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Recent Articles in Microbiology and Molecular Biology Reviews

Gelvin SB
Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool.
Microbiol Mol Biol Rev. 2003 Mar;67(1):16-37, table of contents.
Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this "natural genetic engineer" for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes. [Abstract/Link to Full Text]

Kohlhaw GB
Leucine biosynthesis in fungi: entering metabolism through the back door.
Microbiol Mol Biol Rev. 2003 Mar;67(1):1-15, table of contents.
After exploring evolutionary aspects of branched-chain amino acid biosynthesis, the review focuses on the extended leucine biosynthetic pathway as it operates in Saccharomyces cerevisiae. First, the genes and enzymes specific for the leucine pathway are considered: LEU4 and LEU9 (encoding the alpha-isopropylmalate synthase isoenzymes), LEU1 (isopropylmalate isomerase), and LEU2 (beta-isopropylmalate dehydrogenase). Emphasis is given to the unusual distribution of the branched-chain amino acid pathway enzymes between mitochondrial matrix and cytosol, on the newly defined role of Leu5p, and on regulatory mechanisms governing gene expression and enzyme activity, including new evidence for the metabolic importance of the regulation of alpha-isopropylmalate synthase by coenzyme A. Next, structure-function relationships of the transcriptional regulator Leu3p are addressed, defining its dual role as activator and repressor and discussing evidence in support of the self-masking model. Recent data pointing at a more extended Leu3p regulon are discussed. An overview of the layered controls of the extended leucine pathway is provided that includes a description of the newly recognized roles of Ilv5p and Bat1p in maintaining mitochondrial integrity. Finally, branched-chain amino acid biosynthesis and its regulation in other fungi are summarized, the question of leucine as metabolic signal is addressed, and possible directions of future research in this area are outlined. [Abstract/Link to Full Text]

Goffin C, Ghuysen JM
Biochemistry and comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent.
Microbiol Mol Biol Rev. 2002 Dec;66(4):702-38, table of contents.
The bacterial acyltransferases of the SxxK superfamily vary enormously in sequence and function, with conservation of particular amino acid groups and all-alpha and alpha/beta folds. They occur as independent entities (free-standing polypeptides) and as modules linked to other polypeptides (protein fusions). They can be classified into three groups. The group I SxxK D,D-acyltransferases are ubiquitous in the bacterial world. They invariably bear the motifs SxxK, SxN(D), and KT(S)G. Anchored in the plasma membrane with the bulk of the polypeptide chain exposed on the outer face of it, they are implicated in the synthesis of wall peptidoglycans of the most frequently encountered (4-->3) type. They are inactivated by penicillin and other beta-lactam antibiotics acting as suicide carbonyl donors in the form of penicillin-binding proteins (PBPs). They are components of a morphogenetic apparatus which, as a whole, controls multiple parameters such as shape and size and allows the bacterial cells to enlarge and duplicate their particular pattern. Class A PBP fusions comprise a glycosyltransferase module fused to an SxxK acyltransferase of class A. Class B PBP fusions comprise a linker, i.e., protein recognition, module fused to an SxxK acyltransferase of class B. They ensure the remodeling of the (4-->3) peptidoglycans in a cell cycle-dependent manner. The free-standing PBPs hydrolyze D,D peptide bonds. The group II SxxK acyltransferases frequently have a partially modified bar code, but the SxxK motif is invariant. They react with penicillin in various ways and illustrate the great plasticity of the catalytic centers. The secreted free-standing PBPs, the serine beta-lactamases, and the penicillin sensors of several penicillin sensory transducers help the D,D-acyltransferases of group I escape penicillin action. The group III SxxK acyltransferases are indistinguishable from the PBP fusion proteins of group I in motifs and membrane topology, but they resist penicillin. They are referred to as Pen(r) protein fusions. Plausible hypotheses are put forward on the roles that the Pen(r) protein fusions, acting as L,D-acyltransferases, may play in the (3-->3) peptidoglycan-synthesizing molecular machines. Shifting the wall peptidoglycan from the (4-->3) type to the (3-->3) type could help Mycobacterium tuberculosis and Mycobacterium leprae survive by making them penicillin resistant. [Abstract/Link to Full Text]

Grkovic S, Brown MH, Skurray RA
Regulation of bacterial drug export systems.
Microbiol Mol Biol Rev. 2002 Dec;66(4):671-701, table of contents.
The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed. [Abstract/Link to Full Text]

