); both the shoulder length and the inactivation rate k(max) were linearly modified as a function of t*. Nevertheless, all conditions tested (t* ranging from 2 to 5 min) made it possible to achieve an abatement of at least 7 decimal logarithm units. Postdischarge treatment was also efficient against bacteria not subjected to direct discharge, and the disinfecting properties of "plasma-activated water" were dependent on the treatment time for the solution. Water treated with plasma for 2 min achieved a 3.7-decimal-logarithm-unit reduction in 20 min after application to cells, and abatement greater than 7 decimal logarithm units resulted from the same contact time with water activated with plasma for 10 min. These disinfecting properties were maintained during storage of activated water for 30 min. After that, they declined as the storage time increased. [Abstract/Link to Full Text]

Kolvenbach B, Schlaich N, Raoui Z, Prell J, Zühlke S, Schäffer A, Guengerich FP, Corvini PF
Degradation pathway of bisphenol A: does ipso substitution apply to phenols containing a quaternary alpha-carbon structure in the para position?
Appl Environ Microbiol. 2007 Aug;73(15):4776-84.
The degradation of bisphenol A and nonylphenol involves the unusual rearrangement of stable carbon-carbon bonds. Some nonylphenol isomers and bisphenol A possess a quaternary alpha-carbon atom as a common structural feature. The degradation of nonylphenol in Sphingomonas sp. strain TTNP3 occurs via a type II ipso substitution with the presence of a quaternary alpha-carbon as a prerequisite. We report here a new degradation pathway of bisphenol A. Consequent to the hydroxylation at position C-4, according to a type II ipso substitution mechanism, the C-C bond between the phenolic moiety and the isopropyl group of bisphenol A is broken. Besides the formation of hydroquinone and 4-(2-hydroxypropan-2-yl)phenol as the main metabolites, further compounds resulting from molecular rearrangements consistent with a carbocationic intermediate were identified. Assays with resting cells or cell extracts of Sphingomonas sp. strain TTNP3 under an (18)O(2) atmosphere were performed. One atom of (18)O(2) was present in hydroquinone, resulting from the monooxygenation of bisphenol A and nonylphenol. The monooxygenase activity was dependent on both NADPH and flavin adenine dinucleotide. Various cytochrome P450 inhibitors had identical inhibition effects on the conversion of both xenobiotics. Using a mutant of Sphingomonas sp. strain TTNP3, which is defective for growth on nonylphenol, we demonstrated that the reaction is catalyzed by the same enzymatic system. In conclusion, the degradation of bisphenol A and nonylphenol is initiated by the same monooxygenase, which may also lead to ipso substitution in other xenobiotics containing phenol with a quaternary alpha-carbon. [Abstract/Link to Full Text]

Schleheck D, Knepper TP, Eichhorn P, Cook AM
Parvibaculum lavamentivorans DS-1T degrades centrally substituted congeners of commercial linear alkylbenzenesulfonate to sulfophenyl carboxylates and sulfophenyl dicarboxylates.
Appl Environ Microbiol. 2007 Aug;73(15):4725-32.
Commercial linear alkylbenzenesulfonate (LAS) contains 20 congeners of linear alkanes (C(10) to C(13)) substituted subterminally with the 4-sulfophenyl moiety in any position from lateral to central. Parvibaculum lavamentivorans DS-1(T) degrades each of eight laterally substituted congeners [e.g., 2-(4-sulfophenyl)decane (2-C10-LAS); herein, compounds are named systematically by chain length (e.g., C(10)) and by the position of the substituent on the chain (e.g., position 2)] to a major sulfophenyl carboxylate [SPC; here 3-(4-sulfophenyl)butyrate (3-C4-SPC)] and two minor products, namely, the alpha,beta-unsaturated SPC (SPC-2H, here 3-C4-SPC-2H) and the SPC+2C (here 5-C6-SPC) species (D. Schleheck, T. P. Knepper, K. Fischer, and A. M. Cook, Appl. Environ. Microbiol. 70:4053-4063). The degradation of centrally substituted congeners by strain DS-1 was examined in this work. 5-C10-LAS yielded not only the predicted 4-C8-SPC, 4-C8-SPC-2H, and 6-C10-SPC (about 70% of products) but also sulfophenyl dicarboxylates (SPdC), i.e., C6-, C8-, and C10-SPdC. These were identified by electrospray ionization-mass spectrometry (ESI-MS) after separation by high-pressure liquid chromatography (HPLC). ESI ion-trap MS and ESI-time of flight-MS were used to confirm the identities of key intermediates. Different mixtures of congeners obtained by separation of commercial LAS by HPLC were degraded, and the degradative products were compared. If a congener carried the sulfophenyl substituent on the 5, 6, or 7 position, SPdCs were formed as well as SPC, SPC-2H, and SPC+2C, whereas the substituent on the 2, 3, or 4 position yielded only SPC, SPC-2H, and SPC+2C. Some 50 products were generated from the 20 LAS congeners: 11 major SPCs, each with an SPC-2H and an SPC+2C (i.e., 33 SPC and SPC-2H species), and about 17 SPdC species. A large array of compounds, many in low quantities, is thus generated by P. lavamentivorans DS-1 during the degradation of commercial LAS. [Abstract/Link to Full Text]

Beutin L, Miko A, Krause G, Pries K, Haby S, Steege K, Albrecht N
Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes.
Appl Environ Microbiol. 2007 Aug;73(15):4769-75.
We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans. [Abstract/Link to Full Text]

Merod RT, Warren JE, McCaslin H, Wuertz S
Toward automated analysis of biofilm architecture: bias caused by extraneous confocal laser scanning microscopy images.
Appl Environ Microbiol. 2007 Aug;73(15):4922-30.
An increasing number of studies utilize confocal laser scanning microscopy (CLSM) for in situ visualization of biofilms and rely on the use of image analysis programs to extract quantitative descriptors of architecture. Recently, designed programs have begun incorporating procedures to automatically determine threshold values for three-dimensional CLSM image stacks. We have found that the automated threshold calculation is biased when a stack contains images lacking pixels of biological significance. Consequently, we have created the novel program Auto PHLIP-ML to resolve this bias by iteratively excluding extraneous images based on their area coverage of biomass. A procedure was developed to identify the optimal percent area coverage value used for extraneous image removal (PACVEIR). The optimal PACVEIR was defined to occur when the standard deviation of mean thickness, determined from replicate image stacks, was at a maximum, because it more accurately reflected inherent structural variation. Ten monoculture biofilms of either Ralstonia eutropha JMP228n::gfp or Acinetobacter sp. strain BD413 were tested to verify the routine. All biofilms exhibited an optimal PACVEIR between 0 and 1%. Prior to the exclusion of extraneous images, JMP228n::gfp appeared to develop more homogeneous biofilms than BD413. However, after the removal of extraneous images, JMP228n::gfp biofilms were found to form more heterogeneous biofilms. Similarly, JMP228n::gfp biofilms grown on glass surfaces vis-à-vis polyethylene membranes produced significantly different architectures after extraneous images had been removed but not when such images were included in threshold calculations. This study shows that the failure to remove extraneous images skewed a seemingly objective analysis of biofilm architecture and significantly altered statistically derived conclusions. [Abstract/Link to Full Text]

Albers E, Larsson C, Andlid T, Walsh MC, Gustafsson L
Effect of nutrient starvation on the cellular composition and metabolic capacity of Saccharomyces cerevisiae.
Appl Environ Microbiol. 2007 Aug;73(15):4839-48.
This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose. [Abstract/Link to Full Text]

Roohparvar R, Huser A, Zwiers LH, De Waard MA
Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters.
Appl Environ Microbiol. 2007 Aug;73(15):5011-9.
Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due to this pathogen on wheat seedlings. In vitro modulation of cyproconazole activity could be demonstrated in paper disk bioassays. Some of the active modulators (amitriptyline, flavanone, and phenothiazines) increased the accumulation of cyproconazole in M. graminicola, suggesting that they reversed cyproconazole efflux. However, synergism between cyproconazole and modulators against M. graminicola on wheat seedlings could not be shown. Despite their low in vitro toxicity to M. graminicola, some modulators (amitriptyline, loperamide, and promazine) did show significant intrinsic disease control activity in preventive and curative foliar spray tests with wheat seedlings. The results suggest that these compounds have indirect disease control activity based on modulation of fungal ABC transporters essential for virulence and constitute a new class of disease control agents. [Abstract/Link to Full Text]

Harris DM, van der Krogt ZA, van Gulik WM, van Dijken JP, Pronk JT
Formate as an auxiliary substrate for glucose-limited cultivation of Penicillium chrysogenum: impact on penicillin G production and biomass yield.
Appl Environ Microbiol. 2007 Aug;73(15):5020-5.
Production of beta-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol.mol(-1), an increasing rate of formate oxidation via a cytosolic NAD(+)-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol.mol(-1), the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as beta-lactams. [Abstract/Link to Full Text]

Lindner SN, Vidaurre D, Willbold S, Schoberth SM, Wendisch VF
NCgl2620 encodes a class II polyphosphate kinase in Corynebacterium glutamicum.
Appl Environ Microbiol. 2007 Aug;73(15):5026-33.
Corynebacterium glutamicum is able to accumulate up to 600 mM cytosolic phosphorus in the form of polyphosphate (poly P). Granular poly P (volutin) can make up to 37% of the internal cell volume. This bacterium lacks the classic enzyme of poly P synthesis, class I polyphosphate kinase (PPK1), but it possesses two genes, ppk2A (corresponds to NCgl0880) and ppk2B (corresponds to NCgl2620), for putative class II (PPK2) PPKs. Deletion of ppk2B decreased PPK activity and cellular poly P content, while overexpression of ppk2B increased both PPK activity and cellular poly P content. Neither deletion nor overexpression of ppk2A changed specific activity of PPK or cellular poly P content significantly. Purified PPK2B of C. glutamicum is active as a homotetramer and formed poly P with an average chain length of about 125, as determined with (31)P nuclear magnetic resonance. The catalytic efficiency of C. glutamicum PPK2B was higher in the poly P-forming direction than for nucleoside triphosphate formation from poly P. The ppk2B deletion mutant, which accumulated very little poly P and grew as C. glutamicum wild type under phosphate-sufficient conditions, showed a growth defect under phosphate-limiting conditions. [Abstract/Link to Full Text]

Koike S, Krapac IG, Oliver HD, Yannarell AC, Chee-Sanford JC, Aminov RI, Mackie RI
Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period.
Appl Environ Microbiol. 2007 Aug;73(15):4813-23.
To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc(r)) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc(r) genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tc(r) genes was quantified by real-time quantitative PCR. To confirm the Tc(r) gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc(r) genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc(r) genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool. [Abstract/Link to Full Text]

Yu M, Faan YW, Chung WY, Tsang JS
Isolation and characterization of a novel haloacid permease from Burkholderia cepacia MBA4.
Appl Environ Microbiol. 2007 Aug;73(15):4874-80.
Burkholderia cepacia MBA4 is a bacterium that can utilize 2-haloacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease mediates the uptake of haloacid. In this paper, we report the first cloning and characterization of such a haloacid permease. The structural gene, designated deh4p, was found 353 bases downstream of the dehalogenase gene deh4a. Quantitative analysis of the expression of deh4p showed that it was induced by monochloroacetate (MCA), to a level similar to the MCA-induced level of deh4a. The nucleotide sequence of deh4p was determined, and an open reading frame of 1,656 bp encoding a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. Deh4p has the signatures of sugar transport proteins and integral membrane proteins of the major facilitator superfamily. Uptake of [(14)C]MCA into the cell was Deh4p dependent. Deh4p has apparent K(m)s of 5.5 and 8.9 muM and V(max)s of 9.1 and 23.1 nmol mg(-1) min(-1) for acetate and MCA, respectively. A mutant with a transposon-inactivated haloacid operon failed to grow on MCA even when deh4a was provided in trans. [Abstract/Link to Full Text]

Elshahed MS, Youssef NH, Luo Q, Najar FZ, Roe BA, Sisk TM, Bühring SI, Hinrichs KU, Krumholz LR
Phylogenetic and metabolic diversity of Planctomycetes from anaerobic, sulfide- and sulfur-rich Zodletone Spring, Oklahoma.
Appl Environ Microbiol. 2007 Aug;73(15):4707-16.
We investigated the phylogenetic diversity and metabolic capabilities of members of the phylum Planctomycetes in the anaerobic, sulfide-saturated sediments of a mesophilic spring (Zodletone Spring) in southwestern Oklahoma. Culture-independent analyses of 16S rRNA gene sequences generated using Planctomycetes-biased primer pairs suggested that an extremely diverse community of Planctomycetes is present at the spring. Although sequences that are phylogenetically affiliated with cultured heterotrophic Planctomycetes were identified, the majority of the sequences belonged to several globally distributed, as-yet-uncultured Planctomycetes lineages. Using complex organic media (aqueous extracts of the spring sediments and rumen fluid), we isolated two novel strains that belonged to the Pirellula-Rhodopirellula-Blastopirellula clade within the Planctomycetes. The two strains had identical 16S rRNA gene sequences, and their closest relatives were isolates from Kiel Fjord (Germany), Keauhou Beach (HI), a marine aquarium, and tissues of marine organisms (Aplysina sp. sponges and postlarvae of the giant tiger prawn Penaeus monodon). The closest recognized cultured relative of strain Zi62 was Blastopirellula marina (93.9% sequence similarity). Detailed characterization of strain Zi62 revealed its ability to reduce elemental sulfur to sulfide under anaerobic conditions, as well as its ability to produce acids from sugars; both characteristics may potentially allow strain Zi62 to survive and grow in the anaerobic, sulfide- and sulfur-rich environment at the spring source. Overall, this work indicates that anaerobic metabolic abilities are widely distributed among all major Planctomycetes lineages and suggests carbohydrate fermentation and sulfur reduction as possible mechanisms employed by heterotrophic Planctomycetes for growth and survival under anaerobic conditions. [Abstract/Link to Full Text]

Wijnands LM, Dufrenne JB, van Leusden FM, Abee T
Germination of Bacillus cereus spores is induced by germinants from differentiated Caco-2 Cells, a human cell line mimicking the epithelial cells of the small intestine.
Appl Environ Microbiol. 2007 Aug;73(15):5052-4.
Spores of 11 enterotoxigenic strains of Bacillus cereus isolated from foods and humans adhered with similar efficiencies to Caco-2 cells, whereas subsequent germination triggering was observed with only 8 of these strains. Notably, Hep-2 cells did not trigger germination, while spores of all strains displayed similar germination efficiencies in brain heart infusion broth. [Abstract/Link to Full Text]

Kitphati W, Ngok-Ngam P, Suwanmaneerat S, Sukchawalit R, Mongkolsuk S
Agrobacterium tumefaciens fur has important physiological roles in iron and manganese homeostasis, the oxidative stress response, and full virulence.
Appl Environ Microbiol. 2007 Aug;73(15):4760-8.
In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4. [Abstract/Link to Full Text]

den Besten HM, Ingham CJ, van Hylckama Vlieg JE, Beerthuyzen MM, Zwietering MH, Abee T
Quantitative analysis of population heterogeneity of the adaptive salt stress response and growth capacity of Bacillus cereus ATCC 14579.
Appl Environ Microbiol. 2007 Aug;73(15):4797-804.
Bacterial populations can display heterogeneity with respect to both the adaptive stress response and growth capacity of individual cells. The growth dynamics of Bacillus cereus ATCC 14579 during mild and severe salt stress exposure were investigated for the population as a whole in liquid culture. To quantitatively assess the population heterogeneity of the stress response and growth capacity at a single-cell level, a direct imaging method was applied to monitor cells from the initial inoculum to the microcolony stage. Highly porous Anopore strips were used as a support for the culturing and imaging of microcolonies at different time points. The growth kinetics of cells grown in liquid culture were comparable to those of microcolonies grown upon Anopore strips, even in the presence of mild and severe salt stress. Exposure to mild salt stress resulted in growth that was characterized by a remarkably low variability of microcolony sizes, and the distributions of the log(10)-transformed microcolony areas could be fitted by the normal distribution. Under severe salt stress conditions, the microcolony sizes were highly heterogeneous, and this was apparently caused by the presence of both a nongrowing and growing population. After discriminating these two subpopulations, it was shown that the variability of microcolony sizes of the growing population was comparable to that of non-salt-stressed and mildly salt-stressed populations. Quantification of population heterogeneity during stress exposure may contribute to an optimized application of preservation factors for controlling growth of spoilage and pathogenic bacteria to ensure the quality and safety of minimally processed foods. [Abstract/Link to Full Text]

Rodríguez-Echeverría S, Crisóstomo JA, Freitas H
Genetic diversity of rhizobia associated with Acacia longifolia in two stages of invasion of coastal sand dunes.
Appl Environ Microbiol. 2007 Aug;73(15):5066-70.
We examined the genetic diversity of root nodule bacteria associated with the Australian legume Acacia longifolia in two stages of invasion of a coastal sand dune system. All isolates belonged to the genus Bradyrhizobium. A higher diversity was found in the long-established trees. The results suggest the introduction of exotic bradyrhizobia with the plant. [Abstract/Link to Full Text]

Wisselink HW, Toirkens MJ, del Rosario Franco Berriel M, Winkler AA, van Dijken JP, Pronk JT, van Maris AJ
Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose.
Appl Environ Microbiol. 2007 Aug;73(15):4881-91.
For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved. [Abstract/Link to Full Text]

Paredes CJ, Senger RS, Spath IS, Borden JR, Sillers R, Papoutsakis ET
A general framework for designing and validating oligomer-based DNA microarrays and its application to Clostridium acetobutylicum.
Appl Environ Microbiol. 2007 Jul;73(14):4631-8.
While DNA microarray analysis is widely accepted as an essential tool for modern biology, its use still eludes many researchers for several reasons, especially when microarrays are not commercially available. In that case, the design, construction, and use of microarrays for a sequenced organism constitute substantial, time-consuming, and expensive tasks. Recently, it has become possible to construct custom microarrays using industrial manufacturing processes, which offer several advantages, including speed of manufacturing, quality control, no up-front setup costs, and need-based microarray ordering. Here, we describe a strategy for designing and validating DNA microarrays manufactured using a commercial process. The 22K microarrays for the solvent producer Clostridium acetobutylicum ATCC 824 are based on in situ-synthesized 60-mers employing the Agilent technology. The strategy involves designing a large library of possible oligomer probes for each target (i.e., gene or DNA sequence) and experimentally testing and selecting the best probes for each target. The degenerate C. acetobutylicum strain M5 lacking the pSOL1 megaplasmid (with 178 annotated open reading frames [genes]) was used to estimate the level of probe cross-hybridization in the new microarrays and to establish the minimum intensity for a gene to be considered expressed. Results obtained using this microarray design were consistent with previously reported results from spotted cDNA-based microarrays. The proposed strategy is applicable to any sequenced organism. [Abstract/Link to Full Text]

Guinane CM, Cotter PD, Lawton EM, Hill C, Ross RP
Insertional mutagenesis to generate lantibiotic resistance in Lactococcus lactis.
Appl Environ Microbiol. 2007 Jul;73(14):4677-80.
While the potential emergence of food spoilage and pathogenic bacteria with resistance to lantibiotics is a concern, the creation of derivatives of starter cultures and adjuncts that can grow in the presence of these antimicrobials may have applications in food fermentations. Here a bank of Lactococcus lactis IL1403 mutants was created and screened, and a number of novel genetic loci involved in lantibiotic resistance were identified. [Abstract/Link to Full Text]

Moran MA, Belas R, Schell MA, González JM, Sun F, Sun S, Binder BJ, Edmonds J, Ye W, Orcutt B, Howard EC, Meile C, Palefsky W, Goesmann A, Ren Q, Paulsen I, Ulrich LE, Thompson LS, Saunders E, Buchan A
Ecological genomics of marine Roseobacters.
Appl Environ Microbiol. 2007 Jul;73(14):4559-69.
Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir-related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation. [Abstract/Link to Full Text]

Atterbury RJ, Van Bergen MA, Ortiz F, Lovell MA, Harris JA, De Boer A, Wagenaar JA, Allen VM, Barrow PA
Bacteriophage therapy to reduce salmonella colonization of broiler chickens.
Appl Environ Microbiol. 2007 Jul;73(14):4543-9.
Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by > or = 4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by > or = 2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens. [Abstract/Link to Full Text]

Gerba CP, Kennedy D
Enteric virus survival during household laundering and impact of disinfection with sodium hypochlorite.
Appl Environ Microbiol. 2007 Jul;73(14):4425-8.
This study was conducted to determine whether enteric viruses (adenovirus, rotavirus, and hepatitis A virus) added to cotton cloth swatches survive the wash cycle, the rinse cycle, and a 28-min permanent press drying cycle as commonly practiced in households in the United States. Detergent with and without bleach (sodium hypochlorite) was added to washing machines containing sterile and virus-inoculated 58-cm2 swatches, 3.2 kg of cotton T-shirts and underwear, and a soiled pillowcase designed to simulate the conditions (pH, organic load, etc.) encountered in soiled laundry. The most important factors for the reduction of virus in laundry were passage through the drying cycle and the addition of sodium hypochlorite. Washing with detergent alone was not found to be effective for the removal or inactivation of enteric viruses, as significant concentrations of virus were found on the swatches (reductions of 92 to 99%). It was also demonstrated that viruses are readily transferred from contaminated cloths to uncontaminated clothes. The use of sodium hypochlorite reduced the number of infectious viruses on the swatches after washing and drying by at least 99.99%. Laundering practices in common use in the United States do not eliminate enteric and respiratory viruses from clothes. The use of bleach can further reduce the numbers of enteric viruses in laundry. [Abstract/Link to Full Text]

Burt SA, van der Zee R, Koets AP, de Graaff AM, van Knapen F, Gaastra W, Haagsman HP, Veldhuizen EJ
Carvacrol induces heat shock protein 60 and inhibits synthesis of flagellin in Escherichia coli O157:H7.
Appl Environ Microbiol. 2007 Jul;73(14):4484-90.
The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as Escherichia coli O157:H7. Carvacrol is one of the major antibacterial components of these oils, and p-cymene is thought to be its precursor in the plant. The effects of carvacrol and p-cymene on protein synthesis in E. coli O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or p-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused E. coli O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (P < 0.05) and inhibited the synthesis of flagellin highly significantly (P < 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected. p-Cymene at 1 mM or 10 mM did not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor p-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in E. coli O157:H7. [Abstract/Link to Full Text]

Hotto AM, Satchwell MF, Boyer GL
Molecular characterization of potential microcystin-producing cyanobacteria in Lake Ontario embayments and nearshore waters.
Appl Environ Microbiol. 2007 Jul;73(14):4570-8.
The distribution and genotypic variation of potential microcystin (MC) producers along the southern and eastern shores of Lake Ontario in 2001 and 2003 were examined using a suite of PCR primers. Cyanobacterial, Microcystis sp., and Microcystis-specific toxin primer sets identified shoreline distribution of cyanobacterial DNA (in 97% of the stations) and MC synthetase genes (in 50% of the stations). Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena, and Planktothrix species indicated that the Microcystis sp. genotype was the dominant MC genotype present and revealed a novel Microcystis-like sequence containing a 6-bp insert. Analysis of the same samples with genus-specific mcyE primers confirmed that the Microcystis sp. genotype was the dominant potential MC producer. Genotype compositions within embayments were relatively homogenous compared to those for shoreline and tributary samples. MC concentrations along the shoreline exhibited both temporal and spatial differences as evidenced by the protein phosphatase inhibition assay, at times exceeding the World Health Organization guideline value for drinking water of 1.0 microg MC-LReq liter(-1). MC genotypes are widespread along the New York State shoreline of Lake Ontario, appear to originate nearshore, and can be carried through the lake via wind and surface water current patterns. [Abstract/Link to Full Text]

Ma YF, Wu JF, Wang SY, Jiang CY, Zhang Y, Qi SW, Liu L, Zhao GP, Liu SJ
Nucleotide sequence of plasmid pCNB1 from comamonas strain CNB-1 reveals novel genetic organization and evolution for 4-chloronitrobenzene degradation.
Appl Environ Microbiol. 2007 Jul;73(14):4477-83.
The nucleotide sequence of a new plasmid pCNB1 from Comamonas sp. strain CNB-1 that degrades 4-chloronitrobenzene (4CNB) was determined. pCNB1 belongs to the IncP-1beta group and is 91,181 bp in length. A total of 95 open reading frames appear to be involved in (i) the replication, maintenance, and transfer of pCNB1; (ii) resistance to arsenate and chromate; and (iii) the degradation of 4CNB. The 4CNB degradative genes and arsenate resistance genes were located on an extraordinarily large transposon (44.5 kb), proposed as TnCNB1. TnCNB1 was flanked by two IS1071 elements and represents a new member of the composite I transposon family. The 4CNB degradative genes within TnCNB1 were separated by various truncated genes and genetic homologs from other DNA molecules. Genes for chromate resistance were located on another transposon that was similar to the Tn21 transposon of the class II replicative family that is frequently responsible for the mobilization of mercury resistance genes. Resistance to arsenate and chromate were experimentally confirmed, and transcriptions of arsenate and chromate resistance genes were demonstrated by reverse transcription-PCR. These results described a new member of the IncP-1beta plasmid family, and the findings suggest that gene deletion and acquisition as well as genetic rearrangement of DNA molecules happened during the evolution of the 4CNB degradation pathway on pCNB1. [Abstract/Link to Full Text]

Woebken D, Fuchs BM, Kuypers MM, Amann R
Potential interactions of particle-associated anammox bacteria with bacterial and archaeal partners in the Namibian upwelling system.
Appl Environ Microbiol. 2007 Jul;73(14):4648-57.
Recent studies have shown that the anaerobic oxidation of ammonium by anammox bacteria plays an important role in catalyzing the loss of nitrogen from marine oxygen minimum zones (OMZ). However, in situ oxygen concentrations of up to 25 microM and ammonium concentrations close to or below the detection limit in the layer of anammox activity are hard to reconcile with the current knowledge of the physiology of anammox bacteria. We therefore investigated samples from the Namibian OMZ by comparative 16S rRNA gene analysis and fluorescence in situ hybridization. Our results showed that "Candidatus Scalindua" spp., the typical marine anammox bacteria, colonized microscopic particles that were likely the remains of either macroscopic marine snow particles or resuspended particles. These particles were slightly but significantly (P < 0.01) enriched in Gammaproteobacteria (11.8% +/- 5.0%) compared to the free-water phase (8.1% +/- 1.8%). No preference for the attachment to particles could be observed for members of the Alphaproteobacteria and Bacteroidetes, which were abundant (12 to 17%) in both habitats. The alphaproteobacterial SAR11 clade, the Euryarchaeota, and group I Crenarchaeota, were all significantly depleted in particles compared to their presence in the free-water phase (16.5% +/- 3.5% versus 2.6% +/- 1.7%, 2.7% +/- 1.9% versus <1%, and 14.9% +/- 4.6% versus 2.2% +/- 1.8%, respectively, all P < 0.001). Sequence analysis of the crenarchaeotal 16S rRNA genes showed a 99% sequence identity to the nitrifying "Nitrosopumilus maritimus." Even though we could not observe conspicuous consortium-like structures of anammox bacteria with particle-enriched bacterioplankton groups, we hypothesize that members of Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes play a critical role in extending the anammox reaction to nutrient-depleted suboxic water layers in the Namibian upwelling system by creating anoxic, nutrient-enriched microniches. [Abstract/Link to Full Text]

Miyake R, Kawamoto J, Wei YL, Kitagawa M, Kato I, Kurihara T, Esaki N
Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as the host.
Appl Environ Microbiol. 2007 Aug;73(15):4849-56.
A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4 degrees C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce beta-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4 degrees C and 139 mg/liter of culture at 18 degrees C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system. [Abstract/Link to Full Text]

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Recent Articles in Applied Microbiology

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Bayne HG, Michener HD
Growth of Staphylococcus and Salmonella on frankfurters with and without sodium nitrite.
Appl Microbiol. 1975 Nov;30(5):844-9.
Conventional and nitrite-free frankfurters in loosely wrapped packages were compared as to their ability to support growth of Salmonella, Staphylococcus, and their naturally occurring spoilage flora at 7 C (simulating refrigerated storage) and 20 C (simulating possible temperature abuse). At 7 C Salmonella did not grow in either type of frankfurter; Staphylococcus and the natural spoilage flora sometimes grew more rapidly in the absence of nitrite, but the difference was not significant. At 20 C growth of Salmonella, Staphylococcus, and of the spoilage flora was, at most, only slightly faster on nitrite-free frankfurters. Salmonella was not suppressed in broth culture experiments the pH and nitrite content found in frankfurters. Although either type of frankfurter can become hazardous due to growth of Salmonella or Staphylococcus, no unusual or additional hazard resulted from the omission of nitrite from frankfurters. [Abstract/Link to Full Text]

Smith JL, Huhtanen CN, Kissinger JC, Palumbo SA
Survival of salmonellae during pepperoni manufacture.
Appl Microbiol. 1975 Nov;30(5):759-63.
Survival of salmonellae in artificially contaminated beef-pork mixtures (approximately 10(4) salmonellae/g) was studied in pepperoni prepared by either a natural flora or lactic starter culture fermentation or in nonfermented sausages. The pepperoni did not become salmonellae free during the usual commercial 15 to 30-day drying period. Salmonella dublin was present in all products, fermented or unfermented, after 42 to 43 days of drying. At a lower level of contamination, 10(3)/g, S. dublin could not be recovered from starter culture-fermented pepperoni after 14 days of drying but persisted in the natural flora-fermented sausage. S. typhimurium (initial count, 10(4)/g) was absent after 42 days of drying when starter culture was used to ferment the pepperoni, but was still present in the natural flora-fermented and unfermented products. S. dublin, host adapted to cattle, or S. choleraesuis, host adapted to swine, had similar survival patterns in beef pork, or beef-pork pepperoni. Heating salmonellae contaminated beef-pork pepperoni (after fermantation but before drying) to an internal temperature of 60 C (trichinae inactivating) eliminated the food-borne pathogen from the sausage product. [Abstract/Link to Full Text]

Humber JY, Denny CB, Bohrer CW
Influence of pH on the heat inactivation of staphylococcal enterotoxin A as determined by monkey feeding and serological assay.
Appl Microbiol. 1975 Nov;30(5):755-8.
The effect of pH on the thermal inactivation of staphylococcal enterotoxin A was investigated. Analysis of heated toxin by immunodiffusion in gel indicated that enterotoxin A in beef bouillon was inactivated faster at pH 5.3 than at pH 6.2. The z values (slopes) for the heat inactivation curves at pH 6.2 and 5.3 were 49.5 and 55 F (about 27 and 30 C), respectively. Enterotoxin produced and heated in dialyzed Casamino Acids medium and assayed by monkey feeding was more easily inactivated by heat at pH 5.3 than at pH 7.8. Thermal inactivation curves for enterotoxin A in beef bouillon (5 mug/ml, pH 5.3) were determined by two methods, monkey feeding and serological assay. The z values for the curves obtained by these two methods were both 55 F, although loss of biological or toxic activity of the enterotoxin occurred before loss of serological activity. [Abstract/Link to Full Text]

Foster TL, Winans L
Psychrophilic microorganisms from areas associated with the Viking spacecraft.
Appl Microbiol. 1975 Oct;30(4):546-50.
Microorganisms capable of growth at 7 C were enumerated and isolated from soil samples from the manufacture and assembly areas of the Viking spacecraft. Populations ranging from 4.2 X 10(3) to 7.7 X 10(6)/g of soil were isolated from the 15 soil samples examined. Temperature requirements were determined, and those growing at 3 C, but not at 32 C, were designated as obligate psychrophiles in this investigation. Populations of soil bacteria, including aerobic sporeformers, ranging from 1.5 X 10(2) to 9.8 X 10(5)/g were capable of growth at 3 C, but not at 32 C. Bacterial isolates were identified to major generic groups. No psychrophilic sporeformers were isolated from soil from the manufacture area, but psychrophilic sporeformers ranged from 0 to 6.1 X 10(3)/g from soil from the assembly area. [Abstract/Link to Full Text]

O'toole DK
Effect of Incubation Conditions at 55 C on Moisture Loss from Agar Plates.
Appl Microbiol. 1975 Oct;30(4):692-693.
The percentage of moisture loss was least from plates incubated in plastic bags, sealed jars, or in a humid chamber. There was a stacking effect noted in plates incubated in plastic bags and sealed jars. [Abstract/Link to Full Text]

Sheldon RB, Boylen CW
Factors Affecting the Contribution by Epiphytic Algae to the Primary Productivity of an Oligotrophic Freshwater Lake.
Appl Microbiol. 1975 Oct;30(4):657-667.
A diatom-dominated population of epiphytic algae was studied in an oligotrophic lake to determine the factors which limit epiphyte growth and to measure their contribution to primary productivity. Algae were collected from plants growing at four sites in Lake George, N.Y., during the spring, summer, and fall of 1974. Samples were taken from 3 m, corresponding to the depth at which macrophytes were most productive. Algae exhibited an optimum temperature for HCO(3) uptake at 30 C, although the summer littoral lake temperature ranged from 18 to 25 C. Light saturation occurred at an intensity of 8,608 lux, approximating the environmental intensity at the depth from which algae were taken. Epiphytes exhibited their maximum photosynthetic capacity of 0.6 mg of carbon fixed/m of macrophyte surface area per h in the early afternoon in mid-August. They assimilated approximately 5% as much inorganic carbon as the macrophytes from which they were taken. Epiphyte population densities followed the seasonal growth patterns of the macrophytes, with maximal leaf colonization remaining essentially constant relative to the leaf position on the plant. There was little change in density between sampling sites at any given time. Productivities of epiphytes from bottom leaves were 10-fold greater than those of epiphytes from top leaves. Addition of PO(4), NO(3), NH(3), Si, and SO(4) had no stimulatory effect on photosynthesis. Addition of HCO(3) stimulated photosynthesis greater than 30%, suggesting that carbon may be a limiting nutrient for epiphytic algae in Lake George. [Abstract/Link to Full Text]

Wardle MD, Renninger GM
Bactericidal effect of hydrogen peroxide on spacecraft isolates.
Appl Microbiol. 1975 Oct;30(4):710-1.
Solutions of 3, 10, and 15% hydrogen peroxide were found to have pronounced bactericidal effects, as a function of time of exposure, on sporeformers and non-sporeformers isolated from spacecraft. [Abstract/Link to Full Text]