Symington LS
Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair.
Microbiol Mol Biol Rev. 2002 Dec;66(4):630-70, table of contents.
The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination. [Abstract/Link to Full Text]

Dubreuil JD, Giudice GD, Rappuoli R
Helicobacter pylori interactions with host serum and extracellular matrix proteins: potential role in the infectious process.
Microbiol Mol Biol Rev. 2002 Dec;66(4):617-29, table of contents.
Helicobacter pylori, a gram-negative spiral-shaped bacterium, specifically colonizes the stomachs of humans. Once established in this harsh ecological niche, it remains there virtually for the entire life of the host. To date, numerous virulence factors responsible for gastric colonization, survival, and tissue damage have been described for this bacterium. Nevertheless, a critical feature of H. pylori is its ability to establish a long-lasting infection. In fact, although good humoral (against many bacterial proteins) and cellular responses are observed, most infected persons are unable to eradicate the infection. A large body of evidence has shown that the interaction between H. pylori and the host is very complex. In addition to the effect of virulence factors on colonization and persistence, binding of specialized bacterial proteins, known as receptins, to certain host molecules (ligands) could explain the success of H. pylori as a chronically persisting pathogen. Some of the reported interactions are of high affinity, as revealed by their calculated dissociation constant. This review examines the binding of host proteins (serum and extracellular matrix proteins) to H. pylori and considers the significance of these interactions in the infectious process. A more thorough understanding of the kinetics of these receptin interactions could provide a new approach to preventing deeper tissue invasion in H. pylori infections and could represent an alternative to antibiotic treatment. [Abstract/Link to Full Text]

Morris CE, Bardin M, Berge O, Frey-Klett P, Fromin N, Girardin H, Guinebreti�re MH, Lebaron P, Thi�ry JM, Troussellier M
Microbial biodiversity: approaches to experimental design and hypothesis testing in primary scientific literature from 1975 to 1999.
Microbiol Mol Biol Rev. 2002 Dec;66(4):592-616, table of contents.
Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved. One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations. This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge. This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food. These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here. Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses. Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity. To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity. [Abstract/Link to Full Text]

Crespo JL, Hall MN
Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae.
Microbiol Mol Biol Rev. 2002 Dec;66(4):579-91, table of contents.
TOR (target of rapamycin) is a phosphatidylinositol kinase-related protein kinase that controls cell growth in response to nutrients. Rapamycin is an immunosuppressive and anticancer drug that acts by inhibiting TOR. The modes of action of TOR and rapamycin are remarkably conserved from S. cerevisiae to humans. The current understanding of TOR and rapamycin is derived largely from studies with S. cerevisiae. In this review, we discuss the contributions made by S. cerevisiae to understanding rapamycin action and TOR function. [Abstract/Link to Full Text]

Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS
Microbial cellulose utilization: fundamentals and biotechnology.
Microbiol Mol Biol Rev. 2002 Sep;66(3):506-77, table of contents.
Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts. [Abstract/Link to Full Text]

Kolenbrander PE, Andersen RN, Blehert DS, Egland PG, Foster JS, Palmer RJ
Communication among oral bacteria.
Microbiol Mol Biol Rev. 2002 Sep;66(3):486-505, table of contents.
Human oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities. [Abstract/Link to Full Text]

Ganoza MC, Kiel MC, Aoki H
Evolutionary conservation of reactions in translation.
Microbiol Mol Biol Rev. 2002 Sep;66(3):460-85, table of contents.
Current X-ray diffraction and cryoelectron microscopic data of ribosomes of eubacteria have shed considerable light on the molecular mechanisms of translation. Structural studies of the protein factors that activate ribosomes also point to many common features in the primary sequence and tertiary structure of these proteins. The reconstitution of the complex apparatus of translation has also revealed new information important to the mechanisms. Surprisingly, the latter approach has uncovered a number of proteins whose sequence and/or structure and function are conserved in all cells, indicating that the mechanisms are indeed conserved. The possible mechanisms of a new initiation factor and two elongation factors are discussed in this context. [Abstract/Link to Full Text]

Calvo AM, Wilson RA, Bok JW, Keller NP
Relationship between secondary metabolism and fungal development.
Microbiol Mol Biol Rev. 2002 Sep;66(3):447-59, table of contents.
Filamentous fungi are unique organisms-rivaled only by actinomycetes and plants-in producing a wide range of natural products called secondary metabolites. These compounds are very diverse in structure and perform functions that are not always known. However, most secondary metabolites are produced after the fungus has completed its initial growth phase and is beginning a stage of development represented by the formation of spores. In this review, we describe secondary metabolites produced by fungi that act as sporogenic factors to influence fungal development, are required for spore viability, or are produced at a time in the life cycle that coincides with development. We describe environmental and genetic factors that can influence the production of secondary metabolites. In the case of the filamentous fungus Aspergillus nidulans, we review the only described work that genetically links the sporulation of this fungus to the production of the mycotoxin sterigmatocystin through a shared G-protein signaling pathway. [Abstract/Link to Full Text]

Pe�alva MA, Arst HN
Regulation of gene expression by ambient pH in filamentous fungi and yeasts.
Microbiol Mol Biol Rev. 2002 Sep;66(3):426-46, table of contents.
Life, as we know it, is water based. Exposure to hydroxonium and hydroxide ions is constant and ubiquitous, and the evolutionary pressure to respond appropriately to these ions is likely to be intense. Fungi respond to their environments by tailoring their output of activities destined for the cell surface or beyond to the ambient pH. We are beginning to glimpse how they sense ambient pH and transmit this information to the transcription factor, whose roles ensure that a suitable collection of gene products will be made. Although relatively little is known about pH signal transduction itself, its consequences for the cognate transcription factor are much clearer. Intriguingly, homologues of components of this system mediating the regulation of fungal gene expression by ambient pH are to be found in the animal kingdom. The potential applied importance of this regulatory system lies in its key role in fungal pathogenicity of animals and plants and in its control of fungal production of toxins, antibiotics, and secreted enzymes. [Abstract/Link to Full Text]

Cong YS, Wright WE, Shay JW
Human telomerase and its regulation.
Microbiol Mol Biol Rev. 2002 Sep;66(3):407-25, table of contents.
The telomere is a special functional complex at the end of linear eukaryotic chromosomes, consisting of tandem repeat DNA sequences and associated proteins. It is essential for maintaining the integrity and stability of linear eukaryotic genomes. Telomere length regulation and maintenance contribute to normal human cellular aging and human diseases. The synthesis of telomeres is mainly achieved by the cellular reverse transcriptase telomerase, an RNA-dependent DNA polymerase that adds telomeric DNA to telomeres. Expression of telomerase is usually required for cell immortalization and long-term tumor growth. In humans, telomerase activity is tightly regulated during development and oncogenesis. The modulation of telomerase activity may therefore have important implications in antiaging and anticancer therapy. This review describes the currently known components of the telomerase complex and attempts to provide an update on the molecular mechanisms of human telomerase regulation. [Abstract/Link to Full Text]

Albrecht B, Lairmore MD
Critical role of human T-lymphotropic virus type 1 accessory proteins in viral replication and pathogenesis.
Microbiol Mol Biol Rev. 2002 Sep;66(3):396-406, table of contents.
Human T-cell lymphotropic virus type 1 (HTLV-1) infection is associated with a diverse range of lymphoproliferative and neurodegenerative diseases, yet pathogenic mechanisms induced by the virus remain obscure. This complex retrovirus contains typical structural and enzymatic genes but also unique regulatory and accessory genes in four open reading frames (ORFs) of the pX region of the viral genome (pX ORFs I to IV). The regulatory proteins encoded by pX ORFs III and IV, Tax and Rex, respectively, have been extensively characterized. In contrast the contribution of the four accessory proteins p12(I), p27(I), p13(II), and p30(II), encoded by pX ORFs I and II, to viral replication and pathogenesis remained unclear. Proviral clones that are mutated in either pX ORF I or II, while fully competent in cell culture, are severely limited in their replicative capacity in a rabbit model. Emerging evidence indicates that the HTLV-1 accessory proteins are critical for establishment of viral infectivity, enhance T-lymphocyte activation, and potentially alter gene transcription and mitochondrial function. HTLV-1 pX ORF I expression is critical to the viral infectivity in resting primary lymphocytes, suggesting a role for p12(I) in lymphocyte activation. The endoplasmic reticulum and cis-Golgi localizing p12(I), encoded from pX ORF I, activates NFAT, a key T-cell transcription factor, through calcium-mediated signaling pathways and may lower the threshold of lymphocyte activation via the JAK/STAT pathway. In contrast p30(II) localizes to the nucleus and represses viral promoter activity, but may regulate cellular gene expression through p300/CBP or related coactivators of transcription. p13(II) targets mitochondrial proteins, where it alters the organelle morphology and may influence energy metabolism. Collectively, studies of the molecular functions of the HTLV-1 accessory proteins provide insight into strategies used by retroviruses that are associated with lymphoproliferative diseases. [Abstract/Link to Full Text]