Oremland RS, Taylor BF
Inhibition of methanogenesis in marine sediments by acetylene and ethylene: validity of the acetylene reduction assay for anaerobic microcosms.
Appl Microbiol. 1975 Oct;30(4):707-9.
Methanogenesis was irreversibly inhibited in sediments by concentrations of acetylene employed in nitrogen fixation assays (1 to 20%, vol/vol). Ethylene, but not ethane, also stopped methane production, and the inhibition was reversed by gassing with hydrogen. [Abstract/Link to Full Text]

Halls NA, Ayres JC
Factors affecting the production of sterigmatocystin in semisynthetic media.
Appl Microbiol. 1975 Oct;30(4):702-3.
Production of sterigmatocystin by Aspergillus versicolor was stimulated by inorganic phosphate when used in conjunction with citric acid cycle compounds. [Abstract/Link to Full Text]

Bukovic JA, Johnson HM
Staphylococcal enterotoxin C: solid-phase radioimmunoassay.
Appl Microbiol. 1975 Oct;30(4):700-1.
A solid-phase radioimmunoassay test employing 125I-labeled enterotoxin C and polystyrene tubes coated with specific antibody was used for the detection and quantitation of entertoxin C in condensed milk, cheddar cheese, custard, and ham salad. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 16% or less. [Abstract/Link to Full Text]

Koltin Y, Day PR
Specificity of Ustilago maydis killer proteins.
Appl Microbiol. 1975 Oct;30(4):694-6.
Bacteria and fungi were tested for sensitivity to Ustilago maydis killer strains carrying virus-like particles. Various species taxonomically related to U. maydis were sensitive. [Abstract/Link to Full Text]

Bauchop T, Clarke RT, Newhook JC
Scanning electron microscope study of bacteria associated with the rumen epithelium of sheep.
Appl Microbiol. 1975 Oct;30(4):668-75.
Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery of bacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed. [Abstract/Link to Full Text]

Schwarz JR, Colwell RR
Heterotrophic activity of deep-sea sediment bacteria.
Appl Microbiol. 1975 Oct;30(4):639-49.
Sediment samples, containing mixed microbial populations that were decompressed during retrieval from 7,750 and 8,130 m in the Puerto Rican Trench, were recompressed and incubated at the approximate in situ temperature (3 C) and pressure (775 or 815 atm) in the presence of 14C-labeled amino acids. Heterotrophic activity (total uptake, CO2 respiration, and cellular assimilation) and cellular-associated "pool" concentrations were measured. Compared with atmospheric controls held at 3 C, the total uptake at elevated pressure at 3 C was reduced, on an average, 55 times, CO2 respiration was reduced 45 times, and cellular assimilation was reduced 69 times. Rate of total uptake at elevated pressure was found to range from 4.0 X 10(-11) mug/cell per h for leucine to 2.61 X 10(-10) mug/cell per h for an amino acid mixture. Also, the percentage of total uptake at elevated pressures, respired as CO2, increased at the expense of cellular assimilation (ca. 22% increase). Two cellular-associated amino acid pools were detected, a large, loosely bound, outer pool and a small, tightly bound internal pool. The loosely bound outer pool was removed by a change in the pH of the incubation medium. Even though heterotrophic uptake and the outer, cellular-associated pool were markedly reduced at an elevated pressure, the percentage of total uptake calculated for the unincorporated, tightly bound, intracellular pool was 2 to 19 times that obtained for cultures held at 1 atm. The results were interpreted as indicating that bacterial metabolism and biosynthesis in the deep sea are markedly reduced, with a greater proportion of metabolic activity devoted to cellular maintenance. [Abstract/Link to Full Text]

Whitt DD, Demoss RD
Effect of microflora on the free amino acid distribution in various regions of the mouse gastrointestinal tract.
Appl Microbiol. 1975 Oct;30(4):609-15.
The distribution of free amino acids in the contents of various regions of the gastrointestinal tract (stomach, upper small intestine, lower small intestine, cecum, upper colon and lower colon) was studied in germfree and conventionalized mice. Particular emphasis was placed on the conversion of tryptophan to indole as a probe for studying intermicrobial interactions and microbe-host interactions in vivo. Great differences were observed in the free amino acid content of the various regions of the digestive tract in each type of mouse and also in any one region between germfree and conventionalized mice. As would be expected, there were fewer differences in amino acid distribution between the types of mice in both regions of the small intestine. This correlates with a much lower population of microorganisms in these regions. The changes in free amino acid content and distribution produced by microflora are great enough to serve as a good probe for studying the interactions of a limited number of species of microbes in gnotobiotic animals and assign possible specific functions to each species. [Abstract/Link to Full Text]

Oremland RS
Methane production in shallow-water, tropical marine sediments.
Appl Microbiol. 1975 Oct;30(4):602-8.
The in situ production of methane was monitored in several types of tropical benthic communities. A bed of Thalassia testudinum located in Caesar Creek (Florida Keys) exhibited the highest methanogenic activity (initial rates = 1.81 to 1.86 mumol CH4/m2 per h) as compared with another seagrass (Syringodium sp., 0.15 to 0.33 mumol/m2 per h) and two coral reef environments (Hydro-Lab, 0.016 to 0.10 mumol/m2 per h; Curacao, 0.14 to 0.47 mumol/m2 per h). The results suggest that a wide variety of benthic metabolic processes (e.g., photosynthetic oxygen production) influences methane production rates. [Abstract/Link to Full Text]

Wells JM, Payne JA
Toxigenic Aspergillus and Penicillium isolates from weevil-damaged chestnuts.
Appl Microbiol. 1975 Oct;30(4):536-40.
Aspergillus and Penicillium were among the most common genera of fungi isolated on malt-salt agar from weevil-damaged Chinese chestnut kernels (16.8 and 40.7% occurrence, respectively). Chloroform extracts of 21 of 50 Aspergillus isolates and 18 of 50 representative Penicillium isolates, grown for 4 weeks at 21.1 C on artificial medium, were toxic to day-old cockerels. Tweleve of the toxic Aspergillus isolates were identified as A. wentii, eight as A. flavus, and one as A. flavus var. columnaris. Nine of the toxic Penicillium isolates were identified as P. terrestre, three as P. steckii, two each as P. citrinum and P. funiculosum, and one each as P. herquei (Series) and P. roqueforti (Series). Acute diarrhea was associated with the toxicity of A. wentii and muscular tremors with the toxicity of P. terrestre, one isolate of P. steckii, and one of P. funiculosum. [Abstract/Link to Full Text]

Oie SH, Fystro D
Efficacy of the inactivation of bacterial spores in white petrolatum and a hydrophilic ointment by gamma irradiation.
Appl Microbiol. 1975 Oct;30(4):514-8.
To evaluate the possibilities of using gamma irradiation for the sterilization of ointments, the effect of irradiation on spores of Bacillus pumilus and Bacillus sphaericus in dry material and in two different kinds of ointments was studied. The results indicate that for sterilization purposes irradiation was less effective in white petrolatum as compared to irradiation in the dry state. No such protective effect was found in a hydrophilic ointment. Accordingly, the sterilization dose needed for the sterilization of an ointment can be decided upon only after inactivation experiments with suitable test organisms in the actual preparation. [Abstract/Link to Full Text]

Green BL, Clausen E, Litsky W
Comparison of the new millipore HC with conventional membrane filters for the enumeration of fecal coliform bacteria.
Appl Microbiol. 1975 Oct;30(4):697-9.
Fecal coliform recoveries were determined for six types of membrane filters using 65 nonchlorinated water samples. Results showed that the membranes could be ranked in order of decreasing recovery as follows: Millipore HC greater than Gelman greater than Johns-Manville approximately Sartorius greater than Millipore HA greater than Schleicher & Schuell. [Abstract/Link to Full Text]

Sladek KJ, Suslavich RV, Sohn BI, Dawson FW
Optimum membrane structures for growth of coliform and fecal coliform organisms.
Appl Microbiol. 1975 Oct;30(4):685-91.
The purpose of this study was to determine the optimum membrane filter structure and characteristics for recovery of coliform organisms. Additionally, other factors such as sterilization method and membrane composition were examined. Fecal coliform growth tests with varied samples indicated that the most critical factor in recovery was surface pore morphology and not other factors previously suspected. Fecal coliform counts showed a dramatic increase, with increasing surface opening sizes. Membrane structures with surface openings large enough to surround the entrapped bacteria are required for optimum growth of fecal coliform organisms. Maximum fecal coliform recoveries are obtained using membranes composed of mixed esters of cellulose exhibiting a surface opening diameter of 2.4 mum and a retention pore size of 0.7 mum. [Abstract/Link to Full Text]

Fliermans CB, Schmidt EL
Autoradiography and immunofluorescence combined for autecological study of single cell activity with Nitrobacter as a model system.
Appl Microbiol. 1975 Oct;30(4):676-84.
Specific detection of a particular bacterium by immunofluorescence was combined with estimation of its metabolic activity by autoradiography. The nitrifying bacteria Nitrobacter agilis and N. winogradskyi were used as a model system. Nitrobacter were incubated with NaH14CO3 and 14CO2 prior to study. The same preparations made for autoradiograms were stained with fluorescent antibodies specific for the Nitrobacter species. Examination by epifluorescence and transmitted dark-field microscopy revealed Nitrobacter cells with and without associated silver grains. Direct detection and simultaneous evaluation of metabolic activity of Nitrobacter was demonstrated in pure cultures, in a simple mixed culture, and in a natural soil. [Abstract/Link to Full Text]

Thomson LA, Hageage GJ
Evaluation of excitation light sources for incident immunofluorescence microscopy.
Appl Microbiol. 1975 Oct;30(4):616-24.
A variety of fluorescent excitation light sources were compared using a standard fluorescein solution or a bacterial conjugate with immunofluorescent microscopy. Quantitative data were obtained with microscope photometric apparatus. Both the quantitative data and comparative conjugate titering suggest that the 450-W xenon arc excited significantly more fluorescence than did the more commonly used 250-W mercury arc or the 100-W halogen lamp. The conjugate could be diluted 4 to 32 times more using the 450-W xenon. Additional advantages of 450-W xenon excitation include sufficient energy of wave lengths between 470 to 490 mm, thus permitting narrow-band excitation resulting in less autofluorescence and the ability to perform fluorescent-antibody procedures without the darkening of ambient room light. [Abstract/Link to Full Text]

Newman JS, O'Brien RT
Gas chromatographic presumptive test for coliform bacteria in water.
Appl Microbiol. 1975 Oct;30(4):584-8.
A gas chromatographic procedure which shows promise as a presumptive test for coliform bacteria in water is described. Total coliform bacteria concentrations were determined from the incubation times at 37 C required for ethanol to be produced. Fecal coliform densities were determined in a similar manner at 44.5 C. The culture medium was filter sterilized M-9 salts supplemented with 1% lactose, 0.1% Casamino Acids, and 0.1% yeast extract. Best results were obtained when the initial total coliform concentrations were 5 per ml or higher and when fecal coliform concentrations were 50 per ml or higher. Minimum detection times at these concentrations were 9 and 12 h, respectively. [Abstract/Link to Full Text]

Thomason BM, Hebert GA, Cherry WB
Evaluation of a semiautomated system for direct fluorescent antibody detection of salmonellae.
Appl Microbiol. 1975 Oct;30(4):557-64.
A semi-automatic system under development by Aerojet Medical and Biological Systems for the direct fluorescent antibody detection of salmonellae was evaluated with various food, feed, and environmental samples. All samples were simultaneously examined by Automated Bioassay System (ABS), manual direct fluorescent antibody procedures and cultural procedures. The ABS gave satisfactory results with the processed samples. It detected all of the culturally positive powdered egg and candy samples with no false-negative results and gave only 6.6 and 5.3% false-positive rates, respectively. With meatmeal samples the ABS failed to detect one culturally positive specimen that was also positive by manual fluorescent antibody and gave one (1.1%) false-positive result. A high rate of false-negative results was obtained by ABS on unprocessed samples of creek water, poultry, and sausage. Adding another enrichment step to the protocol reduced the false-negative rate considerably but severely increased the false-positive rate. The instruments worked reasonably well, but research is needed to improve enrichment procedures for samples to be processed by the system. [Abstract/Link to Full Text]

Voss JG
Effects of an antibacterial soap on the ecology of aerobic bacterial flora of human skin.
Appl Microbiol. 1975 Oct;30(4):551-6.
The effects of ad lib use of an antibacterial soap containing 1.0% trichlorocarbanilide and 0.5% trifluoromethyldichlorocarbanilide on the bacterial flora of six skin sites of 132 subjects were measured by comparison with the flora of 93 control subjects who avoided the use of topical antibacterials. Each subject was examined once. The test soap produced significant reductions in geometric mean counts of the total aerobic flora on the back, chest, forearm, calf, and foot; counts were also reduced in the axilla, but not to a significant extent. The overall reduction by the test soap on all sites was 62% (P less than 0.001). Neither age nor sex influenced the effect of the soap on the flora. The antibacterial soap also reduced the prevalence of Staphylococcus aureus on the skin, mostly by virtually eliminating it from areas other than the axilla. Partial inhibition of the gram-positive flora was not accompanied by an increase in gram-negative species. The latter were found principally in the axilla; Klebsiella pneumoniae and Enterobacter aerogenes were the species most frequently found. [Abstract/Link to Full Text]

Sharpe AN, Michaud GL
Enumeration of high numbers of bacteria using hydrophobic grid-membrane filters.
Appl Microbiol. 1975 Oct;30(4):519-24.
Printing a wax grid on a conventional membrane filter yields a device functioning as a most probable number apparatus (MPN), used at a single dilution but with a very large number of growth compartments (e.g., 3,650). By restraining the lateral spread and confluence of colonies, the hydrophobic grid-membrane filter (HGMF) allows growth- or colony-forming units (GU) to be resolved at levels far above those which produce an uncountable lawn on a conventional membrane filter. It also eliminates the size variation of normal bacterial colonies. As a result, the HGMF can give more accurate estimates of the concentration of GU. The method by which grid-cell count observations can be used to obtain MPN estimates of the number of GUs is described, and estimates obtained using the MPN method on the HGMF are compared with those resulting from conventional colony count procedures on membrane filters. A linear relation was observed between MPNGU and the number of GUs, at levels up to 30,000 GUs, for pure cultures of bacteria and for samples of natural waters. The HGMF has great potential for reducing the labor required in quantitative microbiology, since it allows, with one filter, enumeration of microorganisms over a very large concentration range and therefore reduces the need to make dilutions. [Abstract/Link to Full Text]

Robern H, Dighton M, Yano Y, Dickie N
Double-antibody radioimmunoassay for staphylococcal enterotoxin C2.
Appl Microbiol. 1975 Oct;30(4):525-9.
A sensitive double-antibody radioimmunoassay for staphylococcal enterotoxin C2 is described. The assay procedure employs anti-rabbit gamma globulin, prepared in goats, to precipitate the antigen-antibody complex of enterotoxin C2 and anti-enterotoxin C2. The test is sensitive to 100 pg of enterotoxin. [Abstract/Link to Full Text]

Kayser WV, Hickman KC, Bond WW, Favero MS, Carson LA
Bacteriological evaluation of an ultra-pure water-distilling system.
Appl Microbiol. 1975 Oct;30(4):704-6.
A prototype distillation and storage system with recycle for producing ultrapure water was monitored for bacteriological contamination during a period of 24 months. Naturally occurring Pseudomonas aeruginosa and P. cepacia were found to grow rapidly to levels of about 10(5)/ml in water taken from the storage reservoir and also in commercially prepared distilled water. The system was found to eliminate bacterial contaminants introduced into the still with the feed water, but the reservoir, once contaminated, remained contaminated during prolonged recycle, After a single treatment with free chlorine, the entire system remained uncontaminated until accidental or purposeful shutdown. [Abstract/Link to Full Text]

Sayler GS, Nelson JD, Justice A, Colwell RR
Distribution and significance of fecal indicator organisms in the Upper Chesapeake Bay.
Appl Microbiol. 1975 Oct;30(4):625-38.
Total viable aerobic, heterotrophic bacteria, total coliforms, fecal coliforms, and fecal streptococci were enumerated in samples collected at five stations located in the Upper Chesapeake Bay, December 1973 through December 1974. Significant levels of pollution indicator organisms were detected at all of the stations sampled. Highest counts were observed in samples collected at the confluence of the Susquehanna River and the Chesapeake Bay. The indicator organisms examined were observed to be quantitatively distributed independently of temperature and salinity. Counts were not found to be correlated with concentration of suspended sediment. However, significant proportions of both the total viable bacteria (53%) and fecal indicator organisms (greater than 80%) were directly associated with suspended sediments. Correlation coefficinets (r) for the indicator organisms examined in this study ranged from r = 0.80 to r = 0.99 for bottom water and suspended sediment, respectively. Prolonged survival of fecal streptococci in most of the sediment samples was observed, with concomitant reduction of the correlation coefficient from r = 0.99, fecal streptocicci to total coliforms in water, to r = 0.01, fecal streptococci to fecal coliforms in sediments. The results of this study compared favorably with fecal coliforms: fecal streptococci ratios for the various sample types. Characterization of organisms beyond the confirmed most-probable-number procedure provided good correlation between bacterial indicator groups. [Abstract/Link to Full Text]

Highsmith AK, Abshire RL
Evaluation of a most-probable-number technique for the enumeration of Pseudomonas aeruginosa.
Appl Microbiol. 1975 Oct;30(4):596-601.
A most-probable-number (MPN) technique was evaluated for detecting and enumerating Pseudomonas aeruginosa in water and wastewater. Both the presumptive and confirmatory media, as described in the 13th edition of Standard Methods for the Examination of Water and Wastewater, as well as modifications of these media were included in evaluations. Various samples of water were tested, namely chlorinated tap water, creek water, and influent to a wastewater treatment plant. Modified media repeatedly gave higher estimated MPNs of P. aeruginosa than media listed in Standard Methods. P. aeruginosa was detected and recovered from all creek water and wastewater samples, but not from tap water samples tested. This organism was determined to be present in as large numbers as the fecal coliforms and in even greater quantities than the fecal streptococci in all samples, whenever MPN estimations were determined from those positive tubes containing the modified confirmatory medium. [Abstract/Link to Full Text]

Wilson DM, Huang LH, Jay E
Survival of Aspergillus flavus and Fusarium moniliforme in high-moisture corn stored under modified atmospheres.
Appl Microbiol. 1975 Oct;30(4):592-5.
Freshly harvested high-moisture corn with 29.4% moisture and corn remoistened to 19.6% moisture were inoculated with Aspergillus flavus Link ex Fr. and stored for 4 weeks at about 27 C in air (0.03% CO2, 21% O2, and 78% N2) and three modified atmospheres: (i) 99.7% N2 and 0.3% O2; (ii) 61.7% CO2, 8.7% O2, and 29.6% N2; and (iii) 13.5% CO2, 0.5% O2, and 84.8% N2. Kernel infections by A. flavus, Fusarium moniliforme (Sheld.) Snyd. et Hans., and other fungi were monitored weekly. The modified-atmosphere treatments delayed deterioration by A. flavus and F. moniliforme, but their growth was not completely stopped. A. flavus survived better in the remoistened than in the freshly harvested corn. F. moniliforme survived in both. A. flavus and F. moniliforme were the dominant fungi in corn removed from the modified atmospheres and exposed to normal air for 1 week. [Abstract/Link to Full Text]

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Recent Articles in Bacteriological Reviews

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Greenawalt JW, Whiteside TL
Mesosomes: membranous bacterial organelles.
Bacteriol Rev. 1975 Dec;39(4):405-63. [Abstract/Link to Full Text]

Lovett JS
Growth and differentiation of the water mold Blastocladiella emersonii: cytodifferentiation and the role of ribonucleic acid and protein synthesis.
Bacteriol Rev. 1975 Dec;39(4):345-404. [Abstract/Link to Full Text]

Wiseman GM
The hemolysins of Staphylococcus aureus.
Bacteriol Rev. 1975 Dec;39(4):317-44. [Abstract/Link to Full Text]

Cronan JE, Gelmann EP
Physical properties of membrane lipids: biological relevance and regulation.
Bacteriol Rev. 1975 Sep;39(3):232-56. [Abstract/Link to Full Text]

Drlica KA, Kado CI
Crown gall tumors: are bacterial nucleic acids involved?
Bacteriol Rev. 1975 Sep;39(3):186-96. [Abstract/Link to Full Text]

Hemphill HE, Whiteley HR
Bacteriophages of Bacillus subtilis.
Bacteriol Rev. 1975 Sep;39(3):257-315. [Abstract/Link to Full Text]

Rattray JB, Schibeci A, Kidby DK
Lipids of yeasts.
Bacteriol Rev. 1975 Sep;39(3):197-231. [Abstract/Link to Full Text]

Hooks JJ, Gibbs CJ
The foamy viruses.
Bacteriol Rev. 1975 Sep;39(3):169-85. [Abstract/Link to Full Text]

Morita RY
Psychrophilic bacteria.
Bacteriol Rev. 1975 Jun;39(2):144-67. [Abstract/Link to Full Text]

Schwab JH
Suppression of the immune response by microorganisms.
Bacteriol Rev. 1975 Jun;39(2):121-43. [Abstract/Link to Full Text]

Crawford IP
Gene rearrangements in the evolution of the tryptophan synthetic pathway.
Bacteriol Rev. 1975 Jun;39(2):87-120. [Abstract/Link to Full Text]

Lacey RW
Antibiotic resistance plasmids of Staphylococcus aureus and their clinical importance.
Bacteriol Rev. 1975 Mar;39(1):1-32. [Abstract/Link to Full Text]

Clarke CH, Shankel DM
Antimutagenesis in microbial systems.
Bacteriol Rev. 1975 Mar;39(1):33-53. [Abstract/Link to Full Text]

Collier RJ
Diphtheria toxin: mode of action and structure.
Bacteriol Rev. 1975 Mar;39(1):54-85. [Abstract/Link to Full Text]

Collins FM
Vaccines and cell-mediated immunity.
Bacteriol Rev. 1974 Dec;38(4):371-402. [Abstract/Link to Full Text]

MacKenzie JS, Houghton M
Influenza infections during pregnancy: association with congenital malformations and with subsequent neoplasms in children, and potential hazards of live virus vaccines.
Bacteriol Rev. 1974 Dec;38(4):356-70. [Abstract/Link to Full Text]

Docherty JJ, Chopan M
The latent herpes simplex virus.
Bacteriol Rev. 1974 Dec;38(4):337-55. [Abstract/Link to Full Text]

Blumberg PM, Strominger JL
Interaction of penicillin with the bacterial cell: penicillin-binding proteins and penicillin-sensitive enzymes.
Bacteriol Rev. 1974 Sep;38(3):291-335. [Abstract/Link to Full Text]

Lanyi JK
Salt-dependent properties of proteins from extremely halophilic bacteria.
Bacteriol Rev. 1974 Sep;38(3):272-90. [Abstract/Link to Full Text]

McClung LS, Meyer KF
Beginnings of bacteriology in California.
Bacteriol Rev. 1974 Sep;38(3):251-71. [Abstract/Link to Full Text]

Preer JR, Preer LB, Jurand A
Kappa and other endosymbionts in Paramecium aurelia.
Bacteriol Rev. 1974 Jun;38(2):113-63. [Abstract/Link to Full Text]

Hartwell LH
Saccharomyces cerevisiae cell cycle.
Bacteriol Rev. 1974 Jun;38(2):164-98. [Abstract/Link to Full Text]

Slater M, Schaechter M
Control of cell division in bacteria.
Bacteriol Rev. 1974 Jun;38(2):199-221. [Abstract/Link to Full Text]

Grappel SF, Bishop CT, Blank F
Immunology of dermatophytes and dermatophytosis.
Bacteriol Rev. 1974 Jun;38(2):222-50. [Abstract/Link to Full Text]


Aldolase of Lactic Acid Bacteria: a Case History in the Use of an Enzyme as an Evolutionary Marker.
Bacteriol Rev. 1974 Mar;38(1):111.
[This corrects the article on p. 453 in vol. 37.]. [Abstract/Link to Full Text]

Costerton JW, Ingram JM, Cheng KJ
Structure and function of the cell envelope of gram-negative bacteria.
Bacteriol Rev. 1974 Mar;38(1):87-110. [Abstract/Link to Full Text]

Stebbing N
Precursor pools and endogenous control of enzyme synthesis and activity in biosynthetic pathways.
Bacteriol Rev. 1974 Mar;38(1):1-28. [Abstract/Link to Full Text]

Fox EN
M proteins of group A streptococci.
Bacteriol Rev. 1974 Mar;38(1):57-86. [Abstract/Link to Full Text]

Lemke PA, Nash CH
Fungal viruses.
Bacteriol Rev. 1974 Mar;38(1):29-56. [Abstract/Link to Full Text]

Pace NR
Structure and synthesis of the ribosomal ribonucleic acid of prokaryotes.
Bacteriol Rev. 1973 Dec;37(4):562-603. [Abstract/Link to Full Text]

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Recent Articles in BMC Immunology

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Moro M, Cecconi V, Martinoli C, Dallegno E, Giabbai B, Degano M, Glaichenhaus N, Protti MP, Dellabona P, Casorati G
Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen- or tumour-derived synthetic peptides.
BMC Immunol. 2005;624.
BACKGROUND: MHC class I-peptide tetramers are currently utilised to characterize CD8+ T cell responses at single cell level. The generation and use of MHC class II tetramers to study antigen-specific CD4+ T cells appears less straightforward. Most MHC class II tetramers are produced with a homogeneously built-in peptide, reducing greatly their flexibility of use. We attempted the generation of "empty" functional HLA-DR*1101 tetramers, receptive for loading with synthetic peptides by incubation. No such reagent is in fact available for this HLA-DR allele, one of the most frequent in the Caucasian population. RESULTS: We compared soluble MHC class II-immunoglobulin fusion proteins (HLA-DR*1101-Ig) with soluble MHC class II protein fused with an optimised Bir site for enzymatic biotynilation (HLA-DR*1101-Bir), both produced in insect cells. The molecules were multimerised by binding fluorochrome-protein A or fluorochrome-streptavidin, respectively. We find that HLA-DR*1101-Bir molecules are superior to the HLA-DR*1101-Ig ones both in biochemical and functional terms. HLA-DR*1101-Bir molecules can be pulsed with at least three different promiscuous peptide epitopes, derived from Tetanus Toxoid, influenza HA and the tumour associated antigen MAGE-3 respectively, to stain specific CD4+ T cells. Both staining temperature and activation state of CD4+ T cells are critical for the binding of peptide-pulsed HLA-DR*1101-Bir to the cognate TCR. CONCLUSION: It is therefore possible to generate a soluble recombinant HLA-DR*1101 backbone that is receptive for loading with different peptides to stain specific CD4+ T cells. As shown for other HLA-DR alleles, we confirm that not all the strategies to produce soluble HLA-DR*1101 multimers are equivalent. [Abstract/Link to Full Text]

Heinemann DE, Peters JH
Follicular dendritic-like cells derived from human monocytes.
BMC Immunol. 2005;623.
BACKGROUND: Follicular dendritic cells (FDCs) play a central role in controlling B-cell response maturation, isotype switching and the maintenance of B-cell memory. These functions are based on prolonged preservation of antigen and its presentation in its native form by FDCs. However, when entrapping entire pathogens, FDCs can turn into dangerous long-term reservoirs that may preserve viruses or prions in highly infectious form.Despite various efforts, the ontogeny of FDCs has remained elusive. They have been proposed to derive either from bone marrow stromal cells, myeloid cells or local mesenchymal precursors. Still, differentiating FDCs from their precursors in vitro may allow addressing many unsolved issues associated with the (patho-) biology of these important antigen-presenting cells. The aim of our study was to demonstrate that FDC-like cells can be deduced from monocytes, and to develop a protocol in order to quantitatively generate them in vitro. RESULTS: Employing highly purified human monocytes as a starter population, low concentrations of Il-4 (25 U/ml) and GM-CSF (3 U/ml) in combination with Dexamethasone (Dex) (0.5 microM) in serum-free medium trigger the differentiation into FDC-like cells. After transient de-novo membrane expression of alkaline phosphatase (AP), such cells highly up-regulate surface expression of complement receptor I (CD35). Co-expression of CD68 confirms the monocytic origin of both, APpos and CD35pos cells. The common leukocyte antigen CD45 is strongly down-regulated. Successive stimulation with TNF-alpha up-regulates adhesion molecules ICAM-1 (CD54) and VCAM (CD106). Importantly, both, APpos as well as APneg FDC-like cells, heterotypically cluster with and emperipolese B cells and exhibit the FDC characteristic ability to entrap functionally preserved antigen for prolonged times. Identical characteristics are found in monocytes which were highly expanded in vitro by higher doses of GM-CSF (25 U/ml) in the absence of Dex and Il-4 before employing the above differentiation cocktail. CONCLUSION: In this work we provide evidence that FDC-like cells can be derived from monocytes in vitro. Monocyte-derived FDC-like cells quantitatively produced offer a broad utility covering basic research as well as clinical application. [Abstract/Link to Full Text]

Khanna AK
Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation.
BMC Immunol. 2005;622.
BACKGROUND: Immune activation that results due to the aberrant proliferation of lymphocytes leads to inflammation and graft rejection in organ transplant recipients. We hypothesize that the cell cycle control and inflammation are parallel events, inhibition of cellular proliferation by cyclin kinase inhibitor specifically p21 will limit inflammation and prevent allograft rejection. METHODS: We performed in vitro and in vivo studies using lymphocytes, and rat heart transplant model to understand the role of cyclins and p21 on mitogen and allo-induced lymphocyte activation and inflammation. Lymphocyte proliferation was studied by 3H-thymidine uptake assay and mRNA expression was studied RT-PCR. RESULTS: Activation of allo- and mitogen stimulated lymphocytes resulted in increased expression of cyclins, IL-2 and pro-inflammatory cytokines, which was inhibited by cyclosporine. The over-expression of p21 prolonged graft survival in a completely mismatched rat heart transplant model resulted by inhibiting circulating and intra-graft expression of proinflammatory cytokines. CONCLUSION: Cyclins play a significant role in transplant-induced immune activation and p21 over-expression has potential to inhibit T cell activation and inflammation. The results from this study will permit the design of alternate strategies by controlling cell cycle progression to achieve immunosuppression in transplantation. [Abstract/Link to Full Text]

Yin X, Knecht DA, Lynes MA
Metallothionein mediates leukocyte chemotaxis.
BMC Immunol. 2005;621.
BACKGROUND: Metallothionein (MT) is a cysteine-rich, metal-binding protein that can be induced by a variety of agents. Modulation of MT levels has also been shown to alter specific immune functions. We have noticed that the MT genes map close to the chemokines Ccl17 and Cx3cl1. Cysteine motifs that characterize these chemokines are also found in the MT sequence suggesting that MT might also act as a chemotactic factor. RESULTS: In the experiments reported here, we show that immune cells migrate chemotactically in the presence of a gradient of MT. This response can be specifically blocked by two different monoclonal anti-MT antibodies. Exposure of cells to MT also leads to a rapid increase in F-actin content. Incubation of Jurkat T cells with cholera toxin or pertussis toxin completely abrogates the chemotactic response to MT. Thus MT may act via G-protein coupled receptors and through the cyclic AMP signaling pathway to initiate chemotaxis. CONCLUSION: These results suggest that, under inflammatory conditions, metallothionein in the extracellular environment may support the beneficial movement of leukocytes to the site of inflammation. MT may therefore represent a "danger signal"; modifying the character of the immune response when cells sense cellular stress. Elevated metallothionein produced in the context of exposure to environmental toxicants, or as a result of chronic inflammatory disease, may alter the normal chemotactic responses that regulate leukocyte trafficking. Thus, MT synthesis may represent an important factor in immunomodulation that is associated with autoimmune disease and toxicant exposure. [Abstract/Link to Full Text]

Wright KO, Murray DA, Crispe NI, Pierce RH
Quantitative PCR for detection of the OT-1 transgene.
BMC Immunol. 2005;620.
BACKGROUND: Transgenic TCR mice are often used experimentally as a source of T cells of a defined specificity. One of the most widely used transgenic TCR models is the OT-1 transgenic mouse in which the CD8+ T cells express a TCR specific for the SIINFEKL peptide of ovalbumin presented on kb. Although OT-1 CD8+ can be used in a variety of different experimental settings, we principally employ adoptive transfer and peptide-driven expansion of OT-1 cells in order to explore the distribution and fate of these antigen-specific OT-1 T cells. We set out to develop a quantitative PCR assay for OT-1 cells in order to assess the distribution of OT-1 CD8+ T cells in tissues that are either intrinsically difficult to dissociate for flow cytometric analysis or rendered incompatible with flow cytometric analysis through freezing or fixation. RESULTS: We show excellent correlation between flow cytometric assessment of OT-1 cells and OT-1 signal by qPCR assays in cell dilutions as well as in in vivo adoptive transfer experiments. We also demonstrate that qPCR can be performed from archival formalin-fixed paraffin-embedded tissue sections. In addition, the non-quantitative PCR using the OT-1-specific primers without the real-time probe is a valuable tool for OT-1 genotyping, obviating the need for peripheral blood collection and subsequent flow cytometric analysis. CONCLUSION: An OT-1 specific qPCR assay has been developed to quantify adoptively transferred OT-1 cells. OT-1 qPCR to determine cell signal is a valuable adjunct to the standard flow cytometric analysis of OT-1 cell number, particularly in experimental settings where tissue disaggregation is not desirable or in tissues which are not readily disassociated. [Abstract/Link to Full Text]