Hengge-Aronis R
Signal transduction and regulatory mechanisms involved in control of the sigma(S) (RpoS) subunit of RNA polymerase.
Microbiol Mol Biol Rev. 2002 Sep;66(3):373-95, table of contents.
The sigma(S) (RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and related bacteria. While rapidly growing cells contain very little sigma(S), exposure to many different stress conditions results in rapid and strong sigma(S) induction. Consequently, transcription of numerous sigma(S)-dependent genes is activated, many of which encode gene products with stress-protective functions. Multiple signal integration in the control of the cellular sigma(S) level is achieved by rpoS transcriptional and translational control as well as by regulated sigma(S) proteolysis, with various stress conditions differentially affecting these levels of sigma(S) control. Thus, a reduced growth rate results in increased rpoS transcription whereas high osmolarity, low temperature, acidic pH, and some late-log-phase signals stimulate the translation of already present rpoS mRNA. In addition, carbon starvation, high osmolarity, acidic pH, and high temperature result in stabilization of sigma(S), which, under nonstress conditions, is degraded with a half-life of one to several minutes. Important cis-regulatory determinants as well as trans-acting regulatory factors involved at all levels of sigma(S) regulation have been identified. rpoS translation is controlled by several proteins (Hfq and HU) and small regulatory RNAs that probably affect the secondary structure of rpoS mRNA. For sigma(S) proteolysis, the response regulator RssB is essential. RssB is a specific direct sigma(S) recognition factor, whose affinity for sigma(S) is modulated by phosphorylation of its receiver domain. RssB delivers sigma(S) to the ClpXP protease, where sigma(S) is unfolded and completely degraded. This review summarizes our current knowledge about the molecular functions and interactions of these components and tries to establish a framework for further research on the mode of multiple signal input into this complex regulatory system. [Abstract/Link to Full Text]

Hohmann S
Osmotic stress signaling and osmoadaptation in yeasts.
Microbiol Mol Biol Rev. 2002 Jun;66(2):300-72.
The ability to adapt to altered availability of free water is a fundamental property of living cells. The principles underlying osmoadaptation are well conserved. The yeast Saccharomyces cerevisiae is an excellent model system with which to study the molecular biology and physiology of osmoadaptation. Upon a shift to high osmolarity, yeast cells rapidly stimulate a mitogen-activated protein (MAP) kinase cascade, the high-osmolarity glycerol (HOG) pathway, which orchestrates part of the transcriptional response. The dynamic operation of the HOG pathway has been well studied, and similar osmosensing pathways exist in other eukaryotes. Protein kinase A, which seems to mediate a response to diverse stress conditions, is also involved in the transcriptional response program. Expression changes after a shift to high osmolarity aim at adjusting metabolism and the production of cellular protectants. Accumulation of the osmolyte glycerol, which is also controlled by altering transmembrane glycerol transport, is of central importance. Upon a shift from high to low osmolarity, yeast cells stimulate a different MAP kinase cascade, the cell integrity pathway. The transcriptional program upon hypo-osmotic shock seems to aim at adjusting cell surface properties. Rapid export of glycerol is an important event in adaptation to low osmolarity. Osmoadaptation, adjustment of cell surface properties, and the control of cell morphogenesis, growth, and proliferation are highly coordinated processes. The Skn7p response regulator may be involved in coordinating these events. An integrated understanding of osmoadaptation requires not only knowledge of the function of many uncharacterized genes but also further insight into the time line of events, their interdependence, their dynamics, and their spatial organization as well as the importance of subtle effects. [Abstract/Link to Full Text]