Ragin MJ, Hu J, Henderson AJ, August A
A role for the Tec family kinase ITK in regulating SEB-induced interleukin-2 production in vivo via c-jun phosphorylation.
BMC Immunol. 2005;619.
BACKGROUND: Exposure to Staphylococcal Enterotoxin B (SEB), a bacterial superantigen secreted by the Gram-positive bacteria Staphyloccocus aureus, results in the expansion and eventual clonal deletion and anergy of Vbeta8+ T cells, as well as massive cytokine release, including Interleukin-2 (IL-2). This IL-2 is rapidly secreted following exposure to SEB and may contribute to the symptoms seen following exposure to this bacterial toxin. The Tec family kinase ITK has been shown to be important for the production of IL-2 by T cells stimulated in vitro and may represent a good target for blocking the production of this cytokine in vivo. In order to determine if ITK represents such a target, mice lacking ITK were analyzed for their response to SEB exposure. RESULTS: It was found that T cells from mice lacking ITK exhibited significantly reduced proliferative responses to SEB exposure in vitro, as well as in vivo. Examination of IL-2 production revealed that ITK null mice produced reduced levels of this cytokine in vitro, and more dramatically, in vivo. In vivo analysis of c-jun phosphorylation, previously shown to be critical for regulating IL-2 production, revealed that this pathway was specifically activated in SEB reactive Vbeta8+ (but not non-reactive Vbeta6+) T cells from WT mice, but not in Vbeta8+ T cells from ITK null mice. However, toxicity analysis indicated that both WT and ITK null animals were similarly affected by SEB exposure. CONCLUSION: These data show that ITK is required for IL-2 production induced by SEB in vivo, and may regulate signals leading IL-2 production, in part by regulating phosphorylation of c-jun. The data also suggest that perturbing T cell activation pathways leading to IL-2 does not necessarily lead to improved responses to SEB toxicity. [Abstract/Link to Full Text]

Fulgenzi A, Dell'Antonio G, Foglieni C, Dal Cin E, Ticozzi P, Franzone JS, Ferrero ME
Inhibition of chemokine expression in rat inflamed paws by systemic use of the antihyperalgesic oxidized ATP.
BMC Immunol. 2005;618.
BACKGROUND: We previously showed that local use of periodate oxidized ATP (oATP, a selective inhibitor of P2X7 receptors for ATP) in rat paw treated with Freund's adjuvant induced a significant reduction of hyperalgesia Herein we investigate the role of oATP, in the rat paws inflamed by carrageenan, which mimics acute inflammation in humans. RESULTS: Local, oral or intravenous administration of a single dose of oATP significantly reduced thermal hyperalgesia in hind paws of rats for 24 hours, and such effect was greater than that induced by diclofenac or indomethacin. Following oATP treatment, the expression of the pro-inflammatory chemokines interferon-gamma-inducible protein-10 (IP-10), mon ocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) within the inflamed tissues markedly decreased on vessels and infiltrated cells. In parallel, the immunohistochemical findings showed an impairment, with respect to the untreated rats, in P2X7 expression, mainly on nerves and vessels close to the site of inflammation. Finally, oATP treatment significantly reduced the presence of infiltrating inflammatory macrophages in the paw tissue. CONCLUSION: Taken together these results clearly show that oATP reduces carrageenan-induced inflammation in rats. [Abstract/Link to Full Text]

Maecker HT, Moon J, Bhatia S, Ghanekar SA, Maino VC, Payne JK, Kuus-Reichel K, Chang JC, Summers A, Clay TM, Morse MA, Lyerly HK, DeLaRosa C, Ankerst DP, Disis ML
Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT.
BMC Immunol. 2005;617.
BACKGROUND: Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors. RESULTS: Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p <or= 0.001). The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p <or= 0.001). Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation) and tetramer staining (p < 0.001). Roughly similar sensitivity and specificity were observed between the three assays and between shipped and cryopreserved samples for each assay. CONCLUSION: We conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems. [Abstract/Link to Full Text]

Islander U, Hasséus B, Erlandsson MC, Jochems C, Skrtic SM, Lindberg M, Gustafsson JA, Ohlsson C, Carlsten H
Estren promotes androgen phenotypes in primary lymphoid organs and submandibular glands.
BMC Immunol. 2005;616.
BACKGROUND: Estrogens and androgens have extensive effects on the immune system, for example they suppress both T and B lymphopoiesis in thymus and bone marrow. Submandibular glands are sexually dimorphic in rodents, resulting in larger granular convoluted tubules in males compared to females. The aim of the present experiments was to investigate the estrogenic and androgenic effects of 4-estren-3alpha,17beta-diol (estren) on thymus, bone marrow and submandibular glands, and compare the effects to those of 17beta-estradiol (E2) and 5alpha-dihydrotestosterone (DHT), respectively. Estrogen receptors (ERs) were blocked by treatment of mice with the ER-antagonist ICI 182,780; also, knock-out mice lacking one or both ERs were used. RESULTS: As expected, the presence of functional ERs was mandatory for all the effects of E2. Similar to DHT-treatment, estren-treatment resulted in decreased thymus weight, as well as decreased frequency of bone marrow B cells. Treatment with estren or DHT also resulted in a shift in submandibular glands towards an androgen phenotype. All the effects of estren and DHT were independent of ERs. CONCLUSION: Our study is the first to show that estren has similar effects as the androgen DHT on lymphopoiesis in thymus and bone marrow, and on submandibular glands, and that these effects are independent of estrogen receptors. This supports the hypothesis of estren being able to signal through the androgen receptor. [Abstract/Link to Full Text]

Lu D, Yuan XJ, Evans RJ, Pappas AT, Wang H, Su EW, Hamdouchi C, Venkataraman C
Cloning and functional characterization of the rabbit C-C chemokine receptor 2.
BMC Immunol. 2005;615.
BACKGROUND: CC-family chemokine receptor 2 (CCR2) is implicated in the trafficking of blood-borne monocytes to sites of inflammation and is implicated in the pathogenesis of several inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. The major challenge in the development of small molecule chemokine receptor antagonists is the lack of cross-species activity to the receptor in the preclinical species. Rabbit models have been widely used to study the role of various inflammatory molecules in the development of inflammatory processes. Therefore, in this study, we report the cloning and characterization of rabbit CCR2. Data regarding the activity of the CCR2 antagonist will provide valuable tools to perform toxicology and efficacy studies in the rabbit model. RESULTS: Sequence alignment indicated that rabbit CCR2 shares 80 % identity to human CCR2b. Tissue distribution indicated that rabbit CCR2 is abundantly expressed in spleen and lung. Recombinant rabbit CCR2 expressed as stable transfectants in U-937 cells binds radiolabeled 125I-mouse JE (murine MCP-1) with a calculated Kd of 0.1 nM. In competition binding assays, binding of radiolabeled mouse JE to rabbit CCR2 is differentially competed by human MCP-1, -2, -3 and -4, but not by RANTES, MIP-1alpha or MIP-1beta. U-937/rabbit CCR2 stable transfectants undergo chemotaxis in response to both human MCP-1 and mouse JE with potencies comparable to those reported for human CCR2b. Finally, TAK-779, a dual CCR2/CCR5 antagonist effectively inhibits the binding of 125I-mouse JE (IC50 = 2.3 nM) to rabbit CCR2 and effectively blocks CCR2-mediated chemotaxis. CONCLUSION: In this study, we report the cloning of rabbit CCR2 and demonstrate that this receptor is a functional chemotactic receptor for MCP-1. [Abstract/Link to Full Text]

Hasan Z, Zaidi I, Jamil B, Khan MA, Kanji A, Hussain R
Elevated ex vivo monocyte chemotactic protein-1 (CCL2) in pulmonary as compared with extra-pulmonary tuberculosis.
BMC Immunol. 2005;614.
BACKGROUND: Tuberculosis causes 3 million deaths annually. The most common site of tuberculosis is pulmonary however; extra-pulmonary forms of the disease also remain prevalent. Restriction of Mycobacterium tuberculosis depends on effective recruitment and subsequent activation of T lymphocytes, mononuclear and polymorphonuclear cells to the site of infection. Tumor necrosis factor (TNF)-alpha is essential for granuloma formation and is a potent activator of monocyte chemotactic protein (MCP-1, CCL2). CCL2 is essential for recruitment of monocytes and T cells and has been shown to play a role in protection against tuberculosis. Interleukin -8 (CXCL8) is a potent activator of neutrophils. Increased levels of CCL2, CXCL8 and TNFalpha are reported in tuberculosis but their significance in different forms of tuberculosis is as yet unclear. We have used an ex vivo assay to investigate differences in immune parameters in patients with either pulmonary or extra-pulmonary tuberculosis. METHODS: Serum levels of CCL2, CXCL8 and TNFalpha were measured in patients with pulmonary tuberculosis (N = 12), extra-pulmonary tuberculosis (N = 8) and BCG-vaccinated healthy volunteers (N = 12). Whole blood cells were stimulated with non-pathogenic Mycobacterium bovis bacille-Calmette Guerin (BCG) vaccine strain or bacterial lipopolysaccharide (LPS) and cyto/chemokines were monitored in supernatants. RESULTS: Circulating serum levels of CXCL8 and TNFalpha were raised in all tuberculosis patients, while CCL2 levels were not. There was no difference in spontaneous cytokine secretion from whole blood cells between patients and controls. M. bovis BCG-induced ex vivo CCL2 secretion was significantly greater in pulmonary as compared with both extra-pulmonary tuberculosis patients and endemic controls. In response to LPS stimulation, patients with pulmonary tuberculosis showed increased CCL2 and TNFalpha responses as compared with the extra-pulmonary group. BCG-, and LPS-induced CXCL8 secretion was comparable between patients and controls. CONCLUSION: CCL2 is activated by TNFalpha and is essential for recruitment of monocytes and T cells to the site of mycobacterial infection. Increased CCL2 activation in pulmonary tuberculosis may result in a stronger cellular response as compared with extra-pulmonary tuberculosis patients, and this may contribute to the localization of infection to the pulmonary site. [Abstract/Link to Full Text]

Maecker HT, Rinfret A, D'Souza P, Darden J, Roig E, Landry C, Hayes P, Birungi J, Anzala O, Garcia M, Harari A, Frank I, Baydo R, Baker M, Holbrook J, Ottinger J, Lamoreaux L, Epling CL, Sinclair E, Suni MA, Punt K, Calarota S, El-Bahi S, Alter G, Maila H, Kuta E, Cox J, Gray C, Altfeld M, Nougarede N, Boyer J, Tussey L, Tobery T, Bredt B, Roederer M, Koup R, Maino VC, Weinhold K, Pantaleo G, Gilmour J, Horton H, Sekaly RP
Standardization of cytokine flow cytometry assays.
BMC Immunol. 2005;613.
BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays. [Abstract/Link to Full Text]

Dreyfus DH, Nagasawa M, Gelfand EW, Ghoda LY
Modulation of p53 activity by IkappaBalpha: evidence suggesting a common phylogeny between NF-kappaB and p53 transcription factors.
BMC Immunol. 2005;612.
BACKGROUND: In this work we present evidence that the p53 tumor suppressor protein and NF-kappaB transcription factors could be related through common descent from a family of ancestral transcription factors regulating cellular proliferation and apoptosis. P53 is a homotetrameric transcription factor known to interact with the ankyrin protein 53BP2 (a fragment of the ASPP2 protein). NF-kappaB is also regulated by ankyrin proteins, the prototype of which is the IkappaB family. The DNA binding sequences of the two transcription factors are similar, sharing 8 out of 10 nucleotides. Interactions between the two proteins, both direct and indirect, have been noted previously and the two proteins play central roles in the control of proliferation and apoptosis. RESULTS: Using previously published structure data, we noted a significant degree of structural alignment between p53 and NF-kappaB p65. We also determined that IkappaBalpha and p53 bind in vitro through a specific interaction in part involving the DNA binding region of p53, or a region proximal to it, and the amino terminus of IkappaBalpha independently or cooperatively with the ankyrin 3 domain of IkappaBalpha In cotransfection experiments, kappaBalpha could significantly inhibit the transcriptional activity of p53. Inhibition of p53-mediated transcription was increased by deletion of the ankyrin 2, 4, or 5 domains of IkappaBalpha Co-precipitation experiments using the stably transfected ankyrin 5 deletion mutant of kappaBalpha and endogenous wild-type p53 further support the hypothesis that p53 and IkappaBalpha can physically interact in vivo. CONCLUSION: The aggregate results obtained using bacterially produced IkappaBalpha and p53 as well as reticulocyte lysate produced proteins suggest a correlation between in vitro co-precipitation in at least one of the systems and in vivo p53 inhibitory activity. These observations argue for a mechanism involving direct binding of IkappaBalpha to p53 in the inhibition of p53 transcriptional activity, analogous to the inhibition of NF-kappaB by kappaBalpha and p53 by 53BP2/ASPP2. These data furthermore suggest a role for ankyrin proteins in the regulation of p53 activity. Taken together, the NFkappaB and p53 proteins share similarities in structure, DNA binding sites and binding and regulation by ankyrin proteins in support of our hypothesis that the two proteins share common descent from an ancestral transcriptional factor. [Abstract/Link to Full Text]

Nygaard UC, Aase A, Løvik M
The allergy adjuvant effect of particles - genetic factors influence antibody and cytokine responses.
BMC Immunol. 2005;611.
BACKGROUND: There is increasing epidemiological and experimental evidence for an aggravating effect of particulate air pollution on asthma and allergic symptoms and, to a lesser extent, on allergic sensitization. Genetic factors appear to influence not only the magnitude, but also the quality of the adjuvant effect of particles with respect to allergen-specific IgE (Th2-associated) and IgG2a (Th1-associated) responses. In the present study, we aimed to investigate how the genetic background influences the responses to the allergen and particles alone and in combination. We examined how polystyrene particles (PSP) affected the IgE and IgG2a responses against the model allergen ovalbumin (OVA), after subcutaneous injection into the footpad of BALB/cA, BALB/cJ, NIH and C3H/HeN mice, Further, ex vivo IL-4, IFN-gamma and IL-10 cytokine secretion by Con A-stimulated cells from the draining popliteal lymph node (PLN) five days after injection of OVA and PSP separately or in combination was determined. RESULTS: PSP injected with OVA increased the levels of OVA-specific IgE antibodies in all strains examined. In contrast, the IgG2a levels were significantly increased only in NIH and C3H/HeN mice. PSP in the presence of OVA increased cell numbers and IL-4, IL-10 and IFN-gamma levels in BALB/cA, NIH and C3H/HeN mice, with the exception of IFN-gamma in NIH mice. However, each mouse strain had their unique pattern of response to OVA+PSP, OVA and PSP, and also their unique background cytokine response (i.e. the cytokine response in cells from mice injected with buffer only). CONCLUSION: Genetic factors (i.e. the strain of mice) influenced the susceptibility to the adjuvant effect of PSP on both secondary antibody responses and primary cellular responses in the lymph node, as well as the cellular responses to both OVA and PSP given separately. Interestingly, PSP alone induced cytokine responses in the lymph node in some of the mouse strains. Furthermore, we found that the ex vivo cytokine patterns did not predict the in vivo Th2- and Th1-associated antibody response patterns in the different mouse strains. The results indicate that insoluble particles act by increasing the inherent response to the allergen, and that the genetic background may determine whether an additional Th1-associated component is added to the response. [Abstract/Link to Full Text]

Mayorov VI, Rogozin IB, Adkison LR, Frahm C, Kunkel TA, Pavlov YI
Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at G-C pairs in immunoglobulin genes.
BMC Immunol. 2005;6(1):10.
BACKGROUND: Antibody genes are diversified by somatic hypermutation (SHM), gene conversion and class-switch recombination. All three processes are initiated by the activation-induced deaminase (AID). According to a DNA deamination model of SHM, AID converts cytosine to uracil in DNA sequences. The initial deamination of cytosine leads to mutation and recombination in pathways involving replication, DNA mismatch repair and possibly base excision repair. The DNA sequence context of mutation hotspots at G-C pairs during SHM is DGYW/WRCH (G-C is a hotspot position, R = A/G, Y = T/C, W = A/T, D = A/G/T). RESULTS: To investigate the mechanisms of AID-induced mutagenesis in a model system, we studied the genetic consequences of AID expression in yeast. We constructed a yeast vector with an artificially synthesized human AID gene insert using codons common to highly expressed yeast genes. We found that expression of the artificial hAIDSc gene was moderately mutagenic in a wild-type strain and highly mutagenic in an ung1 uracil-DNA glycosylase-deficient strain. A majority of mutations were at G-C pairs. In the ung1 strain, C-G to T-A transitions were found almost exclusively, while a mixture of transitions with 12% transversions was characteristic in the wild-type strain. In the ung1 strain mutations that could have originated from deamination of the transcribed stand were found more frequently. In the wild-type strain, the strand bias was reversed. DGYW/WRCH motifs were preferential sites of mutations. CONCLUSION: The results are consistent with the hypothesis that AID-mediated deamination of DNA is a major cause of mutations at G-C base pairs in immunoglobulin genes during SHM. The sequence contexts of mutations in yeast induced by AID and those of somatic mutations at G-C pairs in immunoglobulin genes are significantly similar. This indicates that the intrinsic substrate specificity of AID itself is a primary determinant of mutational hotspots at G-C base pairs during SHM. [Abstract/Link to Full Text]

Kersten C, Sivertsen EA, Hystad ME, Forfang L, Smeland EB, Myklebust JH
BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1.
BMC Immunol. 2005;6(1):9.
BACKGROUND: Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and are secreted proteins with pleiotropic roles in many different cell types. A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases. In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells. RESULTS: The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8. Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected. Furthermore, we found that BMP-6 had an antiproliferative effect both in naive (CD19+CD27-) and memory B cells (CD19+CD27+) stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induced cell death in activated memory B cells. Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin. Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM. CONCLUSION: In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1. [Abstract/Link to Full Text]

Ferguson AR, Corley RB
Accumulation of marginal zone B cells and accelerated loss of follicular dendritic cells in NF-kappaB p50-deficient mice.
BMC Immunol. 2005;6(1):8.
BACKGROUND: Marginal zone (MZ) B cells play important roles in the early phases of humoral immune responses. In addition to possessing an inherent capacity to rapidly differentiate into antibody secreting cells, MZ B cells also help to regulate the fate of both T-independent and T-dependent blood-borne antigens in the spleen. For T-dependent antigens, MZ B cells bind IgM-antigen complexes in a complement-dependent manner. Once MZ B cells bind IgM-containing immune complexes (IgM-IC), they transport them into B cell follicles for deposition onto follicular dendritic cells (FDCs), an important component of secreted IgM's ability to enhance adaptive immune responses. To further define the requirement for MZ B cells in IgM-IC deposition, mice deficient in the NF-kappaB protein p50, which have been reported to lack MZ B cells, were analyzed for their ability to trap IgM-IC onto FDCs. RESULTS: Mice (2 months of age) deficient in p50 (p50-/-) had small numbers of MZ B cells, as determined by cell surface phenotype and localization in the splenic MZ. These cells bound high levels of IgM-IC both in vivo and in vitro. Subsequent to the binding of IgM-IC by the MZ B cells in p50-/- mice, small amounts of IgM-IC were found localized on FDCs, suggesting that the MZ B cells retained their ability to transport these complexes into splenic follicles. Strikingly, MZ B cells accumulated with age in p50-/- mice. By 6 months of age, p50-/- mice contained normal numbers of these cells as defined by CD21/CD23 profile and high level expression of CD1d, CD9, and IgM, and by their positioning around the marginal sinus. However, FDCs from these older p50-/- mice exhibited a reduced capacity to trap IgM-IC and retain complement components. CONCLUSION: These results demonstrate that while the p50 component of the NF-kappaB transcription complex plays an important role in the early development of MZ B cells, MZ B cells can develop and accumulate in mice lacking this protein. These results highlight the interface between genetic deficiencies and age, and suggest that different transcription factors may play distinct roles in the development and maintenance of cell populations at different ages. [Abstract/Link to Full Text]

Zimmermann N, Colyer JL, Koch LE, Rothenberg ME
Analysis of the CCR3 promoter reveals a regulatory region in exon 1 that binds GATA-1.
BMC Immunol. 2005;6(1):7.
BACKGROUND: CC Chemokine Receptor 3 (CCR3), the major chemokine receptor expressed on eosinophils, binds promiscuously to several ligands including eotaxins 1, 2, and 3. Even though the only cells that consistently accumulate following eotaxin administration in vivo are myeloid cells (primarily eosinophils), other cell types have recently been shown to express CCR3. It is therefore important to elucidate the molecular mechanisms regulating receptor expression. RESULTS: In order to define regions responsible for CCR3 transcription, a DNAse hypersensitive site was identified in the vicinity of exon 1. Coupled with our previous data implicating exon 1 in CCR3 transcription, we hypothesized that transcription factors bind to exon-1. Electrophoretic mobility shift analysis revealed that nuclear proteins in eosinophilic cells bound to exon 1. Furthermore, antibody interference and mutation studies demonstrated GATA-1 binding to exon 1. In order to test the 1.6-kb CCR3 promoter element (that includes exon 1) for in vivo function, this region was used to generate transgenic mice that expressed a reporter protein. Strong transgene expression was achieved, with the pattern of expression suggesting a broad acting promoter. CONCLUSION: The transcription factor GATA-1 binds to CCR3 exon 1. The 1.6-kb CCR3 promoter element, that includes exon 1, is a strong promoter in vivo. [Abstract/Link to Full Text]

Pablos JL, Santiago B, Tsay D, Singer MS, Palao G, Galindo M, Rosen SD
A HEV-restricted sulfotransferase is expressed in rheumatoid arthritis synovium and is induced by lymphotoxin-alpha/beta and TNF-alpha in cultured endothelial cells.
BMC Immunol. 2005;6(1):6.
BACKGROUND: The recruitment of lymphocytes to secondary lymphoid organs relies on interactions of circulating cells with high endothelial venules (HEV). HEV are exclusive to these organs under physiological conditions, but they can develop in chronically-inflamed tissues. The interaction of L-selectin on lymphocytes with sulfated glycoprotein ligands on HEV results in lymphocyte rolling, which represents the initial step in lymphocyte homing. HEV expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, GST-3, LSST or CHST4), an HEV-restricted sulfotransferase, is essential for the elaboration of L-selectin functional ligands as well as a critical epitope recognized by MECA-79 mAb. RESULTS: We examined the expression of GlcNAc6ST-2 in relationship to the MECA-79 epitope in rheumatoid arthritis (RA) synovial vessels. Expression of GlcNAc6ST-2 was specific to RA synovial tissues as compared to osteoarthritis synovial tissues and localized to endothelial cells of HEV-like vessels and small flat-walled vessels. Double MECA-79 and GlcNAc6ST-2 staining showed colocalization of the MECA-79 epitope and GlcNAc6ST-2. We further found that both TNF-alpha and lymphotoxin-alphabeta induced GlcNAc6ST-2 mRNA and protein in cultured human umbilical vein endothelial cells. CONCLUSION: These observations demonstrate that GlcNAc6ST-2 is induced in RA vessels and provide potential cytokine pathways for its induction. GlcNAc6ST-2 is a novel marker of activated vessels within RA ectopic lymphoid aggregates. This enzyme represents a potential therapeutic target for RA. [Abstract/Link to Full Text]

Boeuf P, Vigan-Womas I, Jublot D, Loizon S, Barale JC, Akanmori BD, Mercereau-Puijalon O, Behr C
CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use.
BMC Immunol. 2005;6(1):5.
BACKGROUND: Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. RESULTS: To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1beta, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-gamma, MIF, TGF-beta1 and TNF-alpha mRNA. We showed that the beta-2 microglobulin (beta2-MG) gene was suitable for data normalisation since the level of beta2-MG transcripts in naive PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-beta1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-beta1 mRNA in response to LPS. CONCLUSION: The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications. [Abstract/Link to Full Text]

Linehan SA
The mannose receptor is expressed by subsets of APC in non-lymphoid organs.
BMC Immunol. 2005;6(1):4.
BACKGROUND: The mannose receptor (MR) is an endocytic receptor of Mphi and endothelial cell subsets whose natural ligands include both self glycoproteins and microbial glycans. It is also expressed by immature cultured dendritic cells (DC), where it mediates high efficiency uptake of glycosylated antigens, yet its role in antigen handling in vivo is unknown. Knowledge of which APC subsets express MR will assist the design of experiments to address its immunological functions. Here the expression of MR by MHC class II positive APC in non-lymphoid organs of the mouse is described. RESULTS: MR positive APC were identified in several peripheral organs: skin, liver, cardiac and skeletal muscle and tongue. MR positive cells in salivary gland, thyroid and pancreas coexpressed MHC class II and the myeloid markers macrosialin and sialoadhesin, but not the dendritic cell markers CD11c or DEC-205. MR and MHC class II colocalised in confocal microscope images, implying that antigen capture may be the primary role of MR in these cells. Distinct ligands of MR were found in salivary gland and pancreas tissue lysates that are candidate physiological ligands of MR positive APC in these organs. CONCLUSIONS: The tissue and subcellular distribution of MR suggest it is appropriately located to serve as a high efficiency antigen uptake receptor of APC. [Abstract/Link to Full Text]

Kim JR, Lim HW, Kang SG, Hillsamer P, Kim CH
Human CD57+ germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination.
BMC Immunol. 2005;6(1):3.
BACKGROUND: The function of CD57+ CD4+ T cells, constituting a major subset of germinal center T (GC-Th) cells in human lymphoid tissues, has been unclear. There have been contradictory reports regarding the B cell helping function of CD57+ GC-Th cells in production of immunoglobulin (Ig). Furthermore, the cytokine and co-stimulation requirement for their helper activity remains largely unknown. To clarify and gain more insight into their function in helping B cells, we systematically investigated the capacity of human tonsil CD57+ GC-Th cells in inducing B cell Ig synthesis. RESULTS: We demonstrated that CD57+ GC-Th cells are highly efficient in helping B cell production of all four subsets of Ig (IgM, IgG, IgA and IgE) compared to other T-helper cells located in germinal centers or interfollicular areas. CD57+ GC-Th cells were particularly more efficient than other T cells in helping GC-B cells but not naive B cells. CD57+ GC-Th cells induced the expression of activation-induced cytosine deaminase (AID) and class switch recombination in developing B cells. IgG1-3 and IgA1 were the major Ig isotypes induced by CD57+ GC-Th cells. CD40L, but not IL-4, IL-10 and IFN-gamma, was critical in CD57+ GC-Th cell-driven B cell production of Ig. However, IL-10, when added exogenously, significantly enhanced the helper activity of CD57+ GC-Th cells, while TGF-beta1 completely and IFN-gamma partially suppressed the CD57+ GC-Th cell-driven Ig production. CONCLUSIONS: CD57+CD4+ T cells in the germinal centers of human lymphoid tissues are the major T helper cell subset for GC-B cells in Ig synthesis. Their helper activity is consistent with their capacity to induce AID and class switch recombination, and can be regulated by CD40L, IL-4, IL-10 and TGF-beta. [Abstract/Link to Full Text]

Reghunathan R, Jayapal M, Hsu LY, Chng HH, Tai D, Leung BP, Melendez AJ
Expression profile of immune response genes in patients with Severe Acute Respiratory Syndrome.
BMC Immunol. 2005;62.
BACKGROUND: Severe acute respiratory syndrome (SARS) emerged in later February 2003, as a new epidemic form of life-threatening infection caused by a novel coronavirus. However, the immune-pathogenesis of SARS is poorly understood. To understand the host response to this pathogen, we investigated the gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from SARS patients, and compared with healthy controls. RESULTS: The number of differentially expressed genes was found to be 186 under stringent filtering criteria of microarray data analysis. Several genes were highly up-regulated in patients with SARS, such as, the genes coding for Lactoferrin, S100A9 and Lipocalin 2. The real-time PCR method verified the results of the gene array analysis and showed that those genes that were up-regulated as determined by microarray analysis were also found to be comparatively up-regulated by real-time PCR analysis. CONCLUSIONS: This differential gene expression profiling of PBMCs from patients with SARS strongly suggests that the response of SARS affected patients seems to be mainly an innate inflammatory response, rather than a specific immune response against a viral infection, as we observed a complete lack of cytokine genes usually triggered during a viral infection. Our study shows for the first time how the immune system responds to the SARS infection, and opens new possibilities for designing new diagnostics and treatments for this new life-threatening disease. [Abstract/Link to Full Text]

Saxinger C, Conrads TP, Goldstein DJ, Veenstra TD
Fully automated synthesis of (phospho)peptide arrays in microtiter plate wells provides efficient access to protein tyrosine kinase characterization.
BMC Immunol. 2005;6(1):1.
BACKGROUND: Synthetic peptides have played a useful role in studies of protein kinase substrates and interaction domains. Synthetic peptide arrays and libraries, in particular, have accelerated the process. Several factors have hindered or limited the applicability of various techniques, such as the need for deconvolution of combinatorial libraries, the inability or impracticality of achieving full automation using two-dimensional or pin solid phases, the lack of convenient interfacing with standard analytical platforms, or the difficulty of compartmentalization of a planar surface when contact between assay components needs to be avoided. This paper describes a process for synthesis of peptides and phosphopeptides on microtiter plate wells that overcomes previous limitations and demonstrates utility in determination of the epitope of an autophosphorylation site phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine kinase, p60c-src. RESULTS: The overall reproducibility of phospho-peptide synthesis and multiplexed EGF receptor (EGFR) autophosphorylation site (pY1173) antibody ELISA (9H2) was within 5.5 to 8.0%. Mass spectrometric analyses of the released (phospho)peptides showed homogeneous peaks of the expected molecular weights. An overlapping peptide array of the complete EGFR cytoplasmic sequence revealed a high redundancy of 9H2 reactive sites. The eight reactive phospopeptides were structurally related and interestingly, the most conserved antibody reactive peptide motif coincided with a subset of other known EGFR autophosphorylation and SH2 binding motifs and an EGFR optimal substrate motif. Finally, peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate array format and were phosphorylated by c-Src with the predicted specificities. The level of phosphorylation was proportional to c-Src concentration with sensitivities below 0.1 Units of enzyme. CONCLUSIONS: The ability of this method to interface with various robotics and instrumentation is highly flexible since the microtiter plate is an industry standard. It is highly scalable by increasing the surface area within the well or the number of wells and does not require specialized robotics. The microtiter plate array system is well suited to the study of protein kinase substrates, antigens, binding molecules, and inhibitors since these all can be quantitatively studied at a single uniform, reproducible interface. [Abstract/Link to Full Text]

Pruett SB, Padgett EL
Thymus-derived glucocorticoids are insufficient for normal thymus homeostasis in the adult mouse.
BMC Immunol. 2004 Nov 2;524.
BACKGROUND: It is unclear if thymus-derived glucocorticoids reach sufficient local concentrations to support normal thymus homeostasis, or if adrenal-derived glucocorticoids from the circulation are required. Modern approaches to this issue (transgenic mice that under or over express glucocorticoid receptor in the thymus) have yielded irreconcilably contradictory results, suggesting fundamental problems with one or more the transgenic mouse strains used. In the present study, a more direct approach was used, in which mice were adrenalectomized with or without restoration of circulating corticosterone using timed release pellets. Reversal of the increased number of thymocytes caused by adrenalectomy following restoration of physiological corticosterone concentrations would indicate that corticosterone is the major adrenal product involved in thymic homeostasis. RESULTS: A clear relationship was observed between systemic corticosterone concentration, thymus cell number, and percentage of apoptotic thymocytes. Physiological concentrations of corticosterone in adrenalectomized mice restored thymus cell number to normal values and revealed differential sensitivity of thymocyte subpopulations to physiological and stress-inducible corticosterone concentrations. CONCLUSION: This indicates that thymus-derived glucocorticoids are not sufficient to maintain normal levels of death by neglect in the thymus, but that apoptosis and possibly other mechanisms induced by physiological, non stress-induced levels of adrenal-derived corticosterone are responsible for keeping the total number of thymocytes within the normal range. [Abstract/Link to Full Text]

Davenport BJ, Willis DG, Prescott J, Farrell RM, Coons TA, Schountz T
Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus).
BMC Immunol. 2004 Sep 30;523.
BACKGROUND: Human infections with Sin Nombre virus (SNV) and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS), a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus) are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. RESULTS: To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC) from deer mouse bone marrow using commercially-available house mouse (Mus musculus) granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. CONCLUSIONS: The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases. [Abstract/Link to Full Text]

Ferrero E, Orciani M, Vacca P, Ortolan E, Crovella S, Titti F, Saccucci F, Malavasi F
Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque.
BMC Immunol. 2004 Sep 21;521.
BACKGROUND: The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. RESULTS: A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of approximately 42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. CONCLUSION: This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme. [Abstract/Link to Full Text]

Chi DS, Fitzgerald SM, Pitts S, Cantor K, King E, Lee SA, Huang SK, Krishnaswamy G
MAPK-dependent regulation of IL-1- and beta-adrenoreceptor-induced inflammatory cytokine production from mast cells: implications for the stress response.
BMC Immunol. 2004 Sep 21;522.
BACKGROUND: Catecholamines, such as epinephrine, are elaborated in stress responses, and mediate vasoconstriction to cause elevation in systemic vascular resistance and blood pressure. Our previous study has shown that IL-1 can induce mast cells to produce proinflammatory cytokines which are involved in atherogenesis. The aim of this study was to determine the effects of epinephrine on IL-1-induced proatherogenic cytokine production from mast cells. RESULTS: Two ml of HMC-1 (0.75 x 106 cells/ml) were cultured with epinephrine (1 x 10-5 M) in the presence or absence of IL-1 beta (10 ng/ml) for 24 hrs. HMC-1 cultured alone produced none to trace amounts of IL-6, IL-8, and IL-13. IL-1 beta significantly induced production of these cytokines in HMC-1, while epinephrine alone did not. However, IL-6, IL-8, and IL-13 production induced by IL-1 beta were significantly enhanced by addition of epinephrine. The enhancing effect appears to involve NF-kappa B and p38 MAPK pathways. Flow cytometry showed the presence of beta1 and beta2 adrenoreceptors on resting mast cells. The enhancing effect of proatherogenic cytokine production by epinephrine was down regulated by the beta1 and beta2 adrenoceptor antagonist, propranolol, but not by the beta1 adrenoceptor antagonist, atenolol, suggesting the effect involved beta2 adrenoceptors. The enhancing effect of epinephrine on proatherogenic cytokine production was also down regulated by the immunosuppressive drug, dexamethasone. CONCLUSIONS: These results not only confirm that an acute phase cytokine, IL-1 beta, regulates mast cell function, but also show that epinephrine up regulates the IL-1 beta induction of proatherogenic cytokines in mast cells. These data provide a novel role for epinephrine, a stress hormone, in inflammation and atherogenesis. [Abstract/Link to Full Text]

Shen Y, Iqbal J, Xiao L, Lynch RC, Rosenwald A, Staudt LM, Sherman S, Dybkaer K, Zhou G, Eudy JD, Delabie J, McKeithan TW, Chan WC
Distinct gene expression profiles in different B-cell compartments in human peripheral lymphoid organs.
BMC Immunol. 2004 Sep 15;520.
BACKGROUND: There are three major B-cell compartments in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MNZ) and the marginal zone (MGZ). Unique sets of B-cells reside in these compartments, and they have specific functional roles in humoral immune response. MNZ B cells are naive cells in a quiescent state and may participate in GC reactions upon proper stimulation. The adult splenic MGZ contains mostly memory B cells and is also known to provide a rapid response to particulate antigens. The GC B-cells proliferate rapidly and undergo selection and affinity maturation. The B-cell maturational process is accompanied by changes in the expression of cell-surface and intracellular proteins and requires signals from the specialized microenvironments. RESULTS: We performed laser microdissection of the three compartments for gene expression profiling by cDNA microarray. The transcriptional program of the GC was dominated by upregulation of genes associated with proliferation and DNA repair or recombination. The MNZ and MGZ showed increased expression of genes promoting cellular quiescence. The three compartments also revealed distinct repertoires of apoptosis-associated genes, chemokines and chemokine receptors. The MNZ and GC showed upregulation of CCL20 and CCL18 respectively. The MGZ was characterized by high expression of many chemokines genes e.g. CXCL12, CCL3, CCL14 and IFN-associated genes, consistent with its role in rapid response to infections. A stromal signature was identified including genes associated with macrophages or with synthesis of extracellular matrix and genes that influenced lymphocyte migration and survival. Differentially expressed genes that did not belong to the above categories include the well characterized BCL6 and CD10 and many others whose function is not known. CONCLUSIONS: Transcriptional profiling of B-cell compartments has identified groups of genes involved in critical molecular and cellular events that affect proliferation, survival migration, and differentiation of the cells. The gene expression study of normal B-cell compartments may additionally contribute to our understanding of the molecular abnormalities of the corresponding lymphoid tumors. [Abstract/Link to Full Text]

Jackson KJ, Gaeta B, Sewell W, Collins AM
Exonuclease activity and P nucleotide addition in the generation of the expressed immunoglobulin repertoire.
BMC Immunol. 2004 Sep 2;519.
BACKGROUND: Immunoglobulin rearrangement involves random and imprecise processes that act to both create and constrain diversity. Two such processes are the loss of nucleotides through the action of unknown exonuclease(s) and the addition of P nucleotides. The study of such processes has been compromised by difficulties in reliably aligning immunoglobulin genes and in the partitioning of nucleotides between segment ends, and between N and P nucleotides. RESULTS: A dataset of 294 human IgM sequences was created and partitioned with the aid of a probabilistic model. Non-random removal of nucleotides is seen between the three IGH gene types with the IGHV gene averaging removals of 1.2 nucleotides compared to 4.7 for the other gene ends (p < 0.001). Individual IGHV, IGHD and IGHJ gene subgroups also display statistical differences in the level of nucleotide loss. For example, within the IGHJ group, IGHJ3 has average removals of 1.3 nucleotides compared to 6.4 nucleotides for IGHJ6 genes (p < 0.002). Analysis of putative P nucleotides within the IgM and pooled datasets revealed only a single putative P nucleotide motif (GTT at the 3' D-REGION end) to occur at a frequency significantly higher then would be expected from random N nucleotide addition. CONCLUSIONS: The loss of nucleotides due to the action of exonucleases is not random, but is influenced by the nucleotide composition of the genes. P nucleotides do not make a significant contribution to diversity of immunoglobulin sequences. Although palindromic sequences are present in 10% of immunologlobulin rearrangements, most of the 'palindromic' nucleotides are likely to have been inserted into the junction during the process of N nucleotide addition. P nucleotides can only be stated with confidence to contribute to diversity of less than 1% of sequences. Any attempt to identify P nucleotides in immunoglobulins is therefore likely to introduce errors into the partitioning of such sequences. [Abstract/Link to Full Text]

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Recent Articles in BMC Infectious Diseases

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Faburay B, Geysen D, Munstermann S, Bell-Sakyi L, Jongejan F
Longitudinal monitoring of Ehrlichia ruminantium infection in Gambian lambs and kids by pCS20 PCR and MAP1-B ELISA.
BMC Infect Dis. 2007;785.
BACKGROUND: The epidemiology of E. ruminantium infection in extensively managed young animals is not adequately understood. Thus in this study, we monitored the onset (age at first infection) and kinetics of E. ruminantium infection and antibody response in extensively managed newborn lambs and kids at three sites in The Gambia. METHODS: We used a nested pCS20 PCR and MAP1-B ELISA in a longitudinal study to monitor the onset (age at first infection) and kinetics of E. ruminantium infection and antibody response respectively, in 77 newborn lambs and kids under a traditional husbandry system at three sites (Kerr Seringe, Keneba, Bansang) in The Gambia where heartwater is known to occur. The animals were monitored for field tick infestation and the comparative performance of the two assays in detecting E. ruminantium infection was also assessed. RESULTS: The infection rate detected by pCS20 PCR varied between 8.6% and 54.8% over the 162-day study period. Nineteen per cent of the animals in week 1 post-partum tested positive by pCS20 PCR with half of these infections (7/14) detected in the first 3 days after birth, suggesting that transmission other than by tick feeding had played a role. The earliest detectable A. variegatum infestation in the animals occurred in week 16 after birth. Antibodies detected by MAP1-B ELISA also varied, between 11.5% and 90%. Although there is considerable evidence that this assay can detect false positives and due to this and other reasons serology is not a reliable predictor of infection at least for heartwater. In contrast to the pCS20 PCR, the serological assay detected the highest proportion of positive animals in week 1 with a gradual decline in seropositivity with increasing age. The pCS20 PCR detected higher E. ruminantium prevalence in the animals with increasing age and both the Spearman's rank test (rs = -0.1512; P = 0.003) and kappa statistic (-0.091 to 0.223) showed a low degree of agreement between the two assays. CONCLUSION: The use of pCS20 PCR supported by transmission studies and clinical data could provide more accurate information on heartwater epidemiology in endemic areas and single-occasion testing of an animal may not reveal its true infection status. The view is supported because both the vector and vertical transmission may play a vital role in the epidemiology of heartwater in young small ruminants; the age range of 4 and 12 weeks corresponds to the period of increased susceptibility to heartwater in traditionally managed small ruminants. [Abstract/Link to Full Text]

Murphy DJ, Brown JR
Identification of gene targets against dormant phase Mycobacterium tuberculosis infections.
BMC Infect Dis. 2007;784.
BACKGROUND: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant M. tuberculosis strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections. METHODS: The availability of M. tuberculosis genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality. RESULTS: Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by devR, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (devR/devS, relA, mprAB), enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability, and drug development. CONCLUSION: Based on our bioinformatics analysis and additional discussion of in-depth biological rationale, several novel anti-TB targets have been proposed as potential opportunities to improve present therapeutic treatments for this disease. [Abstract/Link to Full Text]

Martinez V, Carcelain G, Badell E, Jouan M, Mauger I, Sellier P, Truffot C, Bricaire F, Arend SM, Ottenhoff T, Autran B, Gicquel B
T-cell and serological responses to Erp, an exported Mycobacterium tuberculosis protein, in tuberculosis patients and healthy individuals.
BMC Infect Dis. 2007;783.
BACKGROUND: The identification of antigens able to differentiate tuberculosis (TB) disease from TB infection would be valuable. Cellular and humoral immune responses to Erp (Exported repetitive protein)--a recently identified M. tuberculosis protein--have not yet been investigated in humans and may contribute to this aim. METHODS: We analyzed the cellular and humoral immune responses to Erp, ESAT-6, Ag85B and PPD in TB patients, in BCG+ individuals without infection, BCG+ individuals with latent TB infection (LTBI) and BCG- controls. We used lymphoproliferation, ELISpot IFN-gamma, cytokine production assays and detection of specific human antibodies against recombinant M. tuberculosis proteins. RESULTS: We included 22 TB patients, 9 BCG+ individuals without TB infection, 7 LTBI and 7 BCG- controls. Erp-specific T cell counts were higher in LTBI than in the other groups. Erp-specific T cell counts were higher in LTBI subjects than TB patients (median positive frequency of 211 SFC/106 PBMC (range 118-2000) for LTBI subjects compared to 80 SFC/106 PBMC (range 50-191), p = 0.019); responses to PPD and ESAT-6 antigens did not differ between these groups. IFN-gamma secretion after Erp stimulation differed between TB patients and LTBI subjects (p = 0.02). Moreover, LTBI subjects but not TB patients or healthy subjects produced IgG3 against Erp. CONCLUSION: The frequencies of IFN-gamma-producing specific T cells, the IFN-gamma secretion and the production of IgG3 after Erp stimulation are higher in LTBI subjects than in TB patients, whereas PPD and ESAT-6 are not. [Abstract/Link to Full Text]

Gordon M, Roberts H, Odeka E
Knowledge and attitudes of parents and professionals to neonatal BCG vaccination in light of recent UK policy changes: a questionnaire study.
BMC Infect Dis. 2007;782.
BACKGROUND: Universal BCG vaccination in the UK ended in 2005. The new vaccination policy instead offers a number of different forms of selective vaccination to newborns based on risk of acquiring TB. We set out to assess the attitudes and knowledge of both parents and professionals to the new policy for neonatal BCG vaccination. METHODS: A short questionnaire was designed, made up of demographic and attitude questions, as well as very basic knowledge questions. The researchers handed out the questionnaire to all parents and professionals in the antenatal and postnatal areas, as well as the paediatric and neonatal units during a 6-week period. The site was the Royal Oldham hospital, a district general hospital with 3250 deliveries per year and multi-ethnic in its population mix. RESULTS: A total of 253 completed questionnaires were collected. The ethnic origin of responders was 50.6% White British, 18.2% Bangladeshi, 8.7% Indian, 4% White/Asian, the remaining 18.5% of other origins. 71.5% of responders said they had heard of BCG vaccine. When asked if they knew the new policy for its use, 33.2% answered yes. 24.5% gave the most accurate response when asked who now receives BCG. CONCLUSION: We have found that amongst parents and professionals alike there is a lack of knowledge of the new policy. This has lead to confusion and as knowledge amongst the professionals who identify neonates for vaccination is low, uptake may be sub-optimal. We suggest that units investigate the issue and ensure that the new policy is understood and implemented correctly. [Abstract/Link to Full Text]

Janjua NZ, Razaq M, Chandir S, Rozi S, Mahmood B
Poor knowledge--predictor of nonadherence to universal precautions for blood borne pathogens at first level care facilities in Pakistan.
BMC Infect Dis. 2007;781.
BACKGROUND: We conducted an assessment of knowledge about blood borne pathogens (BBP) and use of universal precautions at first level care facilities (FLCF) in two districts of Pakistan. METHODS: We conducted a cross-sectional survey and selected three different types of FLCFs ; public, general practitioners and unqualified practitioners through stratified random sampling technique. At each facility, we interviewed a prescriber, a dispenser, and a housekeeper for knowledge of BBPs transmission and preventive practices, risk perception, and use of universal precautions. We performed multiple linear regression to assess the effect of knowledge score (11 items) on the practice of universal precautions score (4 items- use of gloves, gown, needle recapping, and HBV vaccination). RESULTS: We interviewed 239 subjects. Most of the participants 128 (53%) were recruited from general practitioners clinics and 166 (69.5%) of them were dispensers. Mean (SD) knowledge score was 3.8 (2.3) with median of 4. MBBS prescribers had the highest knowledge score while the housekeepers had the lowest. Mean universal precautions use score was 2.7 +/- 2.1. Knowledge about mode of transmission and the work experience alone, significantly predicted universal precaution use in multiple linear regression model (adR2 = 0.093). CONCLUSION: Knowledge about mode of transmission of blood borne pathogens is very low. Use of universal precautions can improve with increase in knowledge. [Abstract/Link to Full Text]

Madani TA, Ghabrah TM
Meningococcal, influenza virus, and hepatitis B virus vaccination coverage level among health care workers in Hajj.
BMC Infect Dis. 2007;780.
BACKGROUND: The objective of this study was to assess the compliance of health care workers (HCWs) employed in Hajj in receiving the meningococcal, influenza, and hepatitis B vaccines. METHODS: A cross-sectional survey of doctors and nurses working in all Mena and Arafat hospitals and primary health care centers who attended Hajj-medicine training programs immediately before the beginning of Hajj of the lunar Islamic year 1423 (2003) using self-administered structured questionnaire which included demographic data and data on vaccination history. RESULTS: A total of 392 HCWs were studied including 215 (54.8%) nurses and 177 (45.2%) doctors. One hundred and sixty four (41.8%) HCWs were from Makkah and the rest were recruited from other regions in Saudi Arabia. Three hundred and twenty three (82.4%) HCWs received the quadrivalent (ACYW135) meningococcal meningitis vaccine with 271 (83.9%) HCWs receiving it at least 2 weeks before coming to Hajj, whereas the remaining 52 (16.1%) HCWs received it within < 2 weeks. Only 23 (5.9%) HCWs received the current year's influenza virus vaccine. Two hundred and sixty (66.3%) of HCWs received the three-dose hepatitis B vaccine series, 19.3% received one or two doses, and 14.3% did not receive any dose. There was no statistically significant difference in compliance with the three vaccines between doctors and nurses. CONCLUSION: The meningococcal and hepatitis B vaccination coverage level among HCWs in Hajj was suboptimal and the influenza vaccination level was notably low. Strategies to improve vaccination coverage among HCWs should be adopted by all health care facilities in Saudi Arabia. [Abstract/Link to Full Text]

Lee SC, Huang SS, Lee CW, Fung CP, Lee N, Shieh WB, Siu LK
Comparative antimicrobial susceptibility of aerobic and facultative bacteria from community-acquired bacteremia to ertapenem in Taiwan.
BMC Infect Dis. 2007;779.
BACKGROUND: Ertapenem is a once-a-day carbapenem and has excellent activity against many gram-positive and gram-negative aerobic, facultative, and anaerobic bacteria. The susceptibility of isolates of community-acquired bacteremia to ertapenem has not been reported yet. The present study assesses the in vitro activity of ertapenem against aerobic and facultative bacterial pathogens isolated from patients with community-acquired bacteremia by determining and comparing the MICs of cefepime, cefoxitin, ceftazidime, ceftriaxone, ertapenem, piperacillin, piperacillin-tazobactam, ciprofloxacin, amikacin and gentamicin. The prevalence of extended broad spectrum beta-lactamases (ESBL) producing strains of community-acquired bacteremia and their susceptibility to these antibiotics are investigated. METHODS: Aerobic and facultative bacteria isolated from blood obtained from hospitalized patients with community-acquired bacteremia within 48 hours of admission between August 1, 2004 and September 30, 2004 in Chang Gung Memorial Hospital at Keelung, Taiwan, were identified using standard procedures. Antimicrobial susceptibility was evaluated by Etest according to the standard guidelines provided by the manufacturer and document M100-S16 Performance Standards of the Clinical Laboratory of Standard Institute. Antimicrobial agents including cefepime, cefoxitin, ceftazidime, ceftriaxone, ertapenem, piperacillin, piperacillin-tazobactam, ciprofloxacin, amikacin and gentamicin were used against the bacterial isolates to test their MICs as determined by Etest. For Staphylococcus aureus isolates, MICs of oxacillin were also tested by Etest to differentiate oxacillin-sensitive and oxacillin-resistant S. aureus. RESULTS: Ertapenem was highly active in vitro against many aerobic and facultative bacterial pathogens commonly recovered from patients with community-acquired bacteremia (128/159, 80.5 %). Ertapenem had more potent activity than ceftriaxone, piperacillin-tazobactam, or ciprofloxacin against oxacillin-susceptible S. aureus (17/17, 100%)and was more active than any of these agents against enterobacteriaceae (82/82, 100%). CONCLUSION: Based on the microbiology pattern of community-acquired bacteremia, initial empiric treatment that requires coverage of a broad spectrum of both gram-negative and gram-positive aerobic bacteria, such as ertapenem, may be justified in moderately severe cases of community-acquired bacteremia in non-immunocompromised hosts. [Abstract/Link to Full Text]

Heiro M, Helenius H, Hurme S, Savunen T, Engblom E, Nikoskelainen J, Kotilainen P
Short-term and one-year outcome of infective endocarditis in adult patients treated in a Finnish teaching hospital during 1980-2004.
BMC Infect Dis. 2007;778.
BACKGROUND: Previous studies on factors predicting the prognosis of infective endocarditis have given somewhat conflicting results. Our aim was to define the factors predicting the outcome of patients treated in a Finnish teaching hospital. METHODS: A total of 326 episodes of infective endocarditis in 303 patients treated during 1980-2004 were evaluated for short-term and 1-year outcome and complications. RESULTS: Infection of 2 native valves and the occurrence of neurological complications, peripheral emboli, or heart failure significantly predicted both in-hospital and 1-year mortality, while age > or =65 years or the presence of a major criterion or vegetation on echocardiography predicted death within 1 year. A significant trend was observed between the level of serum C-reactive protein (CRP) on admission and both the short-term and 1-year outcome. In the patients who had CRP values > or =100 mg/l on admission, the hazard ratio for in-hospital death was 2.9-fold and the hazard ratio for 1-year death was 3.9-fold as compared to those with lower CRP values. Male sex and age < 64 years significantly predicted a need for both in-hospital and 1-year surgery, as did the development of heart failure or the presence of a major criterion or vegetation on echocardiography. Peripheral emboli were associated with a need for in-hospital surgery, while Streptococcus pneumoniae as the causative agent or infection of 2 native valves predicted a need for surgery within 1 year from admission. CONCLUSION: Some of the factors (e.g. heart failure, neurological complications, peripheral emboli) predicting a poor prognosis and/or need for surgery were the same observed in previous studies. A new finding was that high CRP values (> or =100 mg/l) on admission significantly predicted both short-term and 1-year mortality. [Abstract/Link to Full Text]

Ng'andwe C, Lowe JJ, Richards PJ, Hause L, Wood C, Angeletti PC
The distribution of sexually-transmitted Human Papillomaviruses in HIV positive and negative patients in Zambia, Africa.
BMC Infect Dis. 2007;777.
BACKGROUND: Human Papillomaviruses (HPV) are double-stranded DNA viruses, considered to be the primary etiological agents in cervical intraepithelial neoplasias and cancers. Approximately 15-20 of the 40 mucosal HPVs confer a high-risk of progression of lesions to invasive cancer. In this study, we investigated the prevalence of sexually transmitted HPVs in Human Immunodeficiency Virus (HIV) positive and negative patients in Zambia, Africa. The rate of high-risk HPV genotypes worldwide varies within each country. Thus, we sought to investigate the rates of HPV infection in sub-Saharan Africa and the potential role of HIV in affecting the HPV genotype distribution. METHODS: This retrospective cross-sectional study reports findings on the association and effects of HIV on HPV infections in an existing cohort of patients at University Teaching Hospital (UTH) Lusaka, Zambia. The objective of this study was to assess HPV prevalence, genotype distribution and to identify co-factors that influence HPV infection. Polymerase chain reaction (PCR) with two standard consensus primer sets (CpI/II and GP5+/6+) was used to test for the presence of HPV DNA. Primers specific for beta-actin were used to monitor DNA quality. Vaginal lavage samples, collected between 1998-1999 from a total of 70 women, were part of a larger cohort that was also analyzed for HIV and human herpesvirus infection. Seventy of the samples yielded usable DNA. HIV status was determined by two rapid assays, Capillus and Determine. The incidence of HIV and HPV infections and HPV genotype distributions were calculated and statistical significance was determined by Chi-Squared test. RESULTS: We determined that most common HPV genotypes detected among these Zambian patients were types 16 and 18 (21.6% each), which is approximately three-fold greater than the rates for HPV16, and ten-fold greater than the rates for HPV18 in the United States. The worldwide prevalence of HPV16 is approximately 14% and HPV18 is 5%. The overall ratio of high-risk (HR) to low-risk (LR) HPVs in the patient cohort was 69% and 31% respectively; essentially identical to that for the HR and LR distributions worldwide. However, we discovered that HIV positive patients were two-times as likely to have an HR HPV as HIV negative individuals, while the distribution of LR HPVs was unaffected by HIV status. Interestingly, we observed a nine-fold increase in HPV18 infection frequency in HIV positive versus HIV negative individuals. CONCLUSION: The rate of oncogenic HPVs (type 16 and 18) in Zambia was much higher than in the U.S., potentially providing an explanation for the high-rates of cervical cancer in Zambia. Surprisingly, we discovered a strong association between positive HIV status and the prevalence of HR HPVs, and specifically HPV18. [Abstract/Link to Full Text]

Duerr HP, Brockmann SO, Piechotowski I, Schwehm M, Eichner M
Influenza pandemic intervention planning using InfluSim: pharmaceutical and non- pharmaceutical interventions.
BMC Infect Dis. 2007;776.
BACKGROUND: Influenza pandemic preparedness plans are currently developed and refined on national and international levels. Much attention has been given to the administration of antiviral drugs, but contact reduction can also be an effective part of mitigation strategies and has the advantage to be not limited per se. The effectiveness of these interventions depends on various factors which must be explored by sensitivity analyses, based on mathematical models. METHODS: We use the freely available planning tool InfluSim to investigate how pharmaceutical and non-pharmaceutical interventions can mitigate an influenza pandemic. In particular, we examine how intervention schedules, restricted stockpiles and contact reduction (social distancing measures and isolation of cases) determine the course of a pandemic wave and the success of interventions. RESULTS: A timely application of antiviral drugs combined with a quick implementation of contact reduction measures is required to substantially protract the peak of the epidemic and reduce its height. Delays in the initiation of antiviral treatment (e.g. because of parsimonious use of a limited stockpile) result in much more pessimistic outcomes and can even lead to the paradoxical effect that the stockpile is depleted earlier compared to early distribution of antiviral drugs. CONCLUSION: Pharmaceutical and non-pharmaceutical measures should not be used exclusively. The protraction of the pandemic wave is essential to win time while waiting for vaccine development and production. However, it is the height of the peak of an epidemic which can easily overtax general practitioners, hospitals or even whole public health systems, causing bottlenecks in basic and emergency medical care. [Abstract/Link to Full Text]

Alvarado-Esquivel C, Mercado-Suarez MF, Rodríguez-Briones A, Fallad-Torres L, Ayala-Ayala JO, Nevarez-Piedra LJ, Duran-Morales E, Estrada-Martínez S, Liesenfeld O, Márquez-Conde JA, Martínez-García SA
Seroepidemiology of infection with Toxoplasma gondii in healthy blood donors of Durango, Mexico.
BMC Infect Dis. 2007;775.
BACKGROUND: Toxoplasma gondii (T. gondii) infection in blood donors could represent a risk for transmission in blood recipients. There is scarce information about the epidemiology of T. gondii infection in blood donors in Mexico. Therefore, we sought to determine the prevalence of T. gondii infection and associated socio-demographic and behavioral characteristics in a population of healthy blood donors of Durango City, Mexico. METHODS: Four hundred and thirty two blood donors in two public blood banks of Durango City, Mexico were examined for T. gondii infection between August to September 2006. Blood donors were tested for anti-T. gondii IgG and IgM antibodies by using enzyme-linked immunoassays (Diagnostic Automation Inc., Calabasas, CA, USA). Socio-demographic and behavioral characteristics from each participant were also obtained. RESULTS: Thirty two (7.4%) of 432 blood donors had IgG anti-T. gondii antibodies. Eight (1.9%) of them had also IgM anti-T. gondii antibodies. Multivariate analysis using logic regression showed that T. gondii infection was associated with the presence of cats at home (adjusted OR = 3.81; 95% CI: 1.45-10.01). The age group of 45-60 years showed a significantly higher frequency of T. gondii infection than the group of 25-34 years (p = 0.02). Blood donors without education had a significantly higher frequency of infection (15.8%) than those with 13-19 years of education (4.5%) (p = 0.04). Other characteristics of blood donors including male gender, consumption of undercooked meat or blood transfusion did not show an association with infection. CONCLUSION: The prevalence of T. gondii infection in healthy blood donors of Durango City, Mexico is lower than those reported in blood donors of south and central Mexico, and is one of the lowest reported in blood donors worldwide. T. gondii infection in our blood donors was most likely acquired by contact with cats. Prevalence of infection increased with age and decreased with educational level. [Abstract/Link to Full Text]

Kashyap RS, Rajan AN, Ramteke SS, Agrawal VS, Kelkar SS, Purohit HJ, Taori GM, Daginawala HF
Diagnosis of tuberculosis in an Indian population by an indirect ELISA protocol based on detection of Antigen 85 complex: a prospective cohort study.
BMC Infect Dis. 2007;774.
BACKGROUND: Diagnosis of tuberculosis (TB) remains problematic despite many new advanced diagnostic methods. A reliable and rapid diagnostic test, which could be performed in any standard pathology laboratory, would help to obtain definitive early diagnoses of TB. In the present study we describe a prospective evaluation for demonstrating Antigen (Ag) 85 complex in the sera from TB patients. METHODS: Indirect ELISA, employing monoclonal antibodies (mAb) against the purified Ag 85 complex, was used to demonstrate Ag 85 complex in sera from TB patients. Serum samples were obtained from 197 different groups of patients: confirmed TB {n = 24}, clinically diagnosed TB {n = 104}, disease controls {n = 49} and healthy controls {n = 20}. Receiver operating curve (ROC) was used to calculate the cut off value and comparison between TB and non-TB groups were done by the chi-square test. RESULTS: The indirect ELISA method, using an mAb against Ag 85 complex, yielded 82% sensitivity (95% confidence interval [CI] 67 to 93%) and 86% specificity (95% CI, 57 to 98%) for the diagnosis of TB. The serum positivities for Ag 85 complex in cases of confirmed and clinically diagnosed TB patients were 96% (23/24) and 79% (82/104) respectively, while the positivity for patients in the non-tuberculosis group was 14% (10/69). CONCLUSION: The detection of Ag 85 complex in sera from TB patients by indirect ELISA using mAb against purified Ag 85 complex gives a reliable diagnosis and can be used to develop an immunodiagnostic assay with increased sensitivity and specificity. [Abstract/Link to Full Text]

Domínguez A, Bruguera M, Plans P, Espuñes J, Costa J, Plasencia A, Salleras L
Declining hepatitis A seroprevalence in adults in Catalonia (Spain): a population-based study.
BMC Infect Dis. 2007;773.
BACKGROUND: One of the main uses of seroprevalence studies it to evaluate vaccination programmes. In 1998, a programme of universal vaccination of preadolescents in schools with the hepatitis A vaccine was begun in Catalonia. The objective of this study was to investigate the prevalence and risk factors of hepatitis A virus infection (HAV) in a sample of the adult population of Catalonia in 2002 and to evaluate the changes with respect to a survey carried out in 1996. METHODS: The prevalence of HAV antibodies was determined by a third generation competitive immunometric assay in a representative sample of 1292 people aged >15 years. The association between the prevalence and different sociodemographic variables was determined by multiple logistic regression analysis. RESULTS: The standardized global prevalence of HAV antibodies in 2002 was 68.2%, increased with age (p < 0.0001) and was associated with being born outside Catalonia (OR: 1.75; 95% CI 1.11-2.76) and lower social class (OR: 1.14; 95% CI 1.05-1.25). Compared with the last survey carried out in 1996 the standardized global prevalence was lower (68.2% vs 77.8%; p < 0.0001) as was the prevalence in people under 45 years. CONCLUSION: The prevalence of the hepatitis A virus is decreasing in the adult population of Catalonia, especially in the younger age groups. The programme of vaccination of adolescents begun in 1998 to control the disease can provide indirect protection to the unvaccinated population. [Abstract/Link to Full Text]

Niedrig M, Donoso Mantke O, Altmann D, Zeller H
First international diagnostic accuracy study for the serological detection of West Nile virus infection.
BMC Infect Dis. 2007;772.
BACKGROUND: The diagnosis of an acute or convalescent West Nile (WN) virus infection can be confirmed by various serological assays such as enzyme immunoassay (EIA), immunofluorescence assay (IFA), or neutralisation test (NT) which are conducted by a growing number of laboratories. However, as the degree of proficiency may vary between laboratories, quality control measures for laboratory diagnostics are essential. METHODS: We have performed an external quality assurance (EQA) programme for the serological detection of WN virus infection to assess the diagnostic quality of laboratories. The participating laboratories received a proficiency panel of 10 coded lyophilised test samples comprising four antisera positive for WN antibodies as positive controls, three antisera positive for antibodies against other heterologous flaviviruses plus one multireactive unspecific serum as specificity controls, and two negative serum samples. RESULTS: Twenty-seven laboratories from 20 different countries in Europe, the Middle East, the Americas and Africa participated in this EQA programme. Applying the proficiency criteria of this study, only eight laboratories correctly analysed all samples with their respective EIA, IFA or NT methods. Eighteen laboratories correctly identified between 77.8 and 90% of the samples, and one laboratory identified only 70% correctly with a clear need to eliminate cross-reactivity with other antisera, particularly those elicited by yellow fever virus. Differentiation between the results for IgM and IgG was considered separately and revealed that IgM-antibodies were detected less frequently than IgG-antibodies (p < 0.001). However, the assay used was not a significant technical factor influencing laboratory performance. CONCLUSION: The EQA programme provides information on the quality of different serological assays used by the participating laboratories and indicates that most need to improve their assays, in particular to avoid cross-reactions with antibodies to heterologous flaviviruses. [Abstract/Link to Full Text]

Colugnati FA, Staras SA, Dollard SC, Cannon MJ
Incidence of cytomegalovirus infection among the general population and pregnant women in the United States.
BMC Infect Dis. 2007;771.
BACKGROUND: Cytomegalovirus (CMV) is a common opportunistic infection among HIV-infected individuals, a major source of serious complications among organ-transplant recipients, and a leading cause of hearing loss, vision loss, and mental retardation among congenitally infected children. Women infected for the first time during pregnancy are especially likely to transmit CMV to their fetuses. More children suffer serious disabilities caused by congenital CMV than by several better-known childhood maladies such as Down syndrome or fetal alcohol syndrome METHODS: Using CMV seroprevalence data from the nationally representative Third National Health and Nutrition Examination Survey, we estimated CMV incidence among the general United States population and among pregnant women. We employed catalytic models that used age-specific CMV seroprevalences as cumulative markers of past infections in order to derive estimates of three basic parameters: the force of infection, the basic reproductive rate, and the average age of infection. Our main focus was the force of infection, an instantaneous per capita rate of acquisition of infection that approximates the incidence of infection in the seronegative population. RESULTS: Among the United States population ages 12-49 the force of infection was 1.6 infections per 100 susceptible persons per year (95% confidence interval: 1.2, 2.4). The associated basic reproductive rate of 1.7 indicates that, on average, an infected person transmits CMV to nearly two susceptible people. The average age of CMV infection was 28.6 years. Force of infection was significantly higher among non-Hispanic Blacks (5.7) and Mexican Americans (5.1) than among non-Hispanic Whites (1.4). Force of infection was significantly higher in the low household income group (3.5) than in the middle (2.1) and upper (1.5) household income groups. Based on these CMV incidence estimates, approximately 27,000 new CMV infections occur among seronegative pregnant women in the United States each year. CONCLUSION: These thousands of CMV infections in pregnant women, along with the sharp racial/ethnic disparities in CMV incidence, are compelling reasons for accelerating research on vaccines and other interventions for preventing congenital CMV disease. Nevertheless, the relatively low force of infection provides encouraging evidence that modestly effective vaccines and rates of vaccination could significantly reduce CMV transmission. [Abstract/Link to Full Text]

Riedel A, Choe L, Inciardi J, Yuen C, Martin T, Guglielmo BJ
Antifungal prophylaxis in chemotherapy-associated neutropenia: a retrospective, observational study.
BMC Infect Dis. 2007;770.
BACKGROUND: In August 2002, the antifungal prophylaxis algorithm for neutropenic hematology/oncology (NHO) patients at the Medical Center was changed from conventional amphotericin (AMB) to an azole (AZ) based regimen (fluconazole [FLU] in low-risk and voriconazole [VOR] in high-risk patients). The aim of our study was to compare outcomes associated with the two regimens, including breakthrough fungal infection, adverse drug events, and costs. METHODS: Adult, non-febrile, NHO patients who received prophylactic AMB from 01 August 2001-30 July 2002 or AZ from 01 August 2002-30 July 2003 were retrospectively evaluated. RESULTS: A total of 370 patients (AMB: n = 181; AZ: n = 216) associated with 580 hospitalizations (AMB: n = 259; AZ: n = 321) were included. The incidence of probable/definite breakthrough Aspergillus infections was similar among regimens (AMB: 1.9% vs AZ: 0.6%; p=0.19). A greater incidence of mild/moderate (24.7% vs. 5.3%; p < 0.0001) and severe renal dysfunction (13.5% vs. 4.4%; p < 0.0012) was observed with AMB. In contrast, patients treated with VOR were found to have an increased rate of severe hepatic toxicity (32.5%) compared with patients treated with either AMB (22.6%) or FLU (21.4%) (p = 0.05). While the AZ period was associated with a >$9,000 increase in mean total costs/hospitalization, the mean acquisition cost associated with AZ was only $947/hospitalization more than AMB. CONCLUSION: While an AZ-based regimen is associated with increased cost, the reduced rate of nephrotoxicity and availability of oral dosage forms, suggests that azoles be used preferentially over AMB. However, an increased rate of severe hepatic toxicity may be associated with VOR. [Abstract/Link to Full Text]