Meidanis J, Braga MD, Verjovski-Almeida S
Whole-genome analysis of transporters in the plant pathogen Xylella fastidiosa.
Microbiol Mol Biol Rev. 2002 Jun;66(2):272-99.
The transport systems of the first completely sequenced genome of a plant parasite, Xylella fastidiosa, were analyzed. In all, 209 proteins were classified here as constitutive members of transport families; thus, we have identified 69 new transporters in addition to the 140 previously annotated. The analysis lead to several hints on potential ways of controlling the disease it causes on citrus trees. An ADP:ATP translocator, previously found in intracellular parasites only, was found in X. fastidiosa. A P-type ATPase is missing-among the 24 completely sequenced eubacteria to date, only three (including X. fastidiosa) do not have a P-type ATPase, and they are all parasites transmitted by insect vectors. An incomplete phosphotransferase system (PTS) was found, without the permease subunits-we conjecture either that they are among the hypothetical proteins or that the PTS plays a solely metabolic regulatory role. We propose that the Ttg2 ABC system might be an import system eventually involved in glutamate import rather than a toluene exporter, as previously annotated. X. fastidiosa exhibits fewer proteins with > or =4 alpha-helical transmembrane spanners than any other completely sequenced prokaryote to date. X. fastidiosa has only 2.7% of all open reading frames identifiable as major transporters, which puts it as the eubacterium having the lowest percentage of open reading frames involved in transport, closer to two archaea, Methanococcus jannaschii (2.4%) and Methanobacterium thermoautotrophicum (2.4%). [Abstract/Link to Full Text]

Bentley R, Chasteen TG
Microbial methylation of metalloids: arsenic, antimony, and bismuth.
Microbiol Mol Biol Rev. 2002 Jun;66(2):250-71.
A significant 19th century public health problem was that the inhabitants of many houses containing wallpaper decorated with green arsenical pigments experienced illness and death. The problem was caused by certain fungi that grew in the presence of inorganic arsenic to form a toxic, garlic-odored gas. The garlic odor was actually put to use in a very delicate microbiological test for arsenic. In 1933, the gas was shown to be trimethylarsine. It was not until 1971 that arsenic methylation by bacteria was demonstrated. Further research in biomethylation has been facilitated by the development of delicate techniques for the determination of arsenic species. As described in this review, many microorganisms (bacteria, fungi, and yeasts) and animals are now known to biomethylate arsenic, forming both volatile (e.g., methylarsines) and nonvolatile (e.g., methylarsonic acid and dimethylarsinic acid) compounds. The enzymatic mechanisms for this biomethylation are discussed. The microbial conversion of sodium arsenate to trimethylarsine proceeds by alternate reduction and methylation steps, with S-adenosylmethionine as the usual methyl donor. Thiols have important roles in the reductions. In anaerobic bacteria, methylcobalamin may be the donor. The other metalloid elements of the periodic table group 15, antimony and bismuth, also undergo biomethylation to some extent. Trimethylstibine formation by microorganisms is now well established, but this process apparently does not occur in animals. Formation of trimethylbismuth by microorganisms has been reported in a few cases. Microbial methylation plays important roles in the biogeochemical cycling of these metalloid elements and possibly in their detoxification. The wheel has come full circle, and public health considerations are again important. [Abstract/Link to Full Text]

Crosa JH, Walsh CT
Genetics and assembly line enzymology of siderophore biosynthesis in bacteria.
Microbiol Mol Biol Rev. 2002 Jun;66(2):223-49.
The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium. [Abstract/Link to Full Text]

Patriarca EJ, Tat� R, Iaccarino M
Key role of bacterial NH(4)(+) metabolism in Rhizobium-plant symbiosis.
Microbiol Mol Biol Rev. 2002 Jun;66(2):203-22.
Symbiotic nitrogen fixation is carried out in specialized organs, the nodules, whose formation is induced on leguminous host plants by bacteria belonging to the family Rhizobiaceae: Nodule development is a complex multistep process, which requires continued interaction between the two partners and thus the exchange of different signals and metabolites. NH(4)(+) is not only the primary product but also the main regulator of the symbiosis: either as ammonium and after conversion into organic compounds, it regulates most stages of the interaction, from the production of nodule inducers to the growth, function, and maintenance of nodules. This review examines the adaptation of bacterial NH(4)(+) metabolism to the variable environment generated by the plant, which actively controls and restricts bacterial growth by affecting oxygen and nutrient availability, thereby allowing a proficient interaction and at the same time preventing parasitic invasion. We describe the regulatory circuitry responsible for the downregulation of bacterial genes involved in NH(4)(+) assimilation occurring early during nodule invasion. This is a key and necessary step for the differentiation of N(2)-fixing bacteroids (the endocellular symbiotic form of rhizobia) and for the development of efficient nodules. [Abstract/Link to Full Text]