Kulwichit W, Nilgate S, Chatsuwan T, Krajiw S, Unhasuta C, Chongthaleong A
Accuracies of Leuconostoc phenotypic identification: a comparison of API systems and conventional phenotypic assays.
BMC Infect Dis. 2007;769.
BACKGROUND: Commercial diagnostics are commonly used to identify gram-positive bacteria. Errors have been reported mostly at the species level. We have found certain phenotypic criteria used in API systems which significantly misidentify Leuconostoc, an emerging human pathogen, at the genus level. We also attempt to find practical, conventional phenotypic assays for accurate identification of this group of bacteria. METHODS: Clinical isolates of catalase-negative, gram-positive coccoid or coccobacillary bacteria with non-beta hemolysis in our institute during 1997-2004 were subject to an identification aid by API 20 STREP, following the instruction manual, as an aid to conventional phenotypic tests. Those identified as Leuconostoc by API 20 STREP were re-examined by the same kit and also by API 50 CHL according to the instruction manuals, by our Leuconostoc conventional phenotypic assays, by Leuconostoc- and Lactobacillus-specific PCR's, and, where possible, by 16S rDNA sequence analysis. In addition, catalase-negative gram-positive isolates during 2005-2006 which were resistant to vancomycin at high levels were also evaluated by the same phenotypic and genotypic assays. RESULTS: Out of several thousands of clinical gram-positive isolates, 26 catalase negative gram-positive isolates initially identified as Leuconostoc by API 20 STREP and 7 vancomycin-resistant gram-positive catalase-negative bacteria entered the study. 11 out of the 26 isolates and all the 7 isolates were identified as Leuconostoc by API 20 STREP. Only 5 isolates, however, were confirmed by both genotypic and all defined conventional phenotypic criteria. API 50 CHL also failed to reliably provide accurate identification of Leuconostoc. We have identified key problem tests in API 20 STREP leading to misidentification of the bacteria. A simple, conventional set of phenotypic tests for Leuconostoc identification is proposed. CONCLUSION: The current API systems cannot accurately identify Leuconostoc. Identification of vancomycin-resistant, catalase-negative gram-positive bacteria should be performed by a few practical phenotypic assays, with assistance of genotypic assays where available. [Abstract/Link to Full Text]

Currie BJ, Gal D, Mayo M, Ward L, Godoy D, Spratt BG, LiPuma JJ
Using BOX-PCR to exclude a clonal outbreak of melioidosis.
BMC Infect Dis. 2007;768.
BACKGROUND: Although melioidosis in endemic regions is usually caused by a diverse range of Burkholderia pseudomallei strains, clonal outbreaks from contaminated potable water have been described. Furthermore B. pseudomallei is classified as a CDC Group B bioterrorism agent. Ribotyping, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) have been used to identify genetically related B. pseudomallei isolates, but they are time consuming and technically challenging for many laboratories. METHODS: We have adapted repetitive sequence typing using a BOX A1R primer for typing B. pseudomallei and compared BOX-PCR fingerprinting results on a wide range of well-characterized B. pseudomallei isolates with MLST and PFGE performed on the same isolates. RESULTS: BOX-PCR typing compared favourably with MLST and PFGE performed on the same isolates, both discriminating between the majority of multilocus sequence types and showing relatedness between epidemiologically linked isolates from various outbreak clusters. CONCLUSION: Our results suggest that BOX-PCR can be used to exclude a clonal outbreak of melioidosis within 10 hours of receiving the bacterial strains. [Abstract/Link to Full Text]

Wilson S, Booth M, Jones FM, Mwatha JK, Kimani G, Kariuki HC, Vennervald BJ, Ouma JH, Muchiri E, Dunne DW
Age-adjusted Plasmodium falciparum antibody levels in school-aged children are a stable marker of microgeographical variations in exposure to Plasmodium infection.
BMC Infect Dis. 2007;767.
BACKGROUND: Amongst school-aged children living in malaria endemic areas, chronic morbidity and exacerbation of morbidity associated with other infections are often not coincident with the presence or levels of Plasmodium parasitaemia, but may result from long-term exposure to the parasite. Studies of hepatosplenomegaly associated with Schistosoma mansoni infection and exposure to Plasmodium infection indicate that differences that occur over 1-2 km in levels of Plasmodium transmission are related to the degree of exacerbation of hepatosplenomegaly and that Plasmodium falciparum schizont antigen (Pfs)-IgG3 levels may be a marker for the differing levels of exposure. METHODS: To investigate the validity of Pfs-IgG3 measurements as a tool to assess these comparative exposure levels on a microgeographical scale, cross-sectional community surveys were conducted over a 10 x 6 km study site in Makueni District, Kenya, during low and high malaria transmission seasons. During both high and low malaria transmission seasons, thick blood smears were examined microscopically and circulating Pfs-IgG3 levels measured from dried blood spot elute. GIS techniques were used to map prevalence of parasitaemia and Pfs-IgG3 levels. RESULTS: Microgeographical variations in prevalence of parasitaemia were observed during the high but not the low transmission season. Pfs-IgG3 levels were stable between high and low transmission seasons, but increased with age throughout childhood before reaching a plateau in adults. Adjusting Pfs-IgG3 levels of school-aged children for age prior to mapping resulted in spatial patterns that reflected the microgeographical variations observed for high season prevalence of parasitaemia, however, Pfs-IgG3 levels of adults did not. The distances over which age-adjusted Pfs-IgG3 of school-aged children fluctuated were comparable with those distances over which chronic morbidity has previous been shown to vary. CONCLUSION: Age-adjusted Pfs-IgG3 levels of school-aged children are stable and when mapped can provide a tool sensitive enough to detect microgeographical variations in malaria exposure, that would be useful for studying the aetiology of morbidities associated with long-term exposure and co-infections. [Abstract/Link to Full Text]

Pizarro JC, Lucero DE, Stevens L
PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans: high rates found in Chuquisaca, Bolivia.
BMC Infect Dis. 2007;766.
BACKGROUND: The Andean valleys of Bolivia are the only reported location of sylvatic Triatoma infestans, the main vector of Chagas disease in this country, and the high human prevalence of Trypanosoma cruzi infection in this region is hypothesized to result from the ability of vectors to persist in domestic, peri-domestic, and sylvatic environments. Determination of the rate of Trypanosoma infection in its triatomine vectors is an important element in programs directed at reducing human infections. Traditionally, T. cruzi has been detected in insect vectors by direct microscopic examination of extruded feces, or dissection and analysis of the entire bug. Although this technique has proven to be useful, several drawbacks related to its sensitivity especially in the case of small instars and applicability to large numbers of insects and dead specimens have motivated researchers to look for a molecular assay based on the polymerase chain reaction (PCR) as an alternative for parasitic detection of T. cruzi infection in vectors. In the work presented here, we have compared a PCR assay and direct microscopic observation for diagnosis of T. cruzi infection in T. infestans collected in the field from five localities and four habitats in Chuquisaca, Bolivia. The efficacy of the methods was compared across nymphal stages, localities and habitats. METHODS: We examined 152 nymph and adult T. infestans collected from rural areas in the department of Chuquisaca, Bolivia. For microscopic observation, a few drops of rectal content obtained by abdominal extrusion were diluted with saline solution and compressed between a slide and a cover slip. The presence of motile parasites in 50 microscopic fields was registered using 400x magnification. For the molecular analysis, dissection of the posterior part of the abdomen of each insect followed by DNA extraction and PCR amplification was performed using the TCZ1 (5' - CGA GCT CTT GCC CAC ACG GGT GCT - 3') and TCZ2 (5' - CCT CCA AGC AGC GGA TAG TTC AGG - 3') primers. Amplicons were chromatographed on a 2% agarose gel with a 100 bp size standard, stained with ethidium bromide and viewed with UV fluorescence.For both the microscopy and PCR assays, we calculated sensitivity (number of positives by a method divided by the number of positives by either method) and discrepancy (one method was negative and the other was positive) at the locality, life stage and habitat level. The degree of agreement between PCR and microscopy was determined by calculating Kappa (k) values with 95% confidence intervals. RESULTS: We observed a high prevalence of T. cruzi infection in T. infestans (81.16% by PCR and 56.52% by microscopy) and discovered that PCR is significantly more sensitive than microscopic observation. The overall degree of agreement between the two methods was moderate (Kappa = 0.43 +/- 0.07). The level of infection is significantly different among communities; however, prevalence was similar among habitats and life stages. CONCLUSION: PCR was significantly more sensitive than microscopy in all habitats, developmental stages and localities in Chuquisaca, Bolivia. Overall we observed a high prevalence of T. cruzi infection in T. infestans in this area of Bolivia; however, microscopy underestimated infection at all levels examined. [Abstract/Link to Full Text]

Wickham ME, Brown NF, Provias J, Finlay BB, Coombes BK
Oral infection of mice with Salmonella enterica serovar Typhimurium causes meningitis and infection of the brain.
BMC Infect Dis. 2007;765.
BACKGROUND: Salmonella meningitis is a rare and serious infection of the central nervous system following acute Salmonella enterica sepsis. For this pathogen, no appropriate model has been reported in which to examine infection kinetics and natural dissemination to the brain. METHODS: Five mouse lines including C57BL/6, Balb/c, 129S6-Slc11a1tm1Mcg, 129S1/SvImJ, B6.129-Inpp5dtm1Rkh were used in the murine typhoid model to examine the dissemination of systemic Salmonella enterica serovar Typhimurium following oral infection. RESULTS: We report data on spontaneous meningitis and brain infection following oral infection of mice with Salmonella enterica serovar Typhimurium. CONCLUSION: This model may provide a system in which dissemination of bacteria through the central nervous system and the influence of host and bacterial genetics can be queried. [Abstract/Link to Full Text]

Alam MM, Zaidi SZ, Malik SA, Naeem A, Shaukat S, Sharif S, Angez M, Khan A, Butt JA
Serology based disease status of Pakistani population infected with hepatitis B virus.
BMC Infect Dis. 2007;764.
BACKGROUND: The infection rate of hepatitis B virus is continuously increasing in Pakistan. Therefore, a comprehensive study of epidemiological data is the need of time. METHODS: A total of 1300 individuals were screened for HBV infection markers including HBsAg, anti-HBsAg, HBeAg and anti-HBcAg. The association of these disease indicators was compared with patients' epidemiological characteristics like age, socio-economic status and residential area to analyze and find out the possible correlation among these variables and the patients disease status. RESULTS: 52 (4%) individuals were found positive for HBsAg with mean age 23.5 +/- 3.7 years. 9.30%, 33.47% and 12% individuals had HBeAg, antibodies for HBsAg, and antibodies for HBcAg respectively. HBsAg seropositivity rate was significantly associated (p = 0.03) with the residing locality indicating high infection in rural areas. Antibodies titer against HBsAg decreased with the increasing age reflecting an inverse correlation. CONCLUSION: Our results indicate high prevalence rate of Hepatitis B virus infection and nationwide vaccination campaigns along with public awareness and educational programs are needed to be practiced urgently. [Abstract/Link to Full Text]

Graham SM, Baeten JM, Richardson BA, Bankson DD, Lavreys L, Ndinya-Achola JO, Mandaliya K, Overbaugh J, McClelland RS
Higher pre-infection vitamin E levels are associated with higher mortality in HIV-1-infected Kenyan women: a prospective study.
BMC Infect Dis. 2007;763.
BACKGROUND: Low vitamin E levels are often found in HIV-1 infection, and studies have suggested that higher levels may decrease the risk of disease progression. However, vitamin E supplementation has also been reported to increase CCR5 expression, which could increase HIV-1 replication. We hypothesized that vitamin E levels at HIV-1 acquisition may influence disease progression. METHODS: Vitamin E status was measured in stored samples from the last pre-infection visit for 67 Kenyan women with reliably estimated dates of HIV-1 acquisition. Regression analyses were used to estimate associations between pre-infection vitamin E and plasma viral load, time to CD4 count <200 cells/muL, and mortality. RESULTS: After controlling for potential confounding factors, each 1 mg/L increase in pre-infection vitamin E was associated with 0.08 log10 copies/mL (95% CI -0.01 to +0.17) higher set point viral load and 1.58-fold higher risk of mortality (95% CI 1.15-2.16). The association between higher pre-infection vitamin E and mortality persisted after adjustment for set point viral load (HR 1.55, 95% CI 1.13-2.13). CONCLUSION: Higher pre-infection vitamin E levels were associated with increased mortality. Further research is needed to elucidate the role vitamin E plays in HIV-1 pathogenesis. [Abstract/Link to Full Text]

Ranjbar R, Aleo A, Giammanco GM, Dionisi AM, Sadeghifard N, Mammina C
Genetic relatedness among isolates of Shigella sonnei carrying class 2 integrons in Tehran, Iran, 2002-2003.
BMC Infect Dis. 2007;762.
BACKGROUND: Shigella spp. are major cause of diarrhoeal disease in both developing and developed countries. Shigella sonnei is the serogroup of Shigella most frequently responsible for sporadic and epidemic enteritis in developed countries. In recent years the emergence and spread of S. sonnei biotype g carrying class 2 integron have been frequently reported in many countries. Recently, S. sonnei has been reported as the prevalent serogroup of Shigella in Iran.The present study was carried out to investigate phenotypic and genetic characteristics of Shigella sonnei isolates identified in the years 2002 and 2003 in Tehran, Iran. METHODS: Biotyping, drug susceptibility testing, pulsed field gel electrophoresis (PFGE) and analysis of class 2 integrons have been carried out on 60 S. sonnei isolates, including 57 sporadic isolates from paediatric cases of shigellosis occurring in 2002 and 2003, two sporadic isolates recovered in 1984 and the ATCC 9290 strain. RESULTS: Biotype g and resistance to streptomycin, sulfamethoxazole-trimethoprim and tetracycline were exhibited by 54 of the 57 recent isolates. Of the 54 biotype g isolates, 28 exhibited a class 2 integron of 2161 bp, and 24 a class 2 integron of 1371 bp, respectively. Class 2 integrons were not detected in four isolates only, including the two endemic isolates recovered in 1984 and two strains from recent sporadic cases. PFGE divided the strains into eight pulsotypes labeled A to H, three major pulsotypes - A to C - including the large majority of the recent sporadic S. sonnei isolates. Pulsotypes A and C were the most prevalent groups, accounting for 41.6% and 35.0%, respectively, of the isolates under study. CONCLUSION: The results suggest that biotype g, class 2 integron carrying S. sonnei are prevalent in our geographic area. S. sonnei isolated in the years 2002 and 2003 could be attributed to a few predominant clusters including, respectively, strains with pulsotypes B and C carrying a 2161 bp class 2 integron, and those having pulsotype A and a 1371 bp class 2 integron. A few epidemic clones are responsible for the apparently endemic occurrence of shigellosis in Tehran, Iran. [Abstract/Link to Full Text]

Eckstein BC, Adams DA, Eckstein EC, Rao A, Sethi AK, Yadavalli GK, Donskey CJ
Reduction of Clostridium Difficile and vancomycin-resistant Enterococcus contamination of environmental surfaces after an intervention to improve cleaning methods.
BMC Infect Dis. 2007;761.
BACKGROUND: Contaminated environmental surfaces may play an important role in transmission of some healthcare-associated pathogens. In this study, we assessed the adequacy of cleaning practices in rooms of patients with Clostridium difficile-associated diarrhea (CDAD) and vancomycin-resistant Enterococcus (VRE) colonization or infection and examined whether an intervention would result in improved decontamination of surfaces. METHODS: During a 6-week period, we cultured commonly touched surfaces (i.e. bedrails, telephones, call buttons, door knobs, toilet seats, and bedside tables) in rooms of patients with CDAD and VRE colonization or infection before and after housekeeping cleaning, and again after disinfection with 10% bleach performed by the research staff. After the housekeeping staff received education and feedback, additional cultures were collected before and after housekeeping cleaning during a 10-week follow-up period. RESULTS: Of the 17 rooms of patients with VRE colonization or infection, 16 (94%) had one or more positive environmental cultures before cleaning versus 12 (71%) after housekeeping cleaning (p = 0.125), whereas none had positive cultures after bleach disinfection by the research staff (p < 0.001). Of the 9 rooms of patients with CDAD, 100% had positive cultures prior to cleaning versus 7 (78%) after housekeeping cleaning (p = 0.50), whereas only 1 (11%) had positive cultures after bleach disinfection by research staff (p = 0.031). After an educational intervention, rates of environmental contamination after housekeeping cleaning were significantly reduced. CONCLUSION: Our findings provide additional evidence that simple educational interventions directed at housekeeping staff can result in improved decontamination of environmental surfaces. Such interventions should include efforts to monitor cleaning and disinfection practices and provide feedback to the housekeeping staff. [Abstract/Link to Full Text]

Newell ML, Huang S, Fiore S, Thorne C, Mandelbrot L, Sullivan JL, Maupin R, Delke I, Watts DH, Gelber RD, Cunningham CK
Characteristics and management of HIV-1-infected pregnant women enrolled in a randomised trial: differences between Europe and the USA.
BMC Infect Dis. 2007;760.
BACKGROUND: Rates of mother-to-child transmission of HIV-1 (MTCT) have historically been lower in European than in American cohort studies, possibly due to differences in population characteristics. The Pediatric AIDS Clinical Trials Group Protocol (PACTG) 316 trial evaluated the effectiveness of the addition of intrapartum/neonatal nevirapine in reducing MTCT in women already receiving antiretroviral prophylaxis. Participation of large numbers of pregnant HIV-infected women from the US and Western Europe enrolling in the same clinical trial provided the opportunity to identify and explore differences in their characteristics and in the use of non-study interventions to reduce MTCT. METHODS: In this secondary analysis, 1350 women were categorized according to enrollment in centres in the USA (n = 978) or in Europe (n = 372). Factors associated with receipt of highly active antiretroviral therapy and with elective caesarean delivery were identified with logistic regression. RESULTS: In Europe, women enrolled were more likely to be white and those of black race were mainly born in Sub-Saharan Africa. Women in the US were younger and more likely to have previous pregnancies and miscarriages and a history of sexually transmitted infections.More than 90% of women did not report symptoms of their HIV infection; however, more women from the US had symptoms (8%), compared to women from Europe (4%). Women in the US were less likely to have HIV RNA levels <400 copies/ml at delivery than women enrolling in Europe, and more likely to receive highly active antiretroviral therapy, and to start therapy earlier in pregnancy. The elective caesarean delivery rate in Europe was 61%, significantly higher than that in the US (22%). Overall, 1.48% of infants were infected and there was no significant difference in the rate of transmission between Europe and the US despite the different approaches to treatment and delivery. CONCLUSION: These findings confirm that there are important historical differences between the HIV-infected pregnant populations in Western Europe and the USA, both in terms of the characteristics of the women and their obstetric and therapeutic management. Although highly active antiretroviral therapy predominates in pregnancy in both settings now, population differences are likely to remain. TRIAL REGISTRATION: NCT00000869. [Abstract/Link to Full Text]

Pérez-Farinós N, Ordobás M, García-Fernández C, García-Comas L, Cañellas S, Rodero I, Gutiérrez-Rodríguez A, García-Gutiérrez J, Ramírez R
Varicella and herpes zoster in Madrid, based on the Sentinel General Practitioner Network: 1997-2004.
BMC Infect Dis. 2007;759.
BACKGROUND: Varicella (chickenpox) is the primary disease caused by varicella-zoster virus. It is extremely contagious and is frequent in children. Indeed, in the absence of vaccination, a high proportion of the population is liable to contract it. Herpes zoster -more frequent among adults- is caused by reactivation of the latent virus. The objective of this study is to describe the status of and time trend for varicella and herpes zoster in the Madrid Autonomous Region prior to the introduction of the vaccine to the general population. METHODS: Data source: individualised varicella and herpes zoster case records kept by the Madrid Autonomous Region Sentinel General Practitioner Network for the period 1997-2004. Cumulative incidences, crude and standardised incidence rates, and age-specific rates of varicella and herpes zoster were calculated for each year. Kendall's Tau-b correlation coefficient was calculated to evaluate whether incidence displayed a time trend. Spectral density in the time series of weekly incidences was estimated using a periodogram. RESULTS: Standardised annual varicella incidence rates ranged from 742.5 (95% CI: 687.2-797.7) to 1239.6 (95% CI: 1164.5-1313.4) cases per 100 000 person-years. Most cases affected children, though complications were more frequent in adults. Varicella incidence displayed an annual periodicity but no trend over time. Most herpes zoster cases occurred at advanced ages, with incidence registering a rising annual trend but no seasonality factor. CONCLUSION: In the absence of vaccination, no significant changes in varicella incidence were in evidence recent years, though these were observed in the incidence of herpes zoster. Sentinel general practitioner networks are a valid instrument for surveillance of diseases such as varicella. Further varicella vaccination-coverage and vaccine-efficacy studies are called for. [Abstract/Link to Full Text]

Haq JA, Khan MA, Afroze N, Haq T
Localized primary renal aspergillosis in a diabetic patient following lithotripsy--a case report.
BMC Infect Dis. 2007;758.
BACKGROUND: Primary renal aspergillosis is rare in diabetic patients. Diagnosis of localized primary renal Aspergillus infection in diabetic patients requires careful investigations due to its benign presentation and lack of associated systemic clinical features. There is also paucity of information on the role of conservative treatment of such localized infection with antifungal agents only. Here, we describe a case of localized renal aspergillosis in a type 2 diabetic patient with a brief review of literature. CASE PRESENTATION: We describe a case of unilateral renal aspergillosis following intracorporeal pneumatic lithotripsy (ICPL) in a type 2 diabetic man. The patient presented with mild pain in the left lumbar region and periodic expulsion of whitish soft masses per urethra, which yielded growth of Aspergillus fumigatus. He was treated initially with amphotericin B; however, it was stopped after 2 weeks, as he could not tolerate the drug. Subsequently, he was successfully treated with oral itraconazole. CONCLUSION: Localized renal aspergillosis may be suspected in diabetic patients having history of urinary tract instrumentation, mild lumbar pain, passage of suspicious masses in urine and persistent pyuria. Examination of the suspicious substances expelled per urethra is essential for diagnosis as routine multiple urine analysis may yield negative results. Conservative treatment with oral itraconazole alone is effective in cases with incomplete obstruction. [Abstract/Link to Full Text]

Jonasson A, Eriksson C, Jenkinson HF, Källestål C, Johansson I, Strömberg N
Innate immunity glycoprotein gp-340 variants may modulate human susceptibility to dental caries.
BMC Infect Dis. 2007;757.
BACKGROUND: Bacterial adhesion is an important determinant of colonization and infection, including dental caries. The salivary scavenger receptor cysteine-rich glycoprotein gp-340, which mediates adhesion of Streptococcus mutans (implicated in caries), harbours three major size variants, designated gp-340 I to III, each specific to an individual saliva. Here we have examined the association of the gp-340 I to III polymorphisms with caries experience and adhesion of S. mutans. METHODS: A case-referent study was performed in 12-year-old Swedish children with high (n = 19) or low (n = 19) caries experiences. We measured the gp-340 I to III saliva phenotypes and correlated those with multiple outcome measures for caries experience and saliva adhesion of S. mutans using the partial least squares (PLS) multivariate projection technique. In addition, we used traditional statistics and 2-year caries increment to verify the established PLS associations, and bacterial adhesion to purified gp-340 I to III proteins to support possible mechanisms. RESULTS: All except one subject were typed as gp-340 I to III (10, 23 and 4, respectively). The gp-340 I phenotype correlated positively with caries experience (VIP = 1.37) and saliva adhesion of S. mutans Ingbritt (VIP = 1.47). The gp-340 II and III phenotypes tended to behave in the opposite way. Moreover, the gp-340 I phenotype tended to show an increased 2-year caries increment compared to phenotypes II/III. Purified gp-340 I protein mediated markedly higher adhesion of S. mutans strains Ingbritt and NG8 and Lactococcus lactis expressing AgI/II adhesins (SpaP or PAc) compared to gp-340 II and III proteins. In addition, the gp-340 I protein appeared over represented in subjects positive for Db, an allelic acidic PRP variant associated with caries, and subjects positive for both gp-340 I and Db tended to experience more caries than those negative for both proteins. CONCLUSION: Gp-340 I behaves as a caries susceptibility protein. [Abstract/Link to Full Text]

Wang TK, Wong SS
Streptobacillus moniliformis septic arthritis: a clinical entity distinct from rat-bite fever?
BMC Infect Dis. 2007;756.
BACKGROUND: Streptobacillus moniliformis is a zoonotic agent associated with rodent contacts. Although it is more commonly reported to cause rat-bite fever with reactive arthritides, it can also lead to pyogenic infection of the joints. CASE PRESENTATION: We present a lady with past history of osteoarthritis developing streptobacillary septic arthritides of the right knee and left wrist, and required antibiotic and arthrotomy for treatment. We also review 11 previously reported cases of streptobacillary septic arthritis to discuss the characteristics, treatment, prognosis of the infection, and illustrates the differences between streptobacillary rat-bite fever and septic arthritis. Among this patient population, most patients had potential contact with rats (91.6%). The knee is the most commonly affected joint (58.3%), and 83.3% patients having polyarticular involvement. As opposed to rat-bite fever, fever and rash was only present in 58.3% and 16.7% of patients respectively. S. moniliformis bacteremia is uncommon (8.4%) and the prognosis is good. CONCLUSION: Arthrocentesis is useful in distinguishing streptobacillary septic arthritis from reactive arthritis of rat-bite fever. The sole use of commercial media containing sodium polyanethol sulfonate may render the bacterial culture negative. A detailed history of possible exposure to rodents should be elicited from patients with arthritis in order to facilitate microbiologic diagnosis. [Abstract/Link to Full Text]

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Recent Articles in BMC Microbiology

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Brillard J, Lereclus D
Characterization of a small PlcR-regulated gene co-expressed with cereolysin O.
BMC Microbiol. 2007;752.
BACKGROUND: In the human pathogen Bacillus cereus, the expression of most extracellular virulence factors is controlled by the transcriptional activator PlcR. Among these virulence factors, cereolysin O (Clo) is an haemolysin belonging to the cholesterol-dependant cytolysins, a protein family extensively studied in Gram-positive bacteria. RESULTS: In the genomes of bacteria belonging to the B. cereus group, including Bacillus anthracis and Bacillus thuringiensis, a small gene encoding a 26-amino acid peptide was present in multicopy. One copy was always found upstream from the gene encoding Clo. In B. cereus ATCC 14579, the small gene and the clo gene are co-transcribed. Transcriptional fusions showed that the three paralogues identified in this strain were expressed in a PlcR-dependent manner. We propose to name these peptides Spp for small PlcR-regulated peptides. We show that a synthetic peptide corresponding to the deduced product of the spp genes displayed antibacterial activity. CONCLUSION: The co-expression of spp, a small PlcR-regulated multicopy gene with clo suggests a yet unidentified relationship between Spp and the cholesterol-dependent cytolysin in bacteria belonging to the B.cereus group. [Abstract/Link to Full Text]

Kibiki GS, Mulder B, Dolmans WM, de Beer JL, Boeree M, Sam N, van Soolingen D, Sola C, van der Zanden AG
M. tuberculosis genotypic diversity and drug susceptibility pattern in HIV-infected and non-HIV-infected patients in northern Tanzania.
BMC Microbiol. 2007;751.
BACKGROUND: Tuberculosis (TB) is a major health problem and HIV is the major cause of the increase in TB. Sub-Saharan Africa is endemic for both TB and HIV infection. Determination of the prevalence of M. tuberculosis strains and their drug susceptibility is important for TB control.TB positive culture, BAL fluid or sputum samples from 130 patients were collected and genotyped. The spoligotypes were correlated with anti-tuberculous drug susceptibility in HIV-infected and non-HIV patients from Tanzania. RESULTS: One-third of patients were TB/HIV co-infected. Forty-seven spoligotypes were identified.Fourteen isolates (10.8%) had new and unique spoligotypes while 116 isolates (89.2%) belonged to 33 known spoligotypes. The major spoligotypes contained nine clusters: CAS1-Kili 30.0%, LAM11- ZWE 14.6%, ND 9.2%, EAI 6.2%, Beijing 5.4%, T-undefined 4.6%, CAS1-Delhi 3.8%, T1 3.8% and LAM9 3.8%. Twelve (10.8%) of the 111 phenotypically tested strains were resistant to anti-TB drugs. Eight (7.2%) were monoresistant strains: 7 to isoniazid (INH) and one to streptomycin. Four strains (3.5%) were resistant to multiple drugs: one (0.9%) was resistant to INH and streptomycin and the other three (2.7%) were MDR strains: one was resistant to INH, rifampicin and ethambutol and two were resistant to all four anti-TB drugs. Mutation in the katG gene codon 315 and the rpoB hotspot region showed a low and high sensitivity, respectively, as predictor of phenotypic drug resistance. CONCLUSION: CAS1-Kili and LAM11-ZWE were the most common families. Strains of the Beijing family and CAS1-Kili were not or least often associated with resistance, respectively. HIV status was not associated with spoligotypes, resistance or previous TB treatment. [Abstract/Link to Full Text]

Parker CT, Miller WG, Horn ST, Lastovica AJ
Common genomic features of Campylobacter jejuni subsp. doylei strains distinguish them from C. jejuni subsp. jejuni.
BMC Microbiol. 2007;750.
BACKGROUND: Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains. RESULTS: A geographically diverse collection of eight Cjd strains was examined by MLST and determined to be phylogenetically distinct from Cjj strains. Microarray-based CGI approach also supported this. We were able to demonstrate that Cjd strains exhibited divergence from Cjj strains NCTC 11168 and RM1221 in many of the intraspecies hypervariable regions. Moreover, multiple metabolic, transport and virulence functions (e.g. cytolethal distending toxin) were shown to be absent in the Cjd strains examined. CONCLUSION: Our data demonstrate that Cjd are phylogenetically distinct from Cjj strains. Using the CGI approach, we identified subsets of absent genes from amongst the C. jejuni genes that provide clues as to the potential evolutionary origin and unusual pathogenicity of Cjd. [Abstract/Link to Full Text]

Kleinschmidt MC, Michaelis M, Ogbomo H, Doerr HW, Cinatl J
Inhibition of apoptosis prevents West Nile virus induced cell death.
BMC Microbiol. 2007;749.
BACKGROUND: West Nile virus (WNV) infection can cause severe meningitis and encephalitis in humans. Apoptosis was recently shown to contribute to the pathogenesis of WNV encephalitis. Here, we used WNV-infected glioma cells to study WNV-replication and WNV-induced apoptosis in human brain-derived cells. RESULTS: T98G cells are highly permissive for lytic WNV-infection as demonstrated by the production of infectious virus titre and the development of a characteristic cytopathic effect. WNV replication decreased cell viability and induced apoptosis as indicated by the activation of the effector caspase-3, the initiator caspases-8 and -9, poly(ADP-ribose)polymerase (PARP) cleavage and the release of cytochrome c from the mitochondria. Truncation of BID indicated cross-talk between the extrinsic and intrinsic apoptotic pathways. Inhibition of the caspases-8 or -9 inhibited PARP cleavage, demonstrating that both caspases are involved in WNV-induced apoptosis. Pan-caspase inhibition prevented WNV-induced apoptosis without affecting virus replication. CONCLUSION: We found that WNV infection induces cell death in the brain-derived tumour cell line T98G by apoptosis under involvement of constituents of the extrinsic as well as the intrinsic apoptotic pathways. Our results illuminate the molecular mechanism of WNV-induced neural cell death. [Abstract/Link to Full Text]

Rolain JM, Fenollar F, Raoult D
False positive PCR detection of Tropheryma whipplei in the saliva of healthy people.
BMC Microbiol. 2007;748.
BACKGROUND: Tropheryma whipplei, the agent of Whipple's disease (WD), has been recently isolated and the genomes of two isolates have been fully sequenced. Previous diagnosis tools for the diagnosis of the disease used sequence analysis of the 16S rRNA gene. Using this target gene, the high percentage of detection of the bacterium in saliva of healthy people was in contrast to the negative results obtained with specific target genes. The aim of our study was to compare previously published primers targeting the 16S rRNA gene to real-time PCR with Taqman* probes targeting specific repeat genes only found in the genome of T. whipplei in a series of 57 saliva from healthy people. RESULTS: Although the specific real-time PCR assays with both primers and probes were negative for all the samples, 13 out of 57 samples were positive with different primers previously reported targeting the 16S rRNA gene. Among the positive samples, 8 yielded a 231-bp sequence that was 99.1% identical to that of Actinomyces odontolyticus, 2 yielded a 226-bp that was 99.6% identical to that of A. turicensis, and 3 yielded a 160-bp sequence that was 98.5% identical to that of Capnocytophaga gingivalis. We found that the C. gingivalis and A. odontolyticus 16S rRNA sequences obtained in our study share more than 80% homology with the corresponding 16S rRNA sequences of the T. whipplei genomes especially at 5' and 3' end. CONCLUSION: Asymptomatic carriers of T. whipplei in saliva may exist but their prevalence is much lower than those previously reported. Testing the specificity of designed primers is critical to avoid false positive detection of T. whipplei. In atypical case we recommend to test two different specific target genes before concluding. [Abstract/Link to Full Text]

Khairnar K, Parija SC
A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples.
BMC Microbiol. 2007;747.
BACKGROUND: E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. RESULTS: The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. CONCLUSION: The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens. [Abstract/Link to Full Text]

Brigante GR, Luzzaro FA, Pini B, Lombardi G, Sokeng G, Toniolo AQ
Drug susceptibility testing of clinical isolates of streptococci and enterococci by the Phoenix automated microbiology system.
BMC Microbiol. 2007;746.
BACKGROUND: Drug resistance is an emerging problem among streptococcal and enterococcal species. Automated diagnostic systems for species identification and antimicrobial susceptibility testing (AST) have become recently available. We evaluated drug susceptibility of clinical isolates of streptococci and enterococci using the recent Phoenix system (BD, Sparks, MD). Diagnostic tools included the new SMIC/ID-2 panel for streptococci, and the PMIC/ID-14 for enterococci. Two-hundred and fifty isolates have been investigated: beta-hemolytic streptococci (n = 65), Streptococcus pneumoniae (n = 50), viridans group streptococci (n = 32), Enterococcus faecium (n = 40), Enterococcus faecalis (n = 43), other catalase-negative cocci (n = 20). When needed, species ID was determined using molecular methods. Test bacterial strains were chosen among those carrying clinically-relevant resistance determinants (penicillin, macrolides, fluoroquinolones, glycopeptides). AST results of the Phoenix system were compared to minimal inhibitory concentration (MIC) values measured by the Etest method (AB Biodisk, Solna, Sweden). RESULTS: Streptococci: essential agreement (EA) and categorical agreement (CA) were 91.9% and 98.8%, respectively. Major (ME) and minor errors (mE) accounted for 0.1% and 1.1% of isolates, respectively. No very major errors (VME) were produced. Enterococci: EA was 97%, CA 96%. Small numbers of VME (0.9%), ME (1.4%) and mE (2.8%) were obtained. Overall, EA and CA rates for most drugs were above 90% for both genera. A few VME were found: a) teicoplanin and high-level streptomycin for E. faecalis, b) high-level gentamicin for E. faecium. The mean time to results (+/- SD) was 11.8 +/- 0.9 h, with minor differences between streptococci and enterococci. CONCLUSION: The Phoenix system emerged as an effective tool for quantitative AST. Panels based on dilution tests provided rapid and accurate MIC values with regard to clinically-relevant streptococcal and enterococcal species. [Abstract/Link to Full Text]

Fothergill JL, Panagea S, Hart CA, Walshaw MJ, Pitt TL, Winstanley C
Widespread pyocyanin over-production among isolates of a cystic fibrosis epidemic strain.
BMC Microbiol. 2007;745.
BACKGROUND: Some isolates of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa exhibit an unusual virulence-related phenotype, characterized by over-production of quorum sensing-regulated exoproducts such as pyocyanin and LasA protease. Our aim was to determine the prevalence of this unusual phenotype amongst isolates of the epidemic strain, and to study other intraclonal phenotypic and genotypic variations. RESULTS: The unusual phenotype was detected in at least one epidemic strain isolate from the majority of cystic fibrosis patients tested, and can be retained for up to seven years during chronic infection. Multiple sequential isolates of the epidemic strain taken from six patients over a period of up to nine years exhibited a wide range of phenotypes, including different antimicrobial susceptibilities. Our data suggest that each sputum sample contains a mixture of phenotypes and genotypes within the epidemic strain population, including within colony morphotypes. Many isolates exhibit premature (during early rather than late exponential growth) and over-production of pyocyanin, which has a number of toxic effects directly relevant to cystic fibrosis. CONCLUSION: The widespread occurrence of this unusual phenotype suggests that it may play an important role in the success of the epidemic strain. [Abstract/Link to Full Text]

Hitchcock RA, Thomas S, Campbell DA, Sturm NR
The promoter and transcribed regions of the Leishmania tarentolae spliced leader RNA gene array are devoid of nucleosomes.
BMC Microbiol. 2007;744.
BACKGROUND: The spliced leader (SL) RNA provides the 5' m7G cap and first 39 nt for all nuclear mRNAs in kinetoplastids. This small nuclear RNA is transcribed by RNA polymerase II from individual promoters. In Leishmania tarentolae the SL RNA genes reside in two multi-copy tandem arrays designated MINA and MINB. The transcript accumulation from the SL promoter on the drug-selected, episomal SL RNA gene cassette pX-tSL is ~10% that of the genomic array in uncloned L. tarentolae transfectants. This disparity is neither sequence- nor copy-number related, and thus may be due to interference of SL promoter function by epigenetic factors. To explore these possibilities we examined the nucleoplasmic localization of the SL RNA genes as well as their nucleosomal architecture. RESULTS: The genomic SL RNA genes and the episome did not co-localize within the nucleus. Each genomic repeat contains one nucleosome regularly positioned within the non-transcribed intergenic region. The 363-bp MINA array was resistant to micrococcal nuclease digestion between the -258 and -72 positions relative to the transcription start point due to nucleosome association, leaving the promoter elements and the entire transcribed region exposed for protein interactions. A pattern of ~164-bp protected segments was observed, corresponding to the amount of DNA typically bound by a nucleosome. By contrast, nucleosomes on the pX-tSL episome were randomly distributed over the episomal SL cassette, reducing transcription factor access to the episomal promoter by approximately 74%. Cloning of the episome transfectants revealed a range of transcriptional activities, implicating a mechanism of epigenetic heredity. CONCLUSION: The disorganized nucleosomes on the pX episome are in a permissive conformation for transcription of the SL RNA cassette approximately 25% of the time within a given parasite. Nucleosome interference is likely the major factor in the apparent transcriptional repression of the SL RNA gene cassette. Coupled with the requirement for run-around transcription that drives expression of the selectable drug marker, transcription of the episomal SL may be reduced even further due to sub-optimal nucleoplasmic localization and initiation complex disruption. [Abstract/Link to Full Text]

Fagerlund A, Brillard J, Fürst R, Guinebretière MH, Granum PE
Toxin production in a rare and genetically remote cluster of strains of the Bacillus cereus group.
BMC Microbiol. 2007;743.
BACKGROUND: Three enterotoxins are implicated in diarrhoeal food poisoning due to Bacillus cereus: Haemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe), and Cytotoxin K (CytK). Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of B. cereus group strains. B. cereus strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other B. cereus group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of cytK expression. To date, only three strains containing cytK-1 have been identified; B. cereus strains NVH 391/98, NVH 883/00, and INRA AF2. RESULTS: A novel gene variant encoding Nhe was identified in these three strains, which had an average of 80% identity in protein sequence with previously identified Nhe toxins. While culture supernatants containing CytK and Nhe from NVH 391/98 and INRA AF2 were highly cytotoxic, NVH 883/00 expressed little or no CytK and Nhe and was non-cytotoxic. Comparative sequence and expression studies indicated that neither the PlcR/PapR quorum sensing system, nor theYvrGH and YvfTU two-component systems, were responsible for the observed difference in toxin production. Additionally, phylogenetic analysis of 13 genes showed that NVH 391/98, NVH 883/00, and INRA AF2 comprise a novel cluster of strains genetically distant from other B. cereus group strains. CONCLUSION: Due to its divergent sequence, the novel nhe operon had previously not been detected in NVH 391/98 using PCR and several monoclonal antibodies. Thus, toxigenic profiling based on the original nhe sequence will fail to detect the toxin in this group of strains. The observation that strain NVH 883/00 carries cytK-1 but is non-cytotoxic indicates that the detection of this gene variant is not a sufficient criterion for identification of highly cytotoxic strains. The presence of the novel nhe operon and the cytK-1 gene variant in this cluster of strains reflect their phylogenetically remote relationship towards other B. cereus group strains. [Abstract/Link to Full Text]

Lee J, Jayaraman A, Wood TK
Indole is an inter-species biofilm signal mediated by SdiA.
BMC Microbiol. 2007;742.
BACKGROUND: As a stationary phase signal, indole is secreted in large quantities into rich medium by Escherichia coli and has been shown to control several genes (e.g., astD, tnaB, gabT), multi-drug exporters, and the pathogenicity island of E. coli; however, its impact on biofilm formation has not been well-studied. RESULTS: Through a series of global transcriptome analyses, confocal microscopy, isogenic mutants, and dual-species biofilms, we show here that indole is a non-toxic signal that controls E. coli biofilms by repressing motility, inducing the sensor of the quorum sensing signal autoinducer-1 (SdiA), and influencing acid resistance (e.g., hdeABD, gadABCEX). Isogenic mutants showed these associated proteins are directly related to biofilm formation (e.g., the sdiA mutation increased biofilm formation 50-fold), and SdiA-mediated transcription was shown to be influenced by indole. The reduction in motility due to indole addition results in the biofilm architecture changing from scattered towers to flat colonies. Additionally, there are 12-fold more E. coli cells in dual-species biofilms grown in the presence of Pseudomonas cells engineered to express toluene o-monooxygenase (TOM, which converts indole to an insoluble indigoid) than in biofilms with pseudomonads that do not express TOM due to a 22-fold reduction in extracellular indole. Also, indole stimulates biofilm formation in pseudomonads. Further evidence that the indole effects are mediated by SdiA and homoserine lactone quorum sensing is that the addition of N-butyryl-, N-hexanoyl-, and N-octanoyl-L-homoserine lactones repress E. coli biofilm formation in the wild-type strain but not with the sdiA mutant. CONCLUSION: Indole is an interspecies signal that decreases E. coli biofilms through SdiA and increases those of pseudomonads. Indole may be manipulated to control biofilm formation by oxygenases of bacteria that do not synthesize it in a dual-species biofilm. Furthermore, E. coli changes its biofilm in response to signals it cannot synthesize (homoserine lactones), and pseudomonads respond to signals they do not synthesize (indole). [Abstract/Link to Full Text]

Parija SC, Khairnar K
Detection of excretory Entamoeba histolytica DNA in the urine, and detection of E. histolytica DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess.
BMC Microbiol. 2007;741.
BACKGROUND: Amoebic liver abscess (ALA) and pyogenic liver abscesses (PLA) appear identical by ultrasound and other imaging techniques. Collection of blood or liver abscess pus for diagnosis of liver abscesses is an invasive procedure, and the procedure requires technical expertise and disposable syringes. Collection of urine is a noninvasive procedure. Therefore, there has been much interest shown towards the use of urine as an alternative clinical specimen for the diagnosis of some parasitic infections. Here, we report for the first time the detection of E. histolytica DNA excreted in the urine for diagnosis of the cases of ALA. RESULTS: E. histolytica DNA was detected in liver abscess pus specimen of 80.4% of ALA patients by a nested multiplex polymerase chain reaction (PCR) targeting 16S-like r RNA gene. The nested PCR detected E. histolytica DNA in all 37 (100%) liver abscess pus specimens collected prior to metronidazole treatment, but were detected in only 53 of 75 (70.6%) pus specimens collected after therapy with metronidazole. Similarly, the PCR detected E. histolytica DNA in 21 of 53 (39.6%) urine specimens of ALA patients. The test detected E. histolytica DNA in only 4 of 23 (17.4%) urine specimens collected prior to metronidazole treatment, but were detected in 17 of 30 (56.7%) urine specimens collected after treatment with metronidazole. The enzyme-linked immunosorbent assay (ELISA) for the detection of lectin E. histolytica antigen in the liver abscess pus showed a sensitivity of 50% and the indirect haemagglutination (IHA) test for detection of amoebic antibodies in the serum showed a sensitivity of 76.8% for the diagnosis of the ALA. CONCLUSION: The present study for the first time shows that the kidney barrier in ALA patients is permeable to E. histolytica DNA molecule resulting in excretion of E. histolytica DNA in urine which can be detected by PCR. The study also shows that the PCR for detection of E. histolytica DNA in urine of patients with ALA can also be used as a prognostic marker to assess the course of the diseases following therapy by metronidazole. The detection of E. histolytica DNA in urine specimen of ALA patients provides a new approach for the diagnosis of ALA. [Abstract/Link to Full Text]

Strych U, Davlieva M, Longtin JP, Murphy EL, Im H, Benedik MJ, Krause KL
Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae.
BMC Microbiol. 2007;740.
BACKGROUND: Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen Streptococcus pneumoniae has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer. RESULTS: The alanine racemases gene from S. pneumoniae (alrSP) was amplified by PCR and cloned and expressed in Escherichia coli. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an alr auxotroph of E. coli growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg-1 were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively. CONCLUSION: We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen S. pneumoniae. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project. [Abstract/Link to Full Text]

Nouvel LX, Dos Vultos T, Kassa-Kelembho E, Rauzier J, Gicquel B
A non-sense mutation in the putative anti-mutator gene ada/alkA of Mycobacterium tuberculosis and M. bovis isolates suggests convergent evolution.
BMC Microbiol. 2007;739.
BACKGROUND: Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP) in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR). RESULTS: In this study, 55 MDR and 194 non-MDR strains were analyzed. Variations in DNA repair genes ada/alkA and ogt were identified. Among them, by comparison to M. tuberculosis published sequences, we found a non-sense variation in ada/alkA gene which was also observed in M. bovis AF2122 strain. SNPs that are present in the adjacent regions to the amber variation are different in M. bovis and in M. tuberculosis strain. CONCLUSION: An Amber codon was found in the ada/alkA locus of clustered M. tuberculosis isolates and in M. bovis strain AF2122. This is likely due to convergent evolution because SNP differences between strains are incompatible with horizontal transfer of an entire gene. This suggests that such a variation may confer a selective advantage and be implicated in hypermutator phenotype expression, which in turn contributes to adaptation to environmental changes. [Abstract/Link to Full Text]

Luo J, Liu G, Zhong Y, Jia T, Liu K, Chen D, Zhong G
Characterization of hypothetical proteins Cpn0146, 0147, 0284 & 0285 that are predicted to be in the Chlamydia pneumoniae inclusion membrane.
BMC Microbiol. 2007;738.
BACKGROUND: Although more than 100 Chlamydia pneumoniae hypothetical proteins have been predicted to be inclusion membrane proteins, only a few have been experimentally demonstrated to be in the inclusion membrane. Using antibodies raised with fusion proteins, we characterized four such hypothetical proteins encoded by two gene clusters (Cpn0146-147 and Cpn0284-285) in the C. pneumoniae genome. RESULTS: Cpn0146 and 0147 were detected in the inclusion membrane while Cpn0284 and 0285 inside inclusion and mainly associated with reticulate bodies although all four proteins contain an N-terminal bi-lobed hydrophobic region, a signature motif assigned to inclusion membrane proteins. These four hypothetical proteins were only detected in cells infected with C. pneumoniae but not other chlamydial species, with Cpn0147 at 6 hours and Cpn0146, 0284 & 0285 at 24 hours after infection. Cpn0146 & 147 but not Cpn0284 and 285 co-localized with a host cell endoplasmic reticulum marker, a property known to be possessed by some chlamydial inclusion membrane proteins, when expressed in the host cell cytosol via transgenes. However, the endoplasmic reticulum localization of the C. pneumoniae inclusion membrane proteins did not result in inhibition of the subsequent C. pneumoniae infection. CONCLUSION: The hypothetical proteins Cpn0146 & 0147 were localized in the C. pneumoniae inclusion membrane while Cpn0284 & 0285 within the inclusion although all four were predicted to be Inc proteins, suggesting the need to experimentally characterize the predicted Inc proteins. [Abstract/Link to Full Text]

Fu LM, Fu-Liu CS
The gene expression data of Mycobacterium tuberculosis based on Affymetrix gene chips provide insight into regulatory and hypothetical genes.
BMC Microbiol. 2007;737.
BACKGROUND: Tuberculosis remains a leading infectious disease with global public health threat. Its control and management have been complicated by multi-drug resistance and latent infection, which prompts scientists to find new and more effective drugs. With the completion of the genome sequence of the etiologic bacterium, Mycobacterium tuberculosis, it is now feasible to search for new drug targets by sieving through a large number of gene products and conduct genome-scale experiments based on microarray technology. However, the full potential of genome-wide microarray analysis in configuring interrelationships among all genes in M. tuberculosis has yet to be realized. To date, it is only possible to assign a function to 52% of proteins predicted in the genome. RESULTS: We conducted a functional-genomics study using the high-resolution Affymetrix oligonucleotide GeneChip. Approximately one-half of the genes were found to be always expressed, including more than 100 predicted conserved hypotheticals, in the genome of M. tuberculosis during the log phase of in vitro growth. The gene expression profiles were analyzed and visualized through cluster analysis to epitomize the full details of genomic behavior. Broad patterns derived from genome-wide expression experiments in this study have provided insight into the interrelationships among genes in the basic cellular processes of M. tuberculosis. CONCLUSION: Our results have confirmed several known gene clusters in energy production, information pathways, and lipid metabolism, and also hinted at potential roles of hypothetical and regulatory proteins. [Abstract/Link to Full Text]

Habimana O, Le Goff C, Juillard V, Bellon-Fontaine MN, Buist G, Kulakauskas S, Briandet R
Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci.
BMC Microbiol. 2007;736.
BACKGROUND: The first step in biofilm formation is bacterial attachment to solid surfaces, which is dependent on the cell surface physico-chemical properties. Cell wall anchored proteins (CWAP) are among the known adhesins that confer the adhesive properties to pathogenic Gram-positive bacteria. To investigate the role of CWAP of non-pathogen Gram-positive bacteria in the initial steps of biofilm formation, we evaluated the physico-chemical properties and adhesion to solid surfaces of Lactococcus lactis. To be able to grow in milk this dairy bacterium expresses a cell wall anchored proteinase PrtP for breakdown of milk caseins. RESULTS: The influence of the anchored cell wall proteinase PrtP on microbial surface physico-chemical properties, and consequently on adhesion, was evaluated using lactococci carrying different alleles of prtP. The presence of cell wall anchored proteinase on the surface of lactococcal cells resulted in an increased affinity to solvents with different physico-chemical properties (apolar and Lewis acid-base solvents). These properties were observed regardless of whether the PrtP variant was biologically active or not, and were not observed in strains without PrtP. Anchored PrtP displayed a significant increase in cell adhesion to solid glass and tetrafluoroethylene surfaces. CONCLUSION: Obtained results indicate that exposure of an anchored cell wall proteinase PrtP, and not its proteolytic activity, is responsible for greater cell hydrophobicity and adhesion. The increased bacterial affinity to polar and apolar solvents indicated that exposure of PrtP on lactococcal cell surface could enhance the capacity to exchange attractive van der Waals interactions, and consequently increase their adhesion to different types of solid surfaces and solvents. [Abstract/Link to Full Text]

Laín A, Elguezabal N, Brena S, García-Ruiz JC, Del Palacio A, Moragues MD, Pontón J
Diagnosis of invasive candidiasis by enzyme-linked immunosorbent assay using the N-terminal fragment of Candida albicans hyphal wall protein 1.
BMC Microbiol. 2007;735.
BACKGROUND: The diagnosis of invasive candidiasis is difficult because there are no specific clinical manifestations of the disease and colonization and infection are difficult to distinguish. In the last decade, much effort has been made to develop reliable tests for rapid diagnosis of invasive candidiasis, but none of them have found widespread clinical use. RESULTS: Antibodies against a recombinant N-terminal fragment of the Candida albicans germ tube-specific antigen hyphal wall protein 1 (Hwp1) generated in Escherichia coli were detected by both immunoblotting and ELISA tests in a group of 36 hematological or Intensive Care Unit patients with invasive candidiasis and in a group of 45 control patients at high risk for the mycosis who did not have clinical or microbiological data to document invasive candidiasis. Results were compared with an immunofluorescence test to detect antibodies to C. albicans germ tubes (CAGT). The sensitivity, specificity, positive and negative predictive values of a diagnostic test based on the detection of antibodies against the N-terminal fragment of Hwp1 by immunoblotting were 27.8 %, 95.6 %, 83.3 % and 62.3 %, respectively. Detection of antibodies to the N-terminal fragment of Hwp1 by ELISA increased the sensitivity (88.9 %) and the negative predictive value (90.2 %) but slightly decreased the specificity (82.6 %) and positive predictive values (80 %). The kinetics of antibody response to the N-terminal fragment of Hwp1 by ELISA was very similar to that observed by detecting antibodies to CAGT. CONCLUSION: An ELISA test to detect antibodies against a recombinant N-terminal fragment of the C. albicans germ tube cell wall antigen Hwp1 allows the diagnosis of invasive candidiasis with similar results to those obtained by detecting antibodies to CAGT but without the need of treating the sera to adsorb the antibodies against the cell wall surface of the blastospore. [Abstract/Link to Full Text]

Whatmore AM, Perrett LL, MacMillan AP
Characterisation of the genetic diversity of Brucella by multilocus sequencing.
BMC Microbiol. 2007;734.
BACKGROUND: Brucella species include economically important zoonotic pathogens that can infect a wide range of animals. There are currently six classically recognised species of Brucella although, as yet unnamed, isolates from various marine mammal species have been reported. In order to investigate genetic relationships within the group and identify potential diagnostic markers we have sequenced multiple genetic loci from a large sample of Brucella isolates representing the known diversity of the genus. RESULTS: Nine discrete genomic loci corresponding to 4,396 bp of sequence were examined from 160 Brucella isolates. By assigning each distinct allele at a locus an arbitrary numerical designation the population was found to represent 27 distinct sequence types (STs). Diversity at each locus ranged from 1.03-2.45% while overall genetic diversity equated to 1.5%. Most loci examined represent housekeeping gene loci and, in all but one case, the ratio of non-synonymous to synonymous change was substantially <1. Analysis of linkage equilibrium between loci indicated a strongly clonal overall population structure. Concatenated sequence data were used to construct an unrooted neighbour-joining tree representing the relationships between STs. This shows that four previously characterized classical Brucella species, B. abortus, B. melitensis, B. ovis and B. neotomae correspond to well-separated clusters. With the exception of biovar 5, B. suis isolates cluster together, although they form a more diverse group than other classical species with a number of distinct STs corresponding to the remaining four biovars. B. canis isolates are located on the same branch very closely related to, but distinguishable from, B. suis biovar 3 and 4 isolates. Marine mammal isolates represent a distinct, though rather weakly supported, cluster within which individual STs display one of three clear host preferences. CONCLUSION: The sequence database provides a powerful dataset for addressing ongoing controversies in Brucella taxonomy and a tool for unambiguously placing atypical, phenotypically discordant or newly emerging Brucella isolates. Furthermore, by using the phylogenetic backbone described here, robust and rationally selected markers for use in diagnostic assay development can be identified. [Abstract/Link to Full Text]

Favre-Bonté S, Chamot E, Köhler T, Romand JA, van Delden C
Autoinducer production and quorum-sensing dependent phenotypes of Pseudomonas aeruginosa vary according to isolation site during colonization of intubated patients.
BMC Microbiol. 2007;733.
BACKGROUND: Pseudomonas aeruginosa frequently colonizes and is responsible for severe ventilator-associated pneumonia in intubated patients. A quorum-sensing (QS) circuit, depending on the production of the two QS-signaling molecules (autoinducers, AIs) 3-oxo-C12-HSL and C4-HSL, regulates the production by P. aeruginosa of several virulence factors and is required for biofilm formation. Therefore QS-inhibition has been suggested as a new target for preventive and/or therapeutic strategies. However the precise role of QS during colonization and subsequent infections of intubated patients remains unclear. RESULTS: We wondered whether QS is active during colonization of intubated patients, and whether P. aeruginosa isolates growing inside the biofilm covering the intubation devices and those resident in the lungs of colonized patients differ in their QS-dependent phenotypes. We collected the intubation devices of eight patients colonized by P. aeruginosa. We detected 3-oxo-C12-HSL on eight, and C4-HSL on six of these devices. In three of these patients we also obtained P. aeruginosa isolates from tracheal aspirates at the time of extubation (n = 18), as well as isolates from the intubation devices (n = 25). We genotyped these isolates, quantified their AIs production, and determined three QS-dependent phenotypes (adherence capacity, biofilm and elastase production). The production of 3-oxo-C12-HSL was consistently increased for isolates from the intubation devices, whereas the production of C4-HSL was significantly higher for isolates from tracheal aspirates. Isolates from tracheal aspirates produced significantly higher amounts of elastase but less biofilm, and had a marginally reduced adhesion capacity than isolates from the intubation devices. Levels of 3-oxo-C12-HSL and elastase production correlated statistically for tracheal intubation isolates, whereas levels of 3-oxo-C12-HSL production and adhesion ability, as well as biofilm production, correlated weakly amongst intubation device isolates. CONCLUSION: Our findings demonstrate that autoinducers are produced during the colonization of intubated patients by P. aeruginosa. The microenvironment, in which P. aeruginosa grows, may select for bacteria with different capacities to produce autoinducers and certain QS-dependent phenotypes. QS-inhibition might therefore affect differently isolates growing inside the biofilm covering intubation devices and those resident in the lungs. [Abstract/Link to Full Text]

Fletcher LA, Gaunt LF, Beggs CB, Shepherd SJ, Sleigh PA, Noakes CJ, Kerr KG
Bactericidal action of positive and negative ions in air.
BMC Microbiol. 2007;732.
BACKGROUND: In recent years there has been renewed interest in the use of air ionisers to control of the spread of airborne infection. One characteristic of air ions which has been widely reported is their apparent biocidal action. However, whilst the body of evidence suggests a biocidal effect in the presence of air ions the physical and biological mechanisms involved remain unclear. In particular, it is not clear which of several possible mechanisms of electrical origin (i.e. the action of the ions, the production of ozone, or the action of the electric field) are responsible for cell death. A study was therefore undertaken to clarify this issue and to determine the physical mechanisms associated with microbial cell death. RESULTS: In the study seven bacterial species (Staphylococcus aureus, Mycobacterium parafortuitum, Pseudomonas aeruginosa, Acinetobacter baumanii, Burkholderia cenocepacia, Bacillus subtilis and Serratia marcescens) were exposed to both positive and negative ions in the presence of air. In order to distinguish between effects arising from: (i) the action of the air ions; (ii) the action of the electric field, and (iii) the action of ozone, two interventions were made. The first intervention involved placing a thin mica sheet between the ionisation source and the bacteria, directly over the agar plates. This intervention, while leaving the electric field unaltered, prevented the air ions from reaching the microbial samples. In addition, the mica plate prevented ozone produced from reaching the bacteria. The second intervention involved placing an earthed wire mesh directly above the agar plates. This prevented both the electric field and the air ions from impacting on the bacteria, while allowing any ozone present to reach the agar plate. With the exception of Mycobacterium parafortuitum, the principal cause of cell death amongst the bacteria studied was exposure to ozone, with electroporation playing a secondary role. However in the case of Mycobacterium parafortuitum, electroporation resulting from exposure to the electric field appears to have been the principal cause of cell inactivation. CONCLUSION: The results of the study suggest that the bactericidal action attributed to negative air ions by previous researchers may have been overestimated. [Abstract/Link to Full Text]

Vilei EM, Correia I, Ferronha MH, Bischof DF, Frey J
Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells.
BMC Microbiol. 2007;731.
BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204. RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204. CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia. [Abstract/Link to Full Text]

Turner KM, Hanage WP, Fraser C, Connor TR, Spratt BG
Assessing the reliability of eBURST using simulated populations with known ancestry.
BMC Microbiol. 2007;730.
BACKGROUND: The program eBURST uses multilocus sequence typing data to divide bacterial populations into groups of closely related strains (clonal complexes), predicts the founding genotype of each group, and displays the patterns of recent evolutionary descent of all other strains in the group from the founder. The reliability of eBURST was evaluated using populations simulated with different levels of recombination in which the ancestry of all strains was known. RESULTS: For strictly clonal simulations, where all allelic change is due to point mutation, the groups of related strains identified by eBURST were very similar to those expected from the true ancestry and most of the true ancestor-descendant relationships (90-98%) were identified by eBURST. Populations simulated with low or moderate levels of recombination showed similarly high performance but the reliability of eBURST declined with increasing recombination to mutation ratio. Populations simulated under a high recombination to mutation ratio were dominated by a single large straggly eBURST group, which resulted from the incorrect linking of unrelated groups of strains into the same eBURST group. The reliability of the ancestor-descendant links in eBURST diagrams was related to the proportion of strains in the largest eBURST group, which provides a useful guide to when eBURST is likely to be unreliable. CONCLUSION: Examination of eBURST groups within populations of a range of bacterial species showed that most were within the range in which eBURST is reliable, and only a small number (e.g. Burkholderia pseudomallei and Enterococcus faecium) appeared to have such high rates of recombination that eBURST is likely to be unreliable. The study also demonstrates how three simple tests in eBURST v3 can be used to detect unreliable eBURST performance and recognise populations in which there appears to be a high rate of recombination relative to mutation. [Abstract/Link to Full Text]

Bastos KP, Bailão AM, Borges CL, Faria FP, Felipe MS, Silva MG, Martins WS, Fiúza RB, Pereira M, Soares CM
The transcriptome analysis of early morphogenesis in Paracoccidioides brasiliensis mycelium reveals novel and induced genes potentially associated to the dimorphic process.
BMC Microbiol. 2007;729.
BACKGROUND: Paracoccidioides brasiliensis is a human pathogen with a broad distribution in Latin America. The fungus is thermally dimorphic with two distinct forms corresponding to completely different lifestyles. Upon elevation of the temperature to that of the mammalian body, the fungus adopts a yeast-like form that is exclusively associated with its pathogenic lifestyle. We describe expressed sequence tags (ESTs) analysis to assess the expression profile of the mycelium to yeast transition. To identify P. brasiliensis differentially expressed sequences during conversion we performed a large-scale comparative analysis between P. brasiliensis ESTs identified in the transition transcriptome and databases. RESULTS: Our analysis was based on 1107 ESTs from a transition cDNA library of P. brasiliensis. A total of 639 consensus sequences were assembled. Genes of primary metabolism, energy, protein synthesis and fate, cellular transport, biogenesis of cellular components were represented in the transition cDNA library. A considerable number of genes (7.51%) had not been previously reported for P. brasiliensis in public databases. Gene expression analysis using in silico EST subtraction revealed that numerous genes were more expressed during the transition phase when compared to the mycelial ESTs 1. Classes of differentially expressed sequences were selected for further analysis including: genes related to the synthesis/remodeling of the cell wall/membrane. Thirty four genes from this family were induced. Ten genes related to signal transduction were increased. Twelve genes encoding putative virulence factors manifested increased expression. The in silico approach was validated by northern blot and semi-quantitative RT-PCR. CONCLUSION: The developmental program of P. brasiliensis is characterized by significant differential positive modulation of the cell wall/membrane related transcripts, and signal transduction proteins, suggesting the related processes important contributors to dimorphism. Also, putative virulence factors are more expressed in the transition process suggesting adaptation to the host of the yeast incoming parasitic phase. Those genes provide ideal candidates for further studies directed at understanding fungal morphogenesis and its regulation. [Abstract/Link to Full Text]

Werner G, Klare I, Witte W
The current MLVA typing scheme for Enterococcus faecium is less discriminatory than MLST and PFGE for epidemic-virulent, hospital-adapted clonal types.
BMC Microbiol. 2007;728.
BACKGROUND: MLVA (multiple-locus variable-number tandem repeat analysis) is a reliable typing technique introduced recently to differentiate also isolates of Enterococcus faecium. We used the established VNTR (variable number of tandem repeats) scheme to test its suitability to differentiate 58 E. faecium isolates representing mainly outbreaks and clusters of infections and colonizations among patients from 31 German hospitals. All isolates were vancomycin-resistant (vanA type). Typing results for MLVA are compared with results of macrorestriction analysis in PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing). RESULTS: All 51 but one hospital isolates from 1996-2006 were assigned to the clonal complex (CC) of epidemic-virulent, hospital-adapted lineages (MLST CC-17; MLVA CC-1) and differed from isolates of sporadic infections and colonizations (n = 7; 1991-1995) and other non-hospital origins (n = 27). Typing of all 58 hospital VRE revealed MLVA as the least discriminatory method (Simpson's diversity index 0.847) when compared to MLST (0.911) and PFGE (0.976). The two most common MLVA types MT-1 (n = 16) and MT-159 (n = 14) combined isolates of several MLST types including also major epidemic, hospital-adapted, clonal types (MT-1: ST-17, ST-18, ST-280, ST-282; MT-159: ST-78, ST-192, ST-203). These data clearly indicate that non-related E. faecium could possess an identical MLVA type being especially critical when MLVA is used to elucidate supposed outbreaks with E. faecium within a single or among different hospitals. Stability of a given MLVA profile MT-12 (ST-117) during an outbreak over a period of five years was also shown. CONCLUSION: MLVA is a suitable method to assign isolates of E. faecium into distinct clonal complexes. To investigate outbreaks the current MLVA typing scheme for E. faecium does not discriminate enough and cannot be recommended as a standard superior to PFGE. [Abstract/Link to Full Text]

Lee L, Tin S, Kelley ST
Culture-independent analysis of bacterial diversity in a child-care facility.
BMC Microbiol. 2007;727.
BACKGROUND: Child-care facilities appear to provide daily opportunities for exposure and transmission of bacteria and viruses. However, almost nothing is known about the diversity of microbial contamination in daycare facilities or its public health implications. Recent culture-independent molecular studies of bacterial diversity in indoor environments have revealed an astonishing diversity of microorganisms, including opportunistic pathogens and many uncultured bacteria. In this study, we used culture and culture-independent methods to determine the viability and diversity of bacteria in a child-care center over a six-month period. RESULTS: We sampled surface contamination on toys and furniture using sterile cotton swabs in four daycare classrooms. Bacteria were isolated on nutrient and blood agar plates, and 16S rRNA gene sequences were obtained from unique (one of a kind) colony morphologies for species identification. We also extracted DNA directly from nine representative swab samples taken over the course of the study from both toy and furniture surfaces, and used "universal" 16S rRNA gene bacterial primers to create PCR-based clone libraries. The rRNA gene clones were sequenced, and the sequences were compared with related sequences in GenBank and subjected to phylogenetic analyses to determine their evolutionary relationships. Culturing methods identified viable bacteria on all toys and furniture surfaces sampled in the study. Bacillus spp. were the most commonly cultured bacteria, followed by Staphylococcus spp., and Microbacterium spp. Culture-independent methods based on 16S rRNA gene sequencing, on the other hand, revealed an entirely new dimension of microbial diversity, including an estimated 190 bacterial species from 15 bacterial divisions. Sequence comparisons and phylogenetic analyses determined that the clone libraries were dominated by a diverse set of sequences related to Pseudomonas spp., as well as uncultured bacteria originally identified on human vaginal epithelium. Other sequences were related to uncultured bacteria from wastewater sludge, and many human-associated bacteria including a number of pathogens and opportunistic pathogens. Our results suggest that the child-care facility provided an excellent habitat for slime-producing Pseudomonads, and that diaper changing contributed significantly to the bacterial contamination. CONCLUSION: The combination of culture and culture-independent methods provided powerful means for determining both viability and diversity of bacteria in child-care facilities. Our results provided insight into the source of contamination and suggested ways in which sanitation might be improved. Although our study identified a remarkable array of microbial diversity present in a single daycare, it also revealed just how little we comprehend the true extent of microbial diversity in daycare centers or other indoor environments. [Abstract/Link to Full Text]

Hovey JG, Watson EL, Langford ML, Hildebrandt E, Bathala S, Bolland JR, Spadafora D, Mendz GL, McGee DJ
Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates.
BMC Microbiol. 2007;726.
BACKGROUND: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. RESULTS: The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. CONCLUSION: These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori. [Abstract/Link to Full Text]

Mittal S, Mallik S, Sharma S, Virdi JS
Characteristics of beta-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii.
BMC Microbiol. 2007;725.
BACKGROUND: The presence of beta-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the beta-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of beta-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India. RESULTS: The enzymes, Bla-A (a constitutive class A penicillinase) and Bla-B (an inducible class C cephalosporinase) were found to be present in all the clinical and non-clinical strains of Y. intermedia and Y. frederiksenii by double disc diffusion method. The results showed differential expression of Bla-A as indicated by presence/absence of synergy whereas expression of Bla-B was quite consistent. The presence of these enzymes was also reflected in the high minimum inhibitory concentrations, MIC50 (126-1024 mg/L) and MIC90 (256-1024 mg/L) of beta-lactam antibiotics against these species. Restriction fragment length polymorphism (RFLP) revealed heterogeneity in both blaA and blaB genes of Y. intermedia and Y. frederiksenii. The blaA gene of Y. intermedia shared significant sequence identity (87-96%) with blaA of Y. enterocolitica biovars 1A, 1B and 4. The sequence identity of blaA of Y. frederiksenii with these biovars was 77-79%. The sequence identity of blaB gene of Y. intermedia and Y. frederiksenii was more (85%) with that of Y. enterocolitica biovars 1A, 1B and 2 compared to other species viz., Y. bercovieri, Y. aldovae and Y. ruckeri. Isoelectric focusing data further revealed that both Y. intermedia and Y. frederiksenii produced Bla-A (pI 8.7) and "Bla-B like" (pI 5.5-7.1) enzymes. CONCLUSION: Both Y. intermedia and Y. frederiksenii showed presence of blaA and blaB genes and unequivocal expression of the two beta-lactamases. Limited heterogeneity was detected in blaA and blaB genes as judged by PCR-RFLP. Phylogenetic relationships showed that the two species shared a high degree of identity in their bla genes. This is the first study reporting characteristics of beta-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii isolated from Asian region. [Abstract/Link to Full Text]