Sullivan CS, Pipas JM
T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis.
Microbiol Mol Biol Rev. 2002 Jun;66(2):179-202.
Simian virus 40 (SV40) is a small DNA tumor virus that has been extensively characterized due to its relatively simple genetic organization and the ease with which its genome is manipulated. The large and small tumor antigens (T antigens) are the major regulatory proteins encoded by SV40. Large T antigen is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle. Deciphering how a single protein can perform such numerous and diverse functions has remained elusive. Recently it was established that the SV40 T antigens, including large T antigen, are molecular chaperones, each with a functioning DnaJ domain. The molecular chaperones were originally identified as bacterial genes essential for bacteriophage growth and have since been shown to be conserved in eukaryotes, participating in an array of both viral and cellular processes. This review discusses the mechanisms of DnaJ/Hsc70 interactions and how they are used by T antigen to control viral replication and tumorigenesis. The use of the DnaJ/Hsc70 system by SV40 and other viruses suggests an important role for these molecular chaperones in the regulation of the mammalian cell cycle and sheds light on the enigmatic SV40 T antigen-a most amazing molecule. [Abstract/Link to Full Text]

Guertin DA, Trautmann S, McCollum D
Cytokinesis in eukaryotes.
Microbiol Mol Biol Rev. 2002 Jun;66(2):155-78.
Cytokinesis is the final event of the cell division cycle, and its completion results in irreversible partition of a mother cell into two daughter cells. Cytokinesis was one of the first cell cycle events observed by simple cell biological techniques; however, molecular characterization of cytokinesis has been slowed by its particular resistance to in vitro biochemical approaches. In recent years, the use of genetic model organisms has greatly advanced our molecular understanding of cytokinesis. While the outcome of cytokinesis is conserved in all dividing organisms, the mechanism of division varies across the major eukaryotic kingdoms. Yeasts and animals, for instance, use a contractile ring that ingresses to the cell middle in order to divide, while plant cells build new cell wall outward to the cortex. As would be expected, there is considerable conservation of molecules involved in cytokinesis between yeast and animal cells, while at first glance, plant cells seem quite different. However, in recent years, it has become clear that some aspects of division are conserved between plant, yeast, and animal cells. In this review we discuss the major recent advances in defining cytokinesis, focusing on deciding where to divide, building the division apparatus, and dividing. In addition, we discuss the complex problem of coordinating the division cycle with the nuclear cycle, which has recently become an area of intense research. In conclusion, we discuss how certain cells have utilized cytokinesis to direct development. [Abstract/Link to Full Text]

McConville MJ, Mullin KA, Ilgoutz SC, Teasdale RD
Secretory pathway of trypanosomatid parasites.
Microbiol Mol Biol Rev. 2002 Mar;66(1):122-54; table of contents.
The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Trypanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles in the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory transport and protein glycosylation that may be exploited in developing new antiparasite drugs. [Abstract/Link to Full Text]

Meeks JC, Elhai J
Regulation of cellular differentiation in filamentous cyanobacteria in free-living and plant-associated symbiotic growth states.
Microbiol Mol Biol Rev. 2002 Mar;66(1):94-121; table of contents.
Certain filamentous nitrogen-fixing cyanobacteria generate signals that direct their own multicellular development. They also respond to signals from plants that initiate or modulate differentiation, leading to the establishment of a symbiotic association. An objective of this review is to describe the mechanisms by which free-living cyanobacteria regulate their development and then to consider how plants may exploit cyanobacterial physiology to achieve stable symbioses. Cyanobacteria that are capable of forming plant symbioses can differentiate into motile filaments called hormogonia and into specialized nitrogen-fixing cells called heterocysts. Plant signals exert both positive and negative regulatory control on hormogonium differentiation. Heterocyst differentiation is a highly regulated process, resulting in a regularly spaced pattern of heterocysts in the filament. The evidence is most consistent with the pattern arising in two stages. First, nitrogen limitation triggers a nonrandomly spaced cluster of cells (perhaps at a critical stage of their cell cycle) to initiate differentiation. Interactions between an inhibitory peptide exported by the differentiating cells and an activator protein within them causes one cell within each cluster to fully differentiate, yielding a single mature heterocyst. In symbiosis with plants, heterocyst frequencies are increased 3- to 10-fold because, we propose, either differentiation is initiated at an increased number of sites or resolution of differentiating clusters is incomplete. The physiology of symbiotically associated cyanobacteria raises the prospect that heterocyst differentiation proceeds independently of the nitrogen status of a cell and depends instead on signals produced by the plant partner. [Abstract/Link to Full Text]