O'Connor EB, Cotter PD, O'Connor P, O'Sullivan O, Tagg JR, Ross RP, Hill C
Relatedness between the two-component lantibiotics lacticin 3147 and staphylococcin C55 based on structure, genetics and biological activity.
BMC Microbiol. 2007;724.
BACKGROUND: Two component lantibiotics, such as the plasmid-encoded lacticin 3147 produced by Lactococcus lactis DPC3147 and staphylococcin C55 produced by Staphylococcus aureus C55, represent an emerging subgroup of bacteriocins. These two bacteriocins are particularly closely related, exhibiting 86% (LtnA1 and C55alpha) and 55% (LtnA2 and C55beta) identity in their component peptides. The aim of this study was to investigate, for the first time for any two component bacteriocins, the significance of the relatedness between these two systems. RESULTS: So close is this relatedness that the hybrid peptide pairs LtnA1:C55beta and C55alpha:LtnA2 were found to have activities in the single nanomolar range, comparing well with the native pairings. To determine whether this flexibility extended to the associated post-translational modification/processing machinery, the staphylococcin C55 structural genes were directly substituted for their lacticin 3147 counterparts in the ltn operon on the large conjugative lactococcal plasmid pMRC01. It was established that the lacticin LtnA1 post-translational and processing machinery could produce functionally active C55alpha, but not C55beta. In order to investigate in closer detail the significance of the differences between LtnA1 and C55alpha, three residues in LtnA1 were replaced with the equivalent residues in C55alpha. Surprisingly, one such mutant LtnA1-Leu21Ala was not produced. This may be significant given the positioning of this residue in a putative lipid II binding loop. CONCLUSION: It is apparent, despite sharing striking similarities in terms of structure and activity, that these two complex bacteriocins display some highly dedicated features particular to either system. [Abstract/Link to Full Text]

U'Ren JM, Schupp JM, Pearson T, Hornstra H, Friedman CL, Smith KL, Daugherty RR, Rhoton SD, Leadem B, Georgia S, Cardon M, Huynh LY, DeShazer D, Harvey SP, Robison R, Gal D, Mayo MJ, Wagner D, Currie BJ, Keim P
Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping.
BMC Microbiol. 2007;723.
BACKGROUND: The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations. RESULTS: B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10-5 per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria. CONCLUSION: The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen. [Abstract/Link to Full Text]

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Recent Articles in Clinical and Diagnostic Laboratory Immunology

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Harimaya A, Tarkkanen J, Mattila P, Fujii N, Ylikoski J, Himi T
Difference in cytokine production and cell activation between adenoidal lymphocytes and peripheral blood lymphocytes of children with otitis media.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1130-4.
We evaluated the immunological potential of adenoidal lymphocytes from children with recurrent otitis media. Interleukin-4 release and CD69 expression were lower in adenoidal lymphocytes than in peripheral blood lymphocytes (PBL). Our results suggest that there may be a difference between the immunological potential of adenoidal lymphocytes and that of PBL in children with otitis. [Abstract/Link to Full Text]

Kirkpatrick BD, Bentley MD, Thern AM, Larsson CJ, Ventrone C, Sreenivasan MV, Bourgeois L
Comparison of the antibodies in lymphocyte supernatant and antibody-secreting cell assays for measuring intestinal mucosal immune response to a novel oral typhoid vaccine (M01ZH09).
Clin Diagn Lab Immunol. 2005 Sep;12(9):1127-9.
Antibody-secreting cell (ASC) and antibodies in lymphocyte supernatant (ALS) assays are used to assess intestinal mucosal responses to enteric infections and vaccines. The ALS assay, performed on cell supernatants, may represent a convenient alternative to the more established ASC assay. The two methods, measuring immunoglobulin A to Salmonella enterica serovar Typhi lipopolysaccharide, were compared in volunteers vaccinated with a live-attenuated typhoid vaccine M01ZH09. The specificity of the ALS assay compared to the ASC assay was excellent (100%), as was sensitivity (82%). The ALS assay was less sensitive than the ASC assay at <or=42 spots/10(6) peripheral blood lymphocytes. [Abstract/Link to Full Text]

Prince HE, Lapé-Nixon M, Busch MP, Tobler LH, Foster GA, Stramer SL
Utilization of follow-up specimens from viremic blood donors to assess the value of west nile virus immunoglobulin G avidity as an indicator of recent infection.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1123-6.
The value of West Nile virus immunoglobulin G avidity for distinguishing recent from past infection was investigated using 348 follow-up specimens from 170 viremic blood donors. Low avidity accurately indicated infection within the previous 4 months. However, due to rapid avidity maturation in some individuals, high avidity did not accurately indicate past infection. [Abstract/Link to Full Text]

Rajagopalan G, Singh M, Sen MM, Murali NS, Nath KA, David CS
Endogenous superantigens shape response to exogenous superantigens.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1119-22.
Endogenous superantigen-mediated thymic negative selection resulted in a paucity of mature T cells bearing T-cell receptor (TCR) Vbeta8 in the periphery. Consequently, the magnitude of immune response to exogenous superantigen staphylococcal enterotoxin B, which activates TCR Vbeta8(+) T cells, was significantly reduced and conferred protection from superantigen-induced mortality. [Abstract/Link to Full Text]

Kandathil AJ, Kannangai R, David S, Selvakumar R, Job V, Abraham OC, Sridharan G
Human immunodeficiency virus infection and levels of Dehydroepiandrosterone sulfate in plasma among Indians.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1117-8.
The shift in cytokine profile during human immunodeficiency virus (HIV) disease progression is influenced by dehydroepiandrosterone sulfate (DHEAS) level. Radioimmunoassay was used to measure plasma DHEAS for 30 treatment-naïve HIV-infected and 30 uninfected individuals. There was a significant negative correlation of viral load with DHEAS level (P<0.05). Further studies of the use of DHEAS levels for monitoring HIV patients economically are warranted. [Abstract/Link to Full Text]

Green BJ, Schmechel D, Tovey ER
Detection of aerosolized Alternaria alternata conidia, hyphae, and fragments by using a novel double-immunostaining technique.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1114-6.
A double-immunostaining halogen immunoassay was developed to identify aerosolized conidia, hyphae, and fragments of Alternaria alternata by using an anti-Alternaria polyclonal antiserum, while, simultaneously, allergy to these components was concurrently determined by using human immunoglobulin E antibodies. [Abstract/Link to Full Text]

Sheoran AS, Feng X, Singh I, Chapman-Bonofiglio S, Kitaka S, Hanawalt J, Nunnari J, Mansfield K, Tumwine JK, Tzipori S
Monoclonal antibodies against Enterocytozoon bieneusi of human origin.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1109-13.
Enterocytozoon bieneusi is clinically the most significant among the microsporidia infecting humans, causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. The lack of immune reagents is largely due to the absence of methods for laboratory propagation of E. bieneusi. We recently described a procedure for the concentration and purification of spores from diarrheic stool of infected humans. Purified spores were used to immunize mice for production and screening of monoclonal antibodies (MAbs) against E. bieneusi. The eight immunoglobulin M MAbs generated and fully characterized did not cross-react with other human microsporidia or with other microorganisms normally present in stool. One of the MAbs, 2G4, reacted with E. bieneusi spores in stools from monkeys and humans, without background fluorescence, which makes it an ideal diagnostic reagent. It also recognizes intracellular stages of the parasite and will be suitable for determining tissue distribution of E. bieneusi in infected hosts. At least two immunodominant antigens of E. bieneusi of 33,000 and 35,000 Da exist, which were recognized by rabbit and mouse antisera. The availability of MAbs against E. bieneusi will simplify considerably the diagnosis of this infection in humans and will provide tools for epidemiologic investigations regarding the true prevalence of the infection in various human and mammalian populations and the environmental sources of infection. [Abstract/Link to Full Text]

Dimech W, Panagiotopoulos L, Marler J, Laven N, Leeson S, Dax EM
Evaluation of three immunoassays used for detection of anti-rubella virus immunoglobulin M antibodies.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1104-8.
Three automated assays (Abbott AxSYM, Bayer ADVIA Centaur, and bioMerieux VIDAS) used for the detection of rubella virus-specific immunoglobulin M were evaluated. A total of 57 samples from individuals with evidence of infection with rubella virus were used to estimate sensitivity, and 220 samples from blood donors and individuals attending an antenatal clinic who had no evidence of recent infection were used to estimate specificity. Seroconversion panels comprising an additional 31 samples from four individuals were used to determine clinical sensitivity. Samples containing potentially cross-reacting substances were also tested. The sensitivities of the three assays ranged from 84.2 to 96.5%, and the specificities ranged from 96.8 to 99.9%. The Abbott AxSYM assay detected more reactive samples than the other two assays when a panel of 57 positive samples was tested. Bayer ADVIA Centaur detected more reactive samples in the seroconversion panels than the other two assays. All three assays evaluated reported a reactive result in 1 or more of the 48 samples containing potentially cross-reacting analytes. The assays demonstrated comparable performance in testing of a well-characterized panel of samples. [Abstract/Link to Full Text]

Puertollano MA, Cruz-Chamorro L, Puertollano E, Pérez-Toscano MT, Alvarez de Cienfuegos G, de Pablo MA
Assessment of interleukin-12, gamma interferon, and tumor necrosis factor alpha secretion in sera from mice fed with dietary lipids during different stages of Listeria monocytogenes infection.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1098-103.
Recent experimental observations have determined that long-chain n-3 polyunsaturated fatty acids suppress immune functions and are involved in the reduction of infectious disease resistance. BALB/c mice were fed for 4 weeks with one of four diets containing either olive oil (OO), fish oil (FO), hydrogenated coconut oil, or a low fat level. Interleukin-12p70 (IL-12p70), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) production in the sera of mice fed these diets and challenged with Listeria monocytogenes were determined by enzyme-linked immunosorbent assay. In addition, bacterial counts from spleens of mice were carried out at 24, 72, or 96 h of infection. Here, we quantified an initial diminution of production of both IL-12p70 and IFN-gamma, which appear to play an important role in the reduction of host resistance to L. monocytogenes infection. In addition, an efficient elimination of L. monocytogenes was observed in spleens of mice fed a diet containing OO at 96 h of infection, despite reductions in IL-12p70 and TNF-alpha production, suggesting an improvement of immune resistance. Overall, our results indicate that the initial reduction of both IL-12 and IFN-gamma production before L. monocytogenes infection represents the most relevant event that corroborates the impairment of immune resistance by n-3 polyunsaturated fatty acids during the different stages of infection. However, we speculate that the modulation of other cytokines must be also involved in this response, because the alteration of cytokine production in mice fed an FO diet in a late phase of L. monocytogenes infection was similar to that in mice fed OO, whereas the ability to eliminate this bacterium from the spleen was improved in the latter group. [Abstract/Link to Full Text]

Falsafi T, Valizadeh N, Sepehr S, Najafi M
Application of a stool antigen test to evaluate the incidence of Helicobacter pylori infection in children and adolescents from Tehran, Iran.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1094-7.
Helicobacter pylori infection is acquired mainly in childhood, especially in developing countries, where a low-cost, rapid diagnostic technique which is reliable for all age groups may be useful for the management of H. pylori infection. For this purpose, we used an HpSA test (Equipar) to detect H. pylori infection in children and adolescents from Tehran, Iran. Thirty-five children who were positive or negative for H. pylori infection by endoscopy-based tests were used as positive and negative controls for the HpSA test. Stools were collected from 430 randomly selected children and adolescents (4 to 18 years old) from southwest, near the center, and northwest of Tehran. A questionnaire that included presence of recurrent abdominal pain (RAP), family history of infection and/or peptic ulcer disease (PUD), and income of parents was completed. A good agreement was found between the results of endoscopy-based tests and those of the HpSA test; the sensitivity and specificity of the Equipar-HpSA test were 100% and 83.4%, respectively. Among 430 children and adolescents, 47% were positive by the HpSA test, of whom 82% had RAP. No difference in incidence was observed between the two sexes; the various categories of age showed an increasing incidence, ranging from 24% (ages 4 to 6) to 58% (ages 16 to 18). The rate of infection in children and adolescents from the southwest was significantly higher (70%) than the rate in those from the northwest (32%), and a family history of H. pylori infection or PUD was observed in 59% of the HpSA positive subjects. The HpSA test is a useful test to detect H. pylori infection in children and adolescents from developing countries. [Abstract/Link to Full Text]

Tavares E, Maldonado R, Ojeda ML, Miñano FJ
Circulating inflammatory mediators during start of fever in differential diagnosis of gram-negative and gram-positive infections in leukopenic rats.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1085-93.
Gram-negative and gram-positive infections have been considered the most important causes of morbidity and mortality in patients with leukopenia following chemotherapy. However, discrimination between bacterial infections and harmless fever episodes is difficult. Because classical inflammatory signs of infection are often absent and fever is frequently the only sign of infection, the aim of this study was to assess the significance of serum interleukin-6 (IL-6), IL-10, macrophage inflammatory protein-2 (MIP-2), procalcitonin (PCT), and C-reactive protein (CRP) patterns in identifying bacterial infections during start of fever in normal and cyclophosphamide-treated (leukopenic) rats following an injection of lipopolysaccharide (LPS) or muramyl dipeptide (MDP) as a model for gram-negative and gram-positive bacterial infections. We found that, compared to normal rats, immunosuppressed animals exhibited significantly higher fevers and lesser production of all mediators, except IL-6, after toxin challenge. Moreover, compared to rats that received MDP, both groups of animals that received an equivalent dose of LPS showed significantly higher fevers and greater increase in serum cytokine levels. Furthermore, in contrast to those in immunocompetent rats, serum levels of IL-6 and MIP-2 were not significantly changed in leukopenic animals after MDP injection. Other serum markers such as PCT and CRP failed to discriminate between bacterial stimuli in both groups of animals. These results suggest that the use of the analyzed serum markers at an early stage of fever could give useful information for the clinician for excluding gram-negative from gram-positive infections. [Abstract/Link to Full Text]

Vinderola G, Matar C, Perdigon G
Role of intestinal epithelial cells in immune effects mediated by gram-positive probiotic bacteria: involvement of toll-like receptors.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1075-84.
The mechanisms by which probiotic bacteria exert their effects on the immune system are not completely understood, but the epithelium may be a crucial player in the orchestration of the effects induced. In a previous work, we observed that some orally administered strains of lactic acid bacteria (LAB) increased the number of immunoglobulin A (IgA)-producing cells in the small intestine without a concomitant increase in the CD4(+) T-cell population, indicating that some LAB strains induce clonal expansion only of B cells triggered to produce IgA. The present work aimed to study the cytokines induced by the interaction of probiotic LAB with murine intestinal epithelial cells (IEC) in healthy animals. We focused our investigation mainly on the secretion of interleukin 6 (IL-6) necessary for the clonal expansion of B cells previously observed with probiotic bacteria. The role of Toll-like receptors (TLRs) in such interaction was also addressed. The cytokines released by primary cultures of IEC in animals fed with Lactobacillus casei CRL 431 or Lactobacillus helveticus R389 were determined. Cytokines were also determined in the supernatants of primary cultures of IEC of unfed animals challenged with different concentrations of viable or nonviable lactobacilli and Escherichia coli, previously blocked or not with anti-TLR2 and anti-TLR4. We concluded that the small intestine is the place where a major distinction would occur between probiotic LAB and pathogens. This distinction comprises the type of cytokines released and the magnitude of the response, cutting across the line that separates IL-6 necessary for B-cell differentiation, which was the case with probiotic lactobacilli, from inflammatory levels of IL-6 for pathogens. [Abstract/Link to Full Text]

Philipp MT, Wormser GP, Marques AR, Bittker S, Martin DS, Nowakowski J, Dally LG
A decline in C6 antibody titer occurs in successfully treated patients with culture-confirmed early localized or early disseminated Lyme Borreliosis.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1069-74.
C(6), a Borrelia burgdorferi-derived peptide, is used as the antigen in the C(6)-Lyme disease diagnostic test. We assessed retrospectively whether a fourfold decrease or a decrease to a negative value in anti-C(6) antibody titer is positively correlated with a positive response to treatment in a sample of culture-confirmed patients with either early localized (single erythema migrans [EM]; n=93) or early disseminated (multiple EM; n=27) disease. All of these patients had been treated with antibiotics and were free of disease within 6 to 12 months of follow-up. Results show that a serum specimen taken at this time was either C(6) negative or had a >or=4-fold decrease in C(6) antibody titer with respect to a specimen taken at baseline (or during the early convalescent period if the baseline specimen was C(6) negative) for all of the multiple-EM patients (P<0.0001) and in 89% of the single-EM patients (P<0.0001). These results indicate that a decline in anti-C(6) antibody titer coincides with effective antimicrobial therapy in patients with early localized or early disseminated Lyme borreliosis. [Abstract/Link to Full Text]

Chaturvedi AK, Kavishwar A, Shiva Keshava GB, Shukla PK
Monoclonal immunoglobulin G1 directed against Aspergillus fumigatus cell wall glycoprotein protects against experimental murine aspergillosis.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1063-8.
Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, which, being the outermost part of the cell, mediates the host-fungus interplay. For these reasons much effort has focused on the discovery of useful inhibitors of cell wall glucan, chitin, and mannoprotein biosynthesis. In the absence of a wide-spectrum, safe, and potent antifungal agent, a new strategy for antifungal therapy is directed towards the development of monoclonal antibodies (MAbs). In the present study the MAb A9 (immunoglobulin G1 [IgG1]) was identified from hybridomas raised in BALB/c mice immunized with cell wall antigen of Aspergillus fumigatus. The immunoreactive epitopes for this IgG1 MAb appeared to be associated with a peptide moiety, and indirect immunofluorescence microscopy revealed its binding to the cell wall surface of hyphae as well as with swollen conidia. MAb A9 inhibited hyphal development as observed by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (25.76%), reduced the duration of spore germination, and exerted an in vitro cidal effect against Aspergillus fumigatus. The in vivo protective efficacy of MAb A9 was also evaluated in a murine model of invasive aspergillosis, where a reduction in CFU (>4 log(10) units) was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2 x 10(5) CFU/ml) and where enhanced mean survival times (19.5 days) compared to the control (7.1 days) and an irrelevant MAb (6.1 days) were also observed. [Abstract/Link to Full Text]

Colombet I, Saguez C, Sanson-Le Pors MJ, Coudert B, Chatellier G, Espinoza P
Diagnosis of tetanus immunization status: multicenter assessment of a rapid biological test.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1057-62.
Diagnosis of tetanus immunization status by medical interview of patients with wounds is poor. Many protected patients receive unnecessary vaccine or immunoglobulin, and unprotected patients may receive nothing. The aim of this study is to evaluate the feasibility and accuracy of the Tetanos Quick Stick (TQS) rapid finger prick stick test in the emergency department for determining immunization status. We designed a prospective multicenter study for blinded comparison of TQS with an enzyme-linked immunosorbent assay (ELISA). Adults referred for open wounds in 37 French hospital emergency departments had the TQS after receiving standard care (emergency-TQS). TQS was also performed in the hospital laboratory on total blood (blood/lab-TQS) and serum (serum/lab-TQS). ELISA was performed with the same blood sample at a central laboratory. We assessed concordance between emergency-TQS and blood/lab-TQS by the kappa test and the diagnostic accuracy (likelihood ratios) of medical interview, emergency-TQS, and lab-TQS. ELISA was positive in 94.6% of the 988 patients included. Concordance between blood/emergency-TQS and blood/lab-TQS results was moderate (kappa=0.6), with a high proportion of inconclusive blood/emergency-TQS tests (9.8%). Likelihood ratios for immunization were 3.0 (95% confidence interval [CI], 1.8 to 5.1), 36.6 (95% CI, 5.3 to 255.3), 89.1 (95% CI, 5.6 to 1,405.0), and 92.7 (95% CI, 5.9 to 1,462.0) for medical interview, blood/emergency-TQS, blood/lab-TQS, and serum/lab-TQS, respectively. The sensitivity of the blood/emergency-TQS was 76.7%, and the specificity was 98% by reference to the ELISA. TQS use in the emergency room could make tetanus prevention more accurate if its technical feasibility were improved, and our assessment will be supplemented by a cost effectiveness study. [Abstract/Link to Full Text]

Hoane JS, Morrow JK, Saville WJ, Dubey JP, Granstrom DE, Howe DK
Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1050-6.
Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM. [Abstract/Link to Full Text]

Atochina O, Harn D
LNFPIII/LeX-stimulated macrophages activate natural killer cells via CD40-CD40L interaction.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1041-9.
Lacto-N-fucopentaose III (LNFPIII) is a human milk sugar containing the biologically active Lewis X (LeX) trisaccharide. LNFPIII/LeX is also expressed by immunosuppressive helminth parasites, by bacteria, and on a number of tumor/cancer cells. In this report, we first demonstrate that LNFPIII activates macrophages in vitro as indicated by upregulation of Gr-1 expression on F4/80(+) cells. Further, we investigated the effect of LNFPIII-activated macrophages on NK cell activity. We found that LNFPIII-stimulated F4/80(+) cells were able to activate NK cells, inducing upregulation of CD69 expression and gamma interferon (IFN-gamma) production. The experiments show that NK cell activation is macrophage dependent, since NK cells alone did not secrete IFN-gamma in response to LNFPIII. Furthermore, we found that activation of NK cells by glycan-stimulated macrophages required cell-cell contact. As part of the cell-cell contact mechanism, we determined that CD40-CD40L interaction was critical for IFN-gamma secretion by NK cells, as the addition of anti-CD40L antibodies to the coculture blocked IFN-gamma production. We also demonstrated that LNFPIII-stimulated macrophages secrete prostaglandin E(2), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha) but a very low level of IL-12. Interestingly, addition of anti-TNF-alpha, anti-IL-10, or anti-IL-12 monoclonal antibodies did not significantly alter NK cell activity. Our data show that these soluble mediators are not critical for LNFPIII-stimulated macrophage activation of NK cells and provide further evidence for the importance of cell-cell contact and CD40-CD40L interactions between macrophages and NK cells. [Abstract/Link to Full Text]

Marques AR, Hornung RL, Dally L, Philipp MT
Detection of immune complexes is not independent of detection of antibodies in Lyme disease patients and does not confirm active infection with Borrelia burgdorferi.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1036-40.
The Borrelia burgdorferi-specific immune complex (IC) test, which uses polyethylene glycol (PEG) precipitation to isolate ICs from serum, has been used as a research test in the laboratory diagnosis of early Lyme disease (LD) and has been proposed as a marker of active infection. We examined whether B. burgdorferi-specific antibodies were present within PEG-precipitated ICs (PEG-ICs) in patients with LD, posttreatment Lyme disease syndrome, and controls, including individuals who received the outer surface protein A (OspA) vaccine. Using a B. burgdorferi whole-cell enzyme-linked immunosorbent assay (ELISA), we obtained positive PEG-IC results not only in patients with a history of LD, but also in individuals vaccinated with OspA vaccine. The frequency of positive PEG-IC ELISAs in OspA vaccinees was significantly higher with ELISA-reactive than with ELISA-negative unprocessed serum samples (P=0.001), demonstrating dependency between the tests. Similar results were found using samples from rhesus macaques infected with B. burgdorferi, uninfected macaques vaccinated with OspA, and controls. Therefore, testing for the presence of antibodies against B. burgdorferi in PEG-IC preparations is not more likely to reflect active infection than testing in unprocessed serum and should not be used in individuals who received the OspA vaccine. [Abstract/Link to Full Text]

Brunner M
Report refuting value of immune complexes to diagnose Lyme disease is invalid.
Clin Vaccine Immunol. 2006 Feb;13(2):304-5; author reply 305-6. [Abstract/Link to Full Text]

Romero-Steiner S, Holder PF, Gomez de Leon P, Spear W, Hennessy TW, Carlone GM
Avidity determinations for Haemophilus influenzae Type b anti-polyribosylribitol phosphate antibodies.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1029-35.
Determination of antibody avidity measurements can be difficult in human serum depending on the population evaluated. We evaluated three approaches for the determination of antibody avidity for immunoglobulin G (IgG). These approaches were (i) elution of bound antibody with increasing concentrations of a chaotropic agent using a single serum dilution, (ii) binding interference of multiple serum dilutions by a single concentration of a chaotrope, and (iii) elution of multiple serum dilutions by a single concentration of a chaotrope. Parameters that affect the determination of avidity measurements and their limitations were evaluated with pre- and post-Haemophilus influenzae type b conjugate vaccination sera (n=89). We determined that elution of low-avidity antibodies present in multiple dilutions of the serum sample by a single concentration of a chaotrope (0.15 M sodium thiocyanate [NaSCN]) was optimal for the determination of avidity measurements throughout a wide range of IgG concentrations (0.94 to 304.6 microg/ml). The percent reduction in concentration as determined by the elution assay with 0.15 M NaSCN correlated highly (r=0.84) with weighted averages obtained by an elution assay with multiple solutions of NaSCN. The correlation (r=0.57) between elution and binding interference, when a single concentration of a chaotrope was used, was lower than the correlation between the two elution methods (r=0.84). We found that the serum dilution, the heterogeneity of the antibody population, and the concentration of the chaotrope were the primary variables affecting avidity determinations. In this study, we present multiple analysis methods depending on the methodology used. We also present the factors that affect the analysis of avidity determinations given the polyclonal nature of human sera. This experimental approach should benefit the evaluation of similar antibodies induced by other bacterial polysaccharide vaccines. [Abstract/Link to Full Text]

Ndung'u T, Gaseitsiwe S, Sepako E, Doualla-Bell F, Peter T, Kim S, Thior I, Novitsky VA, Essex M
Major histocompatibility complex class II (HLA-DRB and -DQB) allele frequencies in Botswana: association with human immunodeficiency virus type 1 infection.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1020-8.
Southern Africa is facing an unprecedented public health crisis due to the high prevalence of human immunodeficiency virus type 1 (HIV-1). Vaccine development and testing efforts, mainly based on elicitation of HIV-specific T cells, are under way. To understand the role of human leukocyte antigen (HLA) class II alleles in HIV pathogenesis and to facilitate HLA-based HIV-1 vaccine design, we analyzed the frequencies of HLA class II alleles within the southern African country of Botswana. Common HLA class II alleles were identified within the Botswana population through the molecular genotyping of DRB and DQB1 loci. The DRB1 allele groups DRB1*01, DRB1*02/15, DRB1*03, DRB1*11, and DRB1*13 were encountered at frequencies above 20%. Within the DQB1 locus, DQB1*06 (47.7%) was the most common allele group, followed by DQB1*03 (39.2%) and DQB1*04 (25.8%). We found that DRB1*01 was more common in HIV-negative than in HIV-positive individuals and that those who expressed DRB1*08 had lower median viral loads. We demonstrate that the frequencies of certain HLA class II alleles in this Botswana population differ substantially from those in North American populations, including African-Americans. Common allele groups within Botswana cover large percentages of other African populations and could be targeted in regional vaccine designs. [Abstract/Link to Full Text]

Bacon MC, von Wyl V, Alden C, Sharp G, Robison E, Hessol N, Gange S, Barranday Y, Holman S, Weber K, Young MA
The Women's Interagency HIV Study: an observational cohort brings clinical sciences to the bench.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1013-9. [Abstract/Link to Full Text]

Weinberg A
Evaluation of three influenza A and B rapid antigen detection kits--update.
Clin Diagn Lab Immunol. 2005 Aug;12(8):1010. [Abstract/Link to Full Text]

Weinberg A, Walker ML
Evaluation of three immunoassay kits for rapid detection of influenza virus A and B.
Clin Diagn Lab Immunol. 2005 Mar;12(3):367-70.
Influenza causes high morbidity and mortality in very young and elderly individuals, which can be controlled with antivirals and/or vaccines. The success of therapeutic measures is predicated on the rapid and precise diagnosis of the infection. We compared three rapid influenza immunoassay (RIIA) kits for the diagnosis of influenza virus A and B using 178 respiratory specimens submitted for routine testing. BD Directigen Flu A+B (Directigen), Directigen EZ Flu A+B (EZ), and NOW Flu A NOW Flu B (NOW; Binax) tests had comparable combined influenza virus A and B specificities, varying from 94 to 98%. In contrast, the sensitivity of EZ was significantly lower (39%) than that of NOW (76%) and marginally lower than that of Directigen (56%). The differences in sensitivity were most evident in patients who were >9 years old (Directigen, 53%; EZ, 32%; and NOW, 69%). Among specimens, bronchoalveolar lavage fluids yielded the most discrepant results, with sensitivities varying from 0 (EZ) to 100% (NOW), followed by nasopharyngeal swabs (sensitivities of 27 to 100%) and nasal washes (50 to 81%). The Directigen kit format allowed for faster completion but more cumbersome performance and more difficult interpretation compared with the other two kits. Overall, NOW provided the most accurate diagnoses and had user-friendly technical characteristics. However, the low overall sensitivity of the RIIAs indicates that these can be used as screening tools only. [Abstract/Link to Full Text]

Kandathil AJ, Kannangai R, David S, Nithyanandam G, Solomon S, Balakrishnan P, Abraham OC, Subramanian S, Rupali P, Verghese VP, Pulimood S, Sridharan G
Comparison of Microcapillary Cytometry Technology and Flow Cytometry for CD4+ and CD8+ T-Cell Estimation.
Clin Diagn Lab Immunol. 2005 Aug;12(8):1006-9.
An alternative technology for the estimation of T cells based on a microcapillary technique (Guava Technologies, Hayward, CA) was compared to FACSCount (Becton Dickinson, San Jose, CA). Samples from 51 human immunodeficiency virus-infected and 21 healthy individuals were tested. The correlation (r) of the two systems for CD4(+) T cells was 0.994, and the coefficient of variation was 6.5%, establishing equable performance between the two technologies. [Abstract/Link to Full Text]

Nakagawa M, Kim KH, Moscicki AB
Patterns of CD8 T-cell epitopes within the human papillomavirus type 16 (HPV 16) E6 protein among young women whose HPV 16 infection has become undetectable.
Clin Diagn Lab Immunol. 2005 Aug;12(8):1003-5.
The patterns of CD8 T-cell epitopes recognized within the E6 protein in women who had cleared their human papillomavirus 16 infection were examined. T-cell lines were established using autologous dendritic cells infected with a recombinant vaccinia virus. Evidence of potential antigenic epitopes was shown in 8 of 23 (34.8%) women. [Abstract/Link to Full Text]

Lopes AI, Quiding-Jarbrink M, Palha A, Ruivo J, Monteiro L, Oleastro M, Santos A, Fernandes A
Cytokine expression in pediatric Helicobacter pylori infection.
Clin Diagn Lab Immunol. 2005 Aug;12(8):994-1002.
Helicobacter pylori infection is one of the most common gastrointestinal infections worldwide and almost invariably causes chronic gastritis in the infected host. A predominant Th1 profile has been demonstrated in H. pylori-infected mucosa from adults, but no previous study has evaluated in situ cytokine expression in children. We therefore examined expression of proinflammatory, anti-inflammatory, and regulatory cytokines by immunohistochemistry in cryopreserved antral biopsy specimens from 10 H. pylori-infected and 10 uninfected children and correlated expression of cytokines with histology scores. Concomitant expression of interleukin-8 (IL-8), gamma interferon (IFN-gamma), IL-4, transforming growth factor beta, and tumor necrosis factor alpha was seen in 8/10 H. pylori-infected cases and in 5/10 noninfected cases; all H. pylori-infected subjects showed staining for at least two of the cytokines. The proportion of epithelial cytokine-specific staining did not differ significantly between the groups, either in surface or glandular epithelium. Furthermore, no significant differences were noticed between intraepithelial or lamina propria lymphocyte staining in the groups. There was, however, a tendency of higher numbers of IFN-gamma- and IL-8-positive cells in the H. pylori-infected group. IFN-gamma and IL-8 lamina propria lymphocyte expression correlated significantly with antrum chronic inflammation, but there was no correlation between histology scores and epithelial cytokine expression. When the same techniques were used, the cytokine response appeared to be smaller in H. pylori-infected children than in adults, and there was no clear Th1 dominance. These results therefore suggest a different mucosal immunopathology in children. It remains to be determined whether the gastric immune response is downregulated in children with H. pylori infection and whether this is relevant to the outcome of infection. [Abstract/Link to Full Text]

Fraser DG, Leib SR, Zhang BS, Mealey RH, Brown WC, McGuire TC
Lymphocyte proliferation responses induced to broadly reactive Th peptides did not protect against equine infectious anemia virus challenge.
Clin Diagn Lab Immunol. 2005 Aug;12(8):983-93.
The effect of immunization with five lipopeptides, three containing T-helper (Th) epitopes and two with both Th and cytotoxic T-lymphocyte (CTL) epitopes, on equine infectious anemia virus (EIAV) challenge was evaluated. Peripheral blood mononuclear cells from EIAV lipopeptide-immunized horses had significant proliferative responses to Th peptides compared with those preimmunization, and the responses were attributed to significant responses to peptides Gag from positions 221 to 245 (Gag 221-245), Gag 250-269, and Pol 326-347; however, there were no consistent CTL responses. The significant proliferative responses in the EIAV lipopeptide-immunized horses allowed testing of the hypothesis that Th responses to immunization would enhance Th and CTL responses following EIAV challenge and lessen the viral load and the severity of clinical disease. The EIAV lipopeptide-immunized group did have a significant increase in proliferative responses to Th peptides 1 week after virus challenge, whereas the control group did not. Two weeks after challenge, a significant CTL response to virus-infected cell targets occurred in the EIAV lipopeptide-immunized group compared to that in the control group. These Th and CTL responses did not significantly alter either the number of viral RNA copies/ml or disease severity. Thus, lipopeptide-induced proliferative responses and enhanced Th and CTL responses early after virus challenge were unable to control challenge virus load and clinical disease. [Abstract/Link to Full Text]

Pfrepper KI, Enders G, Gohl M, Krczal D, Hlobil H, Wassenberg D, Soutschek E
Seroreactivity to and avidity for recombinant antigens in toxoplasmosis.
Clin Diagn Lab Immunol. 2005 Aug;12(8):977-82.
To improve serodiagnostic methods for the diagnosis of acute toxoplasmosis during pregnancy, a new test system has been developed and evaluated based on the use of recombinant antigens. Five recombinant Toxoplasma gondii antigens (ROP1, MAG1, SAG1, GRA7, and GRA8) were cloned in Escherichia coli, purified, and applied directly onto nitrocellulose membranes in a line assay (recomLine Toxoplasma). A panel of 102 sera from 25 pregnant women with supposed recent toxoplasmosis and from two symptomatic children was compared to a panel of 71 sera from individuals with past infection. Both panels were analyzed using a recombinant line assay for immunoglobulin G (IgG), IgM, and IgA antibodies and a reference enzyme-linked immunosorbent assay. Within the IgM-positive samples, antibodies against ROP1 were predominant regardless of the infection state. In IgG analysis a characteristic antibody pattern was found for very recent infections. This pattern changed to a different one during the time course of infection: antibodies against GRA7 and GRA8 were characteristic for very early IgG, whereas antibodies against SAG1 and MAG1 appeared significantly later. These results were further confirmed by determination of the IgG antibody avidity for every single recombinant antigen. In the time course of infection, IgG antibodies against the early recognized antigens matured significantly earlier than those directed against the later antigens did. The IgA patterns did not give reliable information about the infection time points. The data revealed that the recombinant line assay provides valuable information on the actual state of infection, especially during the early infection time points. [Abstract/Link to Full Text]

Borrow R, Aaberge IS, Santos GF, Eudey TL, Oster P, Glennie A, Findlow J, Høiby EA, Rosenqvist E, Balmer P, Martin D
Interlaboratory standardization of the measurement of serum bactericidal activity by using human complement against meningococcal serogroup b, strain 44/76-SL, before and after vaccination with the Norwegian MenBvac outer membrane vesicle vaccine.
Clin Diagn Lab Immunol. 2005 Aug;12(8):970-6.
There is currently no standardized serum bactericidal antibody (SBA) assay for evaluating immune responses to meningococcal outer membrane vesicle or protein vaccines. Four laboratories, Manchester Health Protection Agency (MC HPA), New Zealand Institute of Environmental Science and Research Limited (NZ ESR), Norwegian Institute of Public Health (NIPH), and Chiron Vaccines (Chiron), measured SBA titers in the same panel of human sera (n=76) from laboratory staff (n=21) vaccinated with MenBvac. Blood samples were collected prevaccination, prior to each of the three doses of MenBvac given at 6-week intervals, and 6 weeks following the third dose. Initial results showed a number of discrepancies in results between the four participating laboratories. The greatest effect on titers appeared to be due to differences among laboratories in the maintenance of the meningococcal serogroup B test strain, 44/76-SL. A repeat study was conducted using the same frozen isolate (meningococcal serogroup B test strain 44/76-SL), freshly distributed to all four laboratories. Using SBA titers from the tilt method for all samples, and using MC HPA as the comparator, the results were as follows for NZ ESR, NIPH, and Chiron, respectively, using log(10) titers: correlation coefficients (r) were 0.966, 0.967, and 0.936; intercepts were 0.08, 0.15, and 0.17; and slopes were 0.930, 0.851, and 0.891. In both prevaccination and postvaccination samples from 15 subjects assayed by all four laboratories, similar increases in SBA (fourfold or greater) were observed (for 11, 11, 9, and 9 subjects for MC HPA, NZ ESR, NIPH, and Chiron, respectively), and similar percentages of subjects with SBA titers of>or=4 p revaccination and 6 weeks following each dose were found. The SBA assay has been harmonized between the four different laboratories with good agreement on seroconversion rates, n-fold changes in titers, and percentages of subjects with SBA titers of >or=4. [Abstract/Link to Full Text]

Dias D, Van Doren J, Schlottmann S, Kelly S, Puchalski D, Ruiz W, Boerckel P, Kessler J, Antonello JM, Green T, Brown M, Smith J, Chirmule N, Barr E, Jansen KU, Esser MT
Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18.
Clin Diagn Lab Immunol. 2005 Aug;12(8):959-69.
A human papillomavirus (HPV) multiplexed competitive Luminex immunoassay first described by Opalka et al. (D. Opalka, C. E. Lachman, S. A. MacMullen, K. U. Jansen, J. F. Smith, N. Chirmule, and M. T. Esser, Clin. Diagn. Lab. Immunol. 10:108--15, 2003) was optimized and validated for use in epidemiology studies and vaccine clinical trials. Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (MAb) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, MAb concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines. [Abstract/Link to Full Text]

Salazar JC, Pope CD, Moore MW, Pope J, Kiely TG, Radolf JD
Lipoprotein-dependent and -independent immune responses to spirochetal infection.
Clin Diagn Lab Immunol. 2005 Aug;12(8):949-58.
In this study, we used the epidermal suction blister technique, in conjunction with multiparameter flow cytometry, to analyze the cellular and cytokine responses elicited by intradermal injection of human volunteers with synthetic analogs for spirochetal lipoproteins and compared the responses to findings previously reported from patients with erythema migrans (EM). Compared with peripheral blood (PB), lipopeptides derived from the N termini of the Borrelia burgdorferi outer surface protein C and the 17-kDa lipoprotein of Treponema pallidum (OspC-L and 17-L, respectively) elicited infiltrates enriched in monocytes/macrophages and dendritic cells (DCs) but also containing substantial percentages of neutrophils and T cells. Monocytoid (CD11c(+)) and plasmacytoid (CD11c(-)) DCs were selectively recruited to the skin in ratios similar to those in PB, but only the former expressed the activation/maturation surface markers CD80, CD83, and DC-SIGN. Monocytes/macrophages and monocytoid DCs, but not plasmacytoid DCs, displayed significant increases in surface expression of Toll-like receptor 1 (TLR1), TLR2, and TLR4. Staining for CD45RO and CD27 revealed that lipopeptides preferentially recruited antigen-experienced T-cell subsets; despite their lack of antigenicity, these agonists induced marked T-cell activation, as evidenced by surface expression of CD69, CD25, and CD71. Lipopeptides also induced significant increases in interleukin 12 (IL-12), IL-10, gamma interferon, and most notably IL-6 without corresponding increases in serum levels of these cytokines. Although lipopeptides and EM lesional infiltrates shared many similarities, differences were noted in a number of immunologic parameters. These studies have provided in situ evidence for a prominent "lipoprotein effect" during human infection while at the same time helping to pinpoint aspects of the cutaneous response that are uniquely driven by spirochetal pathogens. [Abstract/Link to Full Text]

Germenis AE, Yiannaki EE, Zachou K, Roka V, Barbanis S, Liaskos C, Adam K, Kapsoritakis AN, Potamianos S, Dalekos GN
Prevalence and clinical significance of immunoglobulin A antibodies against tissue transglutaminase in patients with diverse chronic liver diseases.
Clin Diagn Lab Immunol. 2005 Aug;12(8):941-8.
The prevalence of celiac disease (CD) and the prevalence and clinical significance of anti-tissue transglutaminase (tTG) antibodies (tTGAbs) in a large series of patients with chronic liver diseases were assessed. We studied 738 patients (462 with chronic viral hepatitis, 117 with autoimmune liver diseases, 113 with alcoholic or nonalcoholic fatty liver disease, and 46 with other liver disorders) and 1,350 healthy controls (HC). Immunoglobulin A (IgA) tTGAbs were measured by enzyme-linked immunosorbent assay and a microsphere-based flow cytometric assay. Positive sera were investigated for IgA antiendomysial antibodies (EmA). IgA tTGAb-positive subjects were invited to undergo a small-intestinal biopsy and HLA-DQ allele typing. Four of 1,350 HC (0.3%) tested tTGAb(+) EmA(+) and underwent a biopsy (CD confirmation in all). Four of 738 liver disease patients tested tTGAbs(+) EmA(+) (0.54%; not statistically significant). Two were HCV infected (1.24%; not statistically significant), and two had transaminasemia of unknown origin. Forty-three patients tested tTGAbs(+) EmA(-) (5.8%; P<0.001 compared to HC). Inhibition experiments verified the existence of specific IgA anti-tTG reactivity. Twenty-six of 43 patients underwent a biopsy (all negative for CD). Binary logistic regression analysis revealed age (P=0.008), cirrhosis (P=0.004), alkaline phosphatase (P=0.026), and antinuclear antibodies (P=0.012) as independent risk factors for tTGAb reactivity among the patients. It was concluded that CD prevalence is the same in HC and patients with chronic liver diseases. The prevalence of tTGAbs is higher in hepatic patients compared to HC, but their specificity for CD diagnosis in this group of patients is low. tTGAbs in patients appear to be associated with the presence of autoimmunity, cirrhosis, and cholestasis, irrespective of the origin of the liver disease. [Abstract/Link to Full Text]

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Recent Articles in Clinical and Molecular Allergy

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Recent Articles in Clinical Microbiology Reviews

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Baughn RE, Musher DM
Secondary syphilitic lesions.
Clin Microbiol Rev. 2005 Jan;18(1):205-16.
An important theme that emerges from all early historical accounts is that in addition to the decreased virulence of Treponema pallidum, the incidence of secondary syphilis has decreased drastically over the past three centuries. Even in the early 20th century, most syphilologists were of the opinion that the disease had undergone changes in its manifestations and that they were dealing with an attenuated form of the spirochete. Such opinions were based primarily on the observations that violent cutaneous reactions and fatalities associated with the secondary stage had become extremely rare. The rate of primary and secondary syphilis in the United States increased in 2002 for the second consecutive year. After a decade-long decline that led to an all-time low in 2000, the recent trend is attributable, to a large extent, by a increase in reported syphilis cases among men, particularly homosexual and bisexual men having sex with men. The present review addresses the clinical and diagnostic criteria for the recognition of secondary syphilis, the clinical course and manifestations of the disease if allowed to proceed past the primary stage of disease in untreated individuals, and the treatment for this stage of the disease. [Abstract/Link to Full Text]

Brecher ME, Hay SN
Bacterial contamination of blood components.
Clin Microbiol Rev. 2005 Jan;18(1):195-204.
Blood for transfusion is a potential source of infection by a variety of known and unknown transmissible agents. Over the last 20 years, astounding reductions in the risk of viral infection via allogeneic blood have been achieved. As a result of this success, bacterial contamination of blood products has emerged as the greatest residual source of transfusion-transmitted disease. This paper summarizes the current status of detection, prevention, and elimination of bacteria in blood products for transfusion. [Abstract/Link to Full Text]

Mukherjee PK, Sheehan DJ, Hitchcock CA, Ghannoum MA
Combination treatment of invasive fungal infections.
Clin Microbiol Rev. 2005 Jan;18(1):163-94.
The persistence of high morbidity and mortality from systemic fungal infections despite the availability of novel antifungals points to the need for effective treatment strategies. Treatment of invasive fungal infections is often hampered by drug toxicity, tolerability, and specificity issues, and added complications often arise due to the lack of diagnostic tests and to treatment complexities. Combination therapy has been suggested as a possible approach to improve treatment outcome. In this article, we undertake a historical review of studies of combination therapy and also focus on recent studies involving newly approved antifungal agents. The limitations surrounding antifungal combinations include nonuniform interpretation criteria, inability to predict the likelihood of clinical success, strain variability, and variations in pharmacodynamic/pharmacokinetic properties of antifungals used in combination. The issue of antagonism between polyenes and azoles is beginning to be addressed, but data regarding other drug combinations are not adequate for us to draw definite conclusions. However, recent data have identified potentially useful combinations. Standardization of assay methods and adoption of common interpretive criteria are essential to avoid discrepancies between different in vitro studies. Larger clinical trials are needed to assess whether combination therapy improves survival and treatment outcome in the most seriously debilitated patients afflicted with life-threatening fungal infections. [Abstract/Link to Full Text]

O'hara CM
Manual and automated instrumentation for identification of Enterobacteriaceae and other aerobic gram-negative bacilli.
Clin Microbiol Rev. 2005 Jan;18(1):147-62.
Identification of gram-negative bacilli, both enteric and nonenteric, by conventional methods is not realistic for clinical microbiology laboratories performing routine cultures in today's world. The use of commercial kits, either manual or automated, to identify these organisms is a common practice. The advent of rapid or "spot" testing has eliminated the need for some commonly isolated organisms to be identified with the systems approach. Commercially available systems provide more in-depth identification to the species level as well as detect new and unusual strains. The answers obtained from these systems may not always be correct and must be interpreted with caution. The patient demographics, laboratory workload and work flow, and technologist's skill levels should dictate the system of choice. Cost considerations introduce another variable into the equation affecting choice. Each system has its own strengths and weaknesses, and each laboratory must decide on the level of sophistication that fulfills its particular needs. [Abstract/Link to Full Text]

Chappuis F, Loutan L, Simarro P, Lejon V, Büscher P
Options for field diagnosis of human african trypanosomiasis.
Clin Microbiol Rev. 2005 Jan;18(1):133-46.
Human African trypanosomiasis (HAT) due to Trypanosoma brucei gambiense or T. b. rhodesiense remains highly prevalent in several rural areas of sub-Saharan Africa and is lethal if left untreated. Therefore, accurate tools are absolutely required for field diagnosis. For T. b. gambiense HAT, highly sensitive tests are available for serological screening but the sensitivity of parasitological confirmatory tests remains insufficient and needs to be improved. Screening for T. b. rhodesiense infection still relies on clinical features in the absence of serological tests available for field use. Ongoing research is opening perspectives for a new generation of field diagnostics. Also essential for both forms of HAT is accurate determination of the disease stage because of the high toxicity of melarsoprol, the drug most widely used during the neurological stage of the illness. Recent studies have confirmed the high accuracy of raised immunoglobulin M levels in the cerebrospinal fluid for the staging of T. b. gambiense HAT, and a promising simple assay (LATEX/IgM) is being tested in the field. Apart from the urgent need for better tools for the field diagnosis of this neglected disease, improved access to diagnosis and treatment for the population at risk remains the greatest challenge for the coming years. [Abstract/Link to Full Text]

Graczyk TK, Knight R, Tamang L
Mechanical transmission of human protozoan parasites by insects.
Clin Microbiol Rev. 2005 Jan;18(1):128-32.
The filthy breeding habits, feeding mechanisms, and indiscriminate travel between filth and food make some groups of synanthropic insects such as nonbiting flies and cockroaches efficient vectors of human enteric protozoan parasites. Twenty-one species of filth flies have been listed by regulatory agencies concerned with sanitation and public health as causative agents of gastrointestinal diseases based on synanthropy, endophily, communicative behavior, and strong attraction to filth and human food. Outbreaks and cases of food-borne diarrheal diseases in urban and rural areas are closely related to the seasonal increase in abundance of filth flies, and enforced fly control is closely related to reductions in the occurrence of such diseases. Mechanical transmission of human parasites by nonbiting flies and epidemiological involvement of other synanthropic insects in human food-borne diseases have not received adequate scientific attention. [Abstract/Link to Full Text]

Lindahl G, Stålhammar-Carlemalm M, Areschoug T
Surface proteins of Streptococcus agalactiae and related proteins in other bacterial pathogens.
Clin Microbiol Rev. 2005 Jan;18(1):102-27.
Streptococcus agalactiae (group B Streptococcus) is the major cause of invasive bacterial disease, including meningitis, in the neonatal period. Although prophylactic measures have contributed to a substantial reduction in the number of infections, development of a vaccine remains an important goal. While much work in this field has focused on the S. agalactiae polysaccharide capsule, which is an important virulence factor that elicits protective immunity, surface proteins have received increasing attention as potential virulence factors and vaccine components. Here, we summarize current knowledge about S. agalactiae surface proteins, with emphasis on proteins that have been characterized immunochemically and/or elicit protective immunity in animal models. These surface proteins have been implicated in interactions with human epithelial cells, binding to extracellular matrix components, and/or evasion of host immunity. Of note, several S. agalactiae surface proteins are related to surface proteins identified in other bacterial pathogens, emphasizing the general interest of the S. agalactiae proteins. Because some S. agalactiae surface proteins elicit protective immunity, they hold promise as components in a vaccine based only on proteins or as carriers in polysaccharide conjugate vaccines. [Abstract/Link to Full Text]

Takayama K, Wang C, Besra GS
Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis.
Clin Microbiol Rev. 2005 Jan;18(1):81-101.
Mycobacterium tuberculosis is known to synthesize alpha-, methoxy-, and keto-mycolic acids. We propose a detailed pathway to the biosynthesis of all mycolic acids in M. tuberculosis. Fatty acid synthetase I provides C(20)-S-coenzyme A to the fatty acid synthetase II system (FAS-IIA). Modules of FAS-IIA and FAS-IIB introduce cis unsaturation at two locations on a growing meroacid chain to yield three different forms of cis,cis-diunsaturated fatty acids (intermediates to alpha-, methoxy-, and keto-meroacids). These are methylated, and the mature meroacids and carboxylated C(26)-S-acyl carrier protein enter into the final Claisen-type condensation with polyketide synthase-13 (Pks13) to yield mycolyl-S-Pks13. We list candidate genes in the genome encoding the proposed dehydrase and isomerase in the FAS-IIA and FAS-IIB modules. We propose that the processing of mycolic acids begins by transfer of mycolic acids from mycolyl-S-Pks13 to d-mannopyranosyl-1-phosphoheptaprenol to yield 6-O-mycolyl-beta-d-mannopyranosyl-1-phosphoheptaprenol and then to trehalose 6-phosphate to yield phosphorylated trehalose monomycolate (TMM-P). Phosphatase releases the phosphate group to yield TMM, which is immediately transported outside the cell by the ABC transporter. Antigen 85 then catalyzes the transfer of a mycolyl group from TMM to the cell wall arabinogalactan and to other TMMs to produce arabinogalactan-mycolate and trehalose dimycolate, respectively. We list candidate genes in the genome that encode the proposed mycolyltransferases I and II, phosphatase, and ABC transporter. The enzymes within this total pathway are targets for new drug discovery. [Abstract/Link to Full Text]

Hambleton S, Gershon AA
Preventing varicella-zoster disease.
Clin Microbiol Rev. 2005 Jan;18(1):70-80.
Varicella-zoster virus (VZV), the cause of chickenpox and shingles, is a pathogen in retreat following the introduction of mass vaccination in the United States in 1995. The live attenuated Oka vaccine, which is safe and immunogenic, gives good protection against both varicella and zoster in the short to medium term. It has undoubtedly been highly effective to date in reducing all forms of varicella, especially severe disease. However, the huge pool of latent wild-type virus in the population represents a continuing threat. Both the biology and the epidemiology of VZV disease suggest that new vaccination strategies will be required over time. [Abstract/Link to Full Text]

Singh N, Paterson DL
Aspergillus infections in transplant recipients.
Clin Microbiol Rev. 2005 Jan;18(1):44-69.
Aspergillus infections are occurring with an increasing frequency in transplant recipients. Notable changes in the epidemiologic characteristics of this infection have occurred; these include a change in risk factors and later onset of infection. Management of invasive aspergillosis continues to be challenging, and the mortality rate, despite the use of newer antifungal agents, remains unacceptably high. Performing molecular studies to discern new targets for antifungal activity, identifying signaling pathways that may be amenable to immunologic interventions, assessing combination regimens of antifungal agents or combining antifungal agents with modulation of the host defense mechanisms, and devising diagnostic assays that can rapidly and reliably diagnose infections represent areas for future investigations that may lead to further improvement in outcomes. [Abstract/Link to Full Text]

Woodfolk JA
Allergy and dermatophytes.
Clin Microbiol Rev. 2005 Jan;18(1):30-43.
Tinea pedis (athlete's foot) and onychomycosis (infection of the toenails) caused by the dermatophyte fungus Trichophyton are highly prevalent in adults. Several Trichophyton allergens have been identified based on elicitation of immunoglobulin E antibody-mediated immediate-hypersensitivity (IH) responses. Evidence of an etiologic role for Trichophyton in asthma in some subjects with IH and chronic dermatophytosis is provided by bronchial reactivity to Trichophyton. Improvement of asthma after systemic antifungal treatment corroborates this link. A unique feature of Trichophyton allergens is the ability of the same antigen to elicit delayed-type hypersensitivity (DTH) in individuals who lack IH reactivity. Delayed responses appear to confer protection, while IH responses do not, based on the association with acute versus chronic skin infection. The amino acid sequence identity of Trichophyton allergens with diverse enzyme families supports a dual role for these proteins in fungal pathogenesis and allergic disease. Characterizing the immunologic properties of Trichophyton allergens and defining immune mechanisms which drive dichotomous responses are pivotal to understanding the dermatophyte-allergy relationship. Recent studies have identified DTH-associated major T-cell epitopes which could facilitate the development of peptide vaccines. Characterization of additional molecular targets by using new techniques may aid not only in the eradication of infection but also in the resolution of allergic symptoms. [Abstract/Link to Full Text]

Schmunis GA, Cruz JR
Safety of the blood supply in Latin America.
Clin Microbiol Rev. 2005 Jan;18(1):12-29.
Appropriate selection of donors, use of sensitive screening tests, and the application of a mandatory quality assurance system are essential to maintain the safety of the blood supply. Laws, decrees, norms, and/or regulations covering most of these aspects of blood transfusion exist in 16 of the 17 countries in Latin America that are the subject of this review. In 17 countries, there is an information system that, although still incomplete (there are no official reports on adverse events and incidents), allows us to establish progress made on the status of the blood supply since 1993. Most advances originated in increased screening coverage for infectious diseases and better quality assurance. However, in 2001 to 2002, tainted blood may have caused infections in 12 of the 17 countries; no country reached the number of donors considered adequate, i.e., 5% of the population, to avoid blood shortages, or decreased significantly the number of blood banks, although larger blood banks are more efficient and take advantage of economies of scale. In those years, paid donors still existed in four countries and replacement donors made up >75% of the blood donors in another eight countries. In addition, countries did not report the number of voluntary donors who were repeat donors, i.e., the healthiest category. In spite of progress made, more improvements are needed. [Abstract/Link to Full Text]

Borkow G, Bentwich Z
Chronic immune activation associated with chronic helminthic and human immunodeficiency virus infections: role of hyporesponsiveness and anergy.
Clin Microbiol Rev. 2004 Oct;17(4):1012-30, table of contents.
Chronic immune activation is one of the hallmarks of human immunodeficiency virus (HIV) infection. It is present also, with very similar characteristics, in very large human populations infested with helminthic infections. We have tried to review the studies addressing the changes in the immune profiles and responses of hosts infected with either one of these two chronic infections. Not surprisingly, several of the immune derangements and impairments seen in HIV infection, and considered by many to be the "specific" effects of HIV, can be found in helminth-infected but HIV-noninfected individuals and can thus be accounted for by the chronic immune activation itself. A less appreciated element in chronic immune activation is the immune suppression and anergy which it may generate. Both HIV and helminth infections represent this aspect in a very wide and illustrative way. Different degrees of anergy and immune hyporesponsiveness are present in these infections and probably have far-reaching effects on the ability of the host to cope with these and other infections. Furthermore, they may have important practical implications, especially with regard to protective vaccinations against AIDS, for populations chronically infected with helminths and therefore widely anergic. The current knowledge of the mechanisms responsible for the generation of anergy by chronic immune activation is thoroughly reviewed. [Abstract/Link to Full Text]

Solomon AW, Peeling RW, Foster A, Mabey DC
Diagnosis and assessment of trachoma.
Clin Microbiol Rev. 2004 Oct;17(4):982-1011, table of contents.
Trachoma is caused by Chlamydia trachomatis. Clinical grading with the WHO simplified system can be highly repeatable provided graders are adequately trained and standardized. At the community level, rapid assessments are useful for confirming the absence of trachoma but do not determine the magnitude of the problem in communities where trachoma is present. New rapid assessment protocols incorporating techniques for obtaining representative population samples (without census preparation) may give better estimates of the prevalence of clinical trachoma. Clinical findings do not necessarily indicate the presence or absence of C. trachomatis infection, particularly as disease prevalence falls. The prevalence of ocular C. trachomatis infection (at the community level) is important because it is infection that is targeted when antibiotics are distributed in trachoma control campaigns. Methods to estimate infection prevalence are required. While culture is a sensitive test for the presence of viable organisms and nucleic acid amplification tests are sensitive and specific tools for the presence of chlamydial nucleic acids, the commercial assays presently available are all too expensive, too complex, or too unreliable for use in national programs. There is an urgent need for a rapid, reliable test for C. trachomatis to assist in measuring progress towards the elimination of trachoma. [Abstract/Link to Full Text]

Edwards JL, Apicella MA
The molecular mechanisms used by Neisseria gonorrhoeae to initiate infection differ between men and women.
Clin Microbiol Rev. 2004 Oct;17(4):965-81, table of contents.
The molecular mechanisms used by the gonococcus to initiate infection exhibit gender specificity. The clinical presentations of disease are also strikingly different upon comparison of gonococcal urethritis to gonococcal cervicitis. An intimate association occurs between the gonococcus and the urethral epithelium and is mediated by the asialoglycoprotein receptor. Gonococcal interaction with the urethral epithelia cell triggers cytokine release, which promotes neutrophil influx and an inflammatory response. Similarly, gonococcal infection of the upper female genital tract also results in inflammation. Gonococci invade the nonciliated epithelia, and the ciliated cells are subjected to the cytotoxic effects of tumor necrosis factor alpha induced by gonococcal peptidoglycan and lipooligosaccharide. In contrast, gonococcal infection of the lower female genital tract is typically asymptomatic. This is in part the result of the ability of the gonococcus to subvert the alternative pathway of complement present in the lower female genital tract. Gonococcal engagement of complement receptor 3 on the cervical epithelia results in membrane ruffling and does not promote inflammation. A model of gonococcal pathogenesis is presented in the context of the male and female human urogenital tracts. [Abstract/Link to Full Text]

Rock RB, Gekker G, Hu S, Sheng WS, Cheeran M, Lokensgard JR, Peterson PK
Role of microglia in central nervous system infections.
Clin Microbiol Rev. 2004 Oct;17(4):942-64, table of contents.
The nature of microglia fascinated many prominent researchers in the 19th and early 20th centuries, and in a classic treatise in 1932, Pio del Rio-Hortega formulated a number of concepts regarding the function of these resident macrophages of the brain parenchyma that remain relevant to this day. However, a renaissance of interest in microglia occurred toward the end of the 20th century, fueled by the recognition of their role in neuropathogenesis of infectious agents, such as human immunodeficiency virus type 1, and by what appears to be their participation in other neurodegenerative and neuroinflammatory disorders. During the same period, insights into the physiological and pathological properties of microglia were gained from in vivo and in vitro studies of neurotropic viruses, bacteria, fungi, parasites, and prions, which are reviewed in this article. New concepts that have emerged from these studies include the importance of cytokines and chemokines produced by activated microglia in neurodegenerative and neuroprotective processes and the elegant but astonishingly complex interactions between microglia, astrocytes, lymphocytes, and neurons that underlie these processes. It is proposed that an enhanced understanding of microglia will yield improved therapies of central nervous system infections, since such therapies are, by and large, sorely needed. [Abstract/Link to Full Text]

Tzipori S, Sheoran A, Akiyoshi D, Donohue-Rolfe A, Trachtman H
Antibody therapy in the management of shiga toxin-induced hemolytic uremic syndrome.
Clin Microbiol Rev. 2004 Oct;17(4):926-41, table of contents.
Hemolytic uremic syndrome (HUS) is a disease that can lead to acute renal failure and often to other serious sequelae, including death. The majority of cases are attributed to infections with Escherichia coli, serotype O157:H7 strains in particular, which cause bloody diarrhea and liberate one or two toxins known as Shiga toxins 1 and 2. These toxins are thought to directly be responsible for the manifestations of HUS. Currently, supportive nonspecific treatment is the only available option for the management of individuals presenting with HUS. The benefit of antimicrobial therapy remains uncertain because of several reports which claim that such intervention can in fact exacerbate the syndrome. There have been only a few specific therapies directed against neutralizing the activities of these toxins, but none so far has been shown to be effective. This article reviews the literature on the mechanism of action of these toxins and the clinical manifestations and current management and treatment of HUS. The major focus of the article, however, is the development and rationale for using neutralizing human antibodies to combat this toxin-induced disease. Several groups are currently pursuing this approach with either humanized, chimeric, or human antitoxin antibodies produced in transgenic mice. They are at different phases of development, ranging from preclinical evaluation to human clinical trials. The information available from preclinical studies indicates that neutralizing specific antibodies directed against the A subunit of the toxin can be highly protective. Such antibodies, even when administered well after exposure to bacterial infection and onset of diarrhea, can prevent the occurrence of systemic complications. [Abstract/Link to Full Text]

Debiasi RL, Tyler KL
Molecular methods for diagnosis of viral encephalitis.
Clin Microbiol Rev. 2004 Oct;17(4):903-25, table of contents.
Hundreds of viruses cause central nervous system (CNS) disease, including meningoencephalitis and postinfectious encephalomyelitis, in humans. The cerebrospinal fluid (CSF) is abnormal in >90% of cases; however, routine CSF studies only rarely lead to identification of a specific etiologic agent. Diagnosis of viral infections of the CNS has been revolutionized by the advent of new molecular diagnostic technologies to amplify viral nucleic acid from CSF, including PCR, nucleic acid sequence-based amplification, and branched-DNA assay. PCR is ideally suited for identifying fastidious organisms that may be difficult or impossible to culture and has been widely applied for detection of both DNA and RNA viruses in CSF. The technique can be performed rapidly and inexpensively and has become an integral component of diagnostic medical practice in the United States and other developed countries. In addition to its use for identification of etiologic agents of CNS disease in the clinical setting, PCR has also been used to quantitate viral load and monitor duration and adequacy of antiviral drug therapy. PCR has also been applied in the research setting to help discriminate active versus postinfectious immune-mediate disease, identify determinants of drug resistance, and investigate the etiology of neurologic disease of uncertain cause. This review discusses general principles of PCR and reverse transcription-PCR, including qualitative, quantitative, and multiplex techniques, with comment on issues of sensitivity, specificity, and positive and negative predictive values. The application of molecular diagnostic methods for diagnosis of specific infectious entities is reviewed in detail, including viruses for which PCR is of proven efficacy and is widely available, viruses for which PCR is less widely available or for which PCR has unproven sensitivity and specificity, and nonviral entities which can mimic viral CNS disease. [Abstract/Link to Full Text]

Fayer R
Sarcocystis spp. in human infections.
Clin Microbiol Rev. 2004 Oct;17(4):894-902, table of contents.
Sarcocystis species are intracellular protozoan parasites with an intermediate-definitive host life cycle based on a prey-predator relationship. Asexual stages develop in intermediate hosts after they ingest the oocyst stage from definitive-host feces and terminate with the formation of intramuscular cysts (sarcocysts). Sarcocysts in meat eaten by a definitive host initiate sexual stages in the intestine that terminate in oocysts excreted in the feces. Most Sarcocystis species infect specific hosts or closely related host species. For example, humans and some primates are definitive hosts for Sarcocystis hominis and S. suihominis after eating raw meat from cattle and pigs, respectively. The prevalence of intestinal sarcocystosis in humans is low and is only rarely associated with illness, except in volunteers who ingest large numbers of sarcocysts. Cases of infection of humans as intermediate hosts, with intramuscular cysts, number less than 100 and are of unknown origin. The asexual stages, including sarcocysts, can stimulate a strong inflammatory response. Livestock have suffered acute debilitating infections, resulting in abortion and death or chronic infections with failure to grow or thrive. This review provides a summary of Sarcocystis biology, including its morphology, life cycle, host specificity, prevalence, diagnosis, treatment, and prevention strategies, for human and food animal infections. [Abstract/Link to Full Text]

Kampf G, Kramer A
Epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs.
Clin Microbiol Rev. 2004 Oct;17(4):863-93, table of contents.
The etiology of nosocomial infections, the frequency of contaminated hands with the different nosocomial pathogens, and the role of health care workers' hands during outbreaks suggest that a hand hygiene preparation should at least have activity against bacteria, yeasts, and coated viruses. The importance of efficacy in choosing the right hand hygiene product is reflected in the new Centers for Disease Control and Prevention guideline on hand hygiene (J. M. Boyce and D. Pittet, Morb. Mortal. Wkly. Rep. 51:1-45, 2002). The best antimicrobial efficacy can be achieved with ethanol (60 to 85%), isopropanol (60 to 80%), and n-propanol (60 to 80%). The activity is broad and immediate. Ethanol at high concentrations (e.g., 95%) is the most effective treatment against naked viruses, whereas n-propanol seems to be more effective against the resident bacterial flora. The combination of alcohols may have a synergistic effect. The antimicrobial efficacy of chlorhexidine (2 to 4%) and triclosan (1 to 2%) is both lower and slower. Additionally, both agents have a risk of bacterial resistance, which is higher for chlorhexidine than triclosan. Their activity is often supported by the mechanical removal of pathogens during hand washing. Taking the antimicrobial efficacy and the mechanical removal together, they are still less effective than the alcohols. Plain soap and water has the lowest efficacy of all. In the new Centers for Disease Control and Prevention guideline, promotion of alcohol-based hand rubs containing various emollients instead of irritating soaps and detergents is one strategy to reduce skin damage, dryness, and irritation. Irritant contact dermatitis is highest with preparations containing 4% chlorhexidine gluconate, less frequent with nonantimicrobial soaps and preparations containing lower concentrations of chlorhexidine gluconate, and lowest with well-formulated alcohol-based hand rubs containing emollients and other skin conditioners. Too few published data from comparative trials are available to reliably rank triclosan. Personnel should be reminded that it is neither necessary nor recommended to routinely wash hands after each application of an alcohol-based hand rub. Long-lasting improvement of compliance with hand hygiene protocols can be successful if an effective and accessible alcohol-based hand rub with a proven dermal tolerance and an excellent user acceptability is supplied, accompanied by education of health care workers and promotion of the use of the product. [Abstract/Link to Full Text]