Narberhaus F
Alpha-crystallin-type heat shock proteins: socializing minichaperones in the context of a multichaperone network.
Microbiol Mol Biol Rev. 2002 Mar;66(1):64-93; table of contents.
Alpha-crystallins were originally recognized as proteins contributing to the transparency of the mammalian eye lens. Subsequently, they have been found in many, but not all, members of the Archaea, Bacteria, and Eucarya. Most members of the diverse alpha-crystallin family have four common structural and functional features: (i) a small monomeric molecular mass between 12 and 43 kDa; (ii) the formation of large oligomeric complexes; (iii) the presence of a moderately conserved central region, the so-called alpha-crystallin domain; and (iv) molecular chaperone activity. Since alpha-crystallins are induced by a temperature upshift in many organisms, they are often referred to as small heat shock proteins (sHsps) or, more accurately, alpha-Hsps. Alpha-crystallins are integrated into a highly flexible and synergistic multichaperone network evolved to secure protein quality control in the cell. Their chaperone activity is limited to the binding of unfolding intermediates in order to protect them from irreversible aggregation. Productive release and refolding of captured proteins into the native state requires close cooperation with other cellular chaperones. In addition, alpha-Hsps seem to play an important role in membrane stabilization. The review compiles information on the abundance, sequence conservation, regulation, structure, and function of alpha-Hsps with an emphasis on the microbial members of this chaperone family. [Abstract/Link to Full Text]

Graves PR, Haystead TA
Molecular biologist's guide to proteomics.
Microbiol Mol Biol Rev. 2002 Mar;66(1):39-63; table of contents.
The emergence of proteomics, the large-scale analysis of proteins, has been inspired by the realization that the final product of a gene is inherently more complex and closer to function than the gene itself. Shortfalls in the ability of bioinformatics to predict both the existence and function of genes have also illustrated the need for protein analysis. Moreover, only through the study of proteins can posttranslational modifications be determined, which can profoundly affect protein function. Proteomics has been enabled by the accumulation of both DNA and protein sequence databases, improvements in mass spectrometry, and the development of computer algorithms for database searching. In this review, we describe why proteomics is important, how it is conducted, and how it can be applied to complement other existing technologies. We conclude that currently, the most practical application of proteomics is the analysis of target proteins as opposed to entire proteomes. This type of proteomics, referred to as functional proteomics, is always driven by a specific biological question. In this way, protein identification and characterization has a meaningful outcome. We discuss some of the advantages of a functional proteomics approach and provide examples of how different methodologies can be utilized to address a wide variety of biological problems. [Abstract/Link to Full Text]

Morrissette NS, Sibley LD
Cytoskeleton of apicomplexan parasites.
Microbiol Mol Biol Rev. 2002 Mar;66(1):21-38; table of contents.
The Apicomplexa are a phylum of diverse obligate intracellular parasites including Plasmodium spp., the cause of malaria; Toxoplasma gondii and Cryptosporidium parvum, opportunistic pathogens of immunocompromised individuals; and Eimeria spp. and Theileria spp., parasites of considerable agricultural importance. These protozoan parasites share distinctive morphological features, cytoskeletal organization, and modes of replication, motility, and invasion. This review summarizes our current understanding of the cytoskeletal elements, the properties of cytoskeletal proteins, and the role of the cytoskeleton in polarity, motility, invasion, and replication. We discuss the unusual properties of actin and myosin in the Apicomplexa, the highly stereotyped microtubule populations in apicomplexans, and a network of recently discovered novel intermediate filament-like elements in these parasites. [Abstract/Link to Full Text]

Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, W�nschiers R, Lindblad P
Hydrogenases and hydrogen metabolism of cyanobacteria.
Microbiol Mol Biol Rev. 2002 Mar;66(1):1-20, table of contents.
Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect--the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included. [Abstract/Link to Full Text]

Goldberg MB
Actin-based motility of intracellular microbial pathogens.
Microbiol Mol Biol Rev. 2001 Dec;65(4):595-626, table of contents.
A diverse group of intracellular microorganisms, including Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, utilize actin-based motility to move within and spread between mammalian host cells. These organisms have in common a pathogenic life cycle that involves a stage within the cytoplasm of mammalian host cells. Within the cytoplasm of host cells, these organisms activate components of the cellular actin assembly machinery to induce the formation of actin tails on the microbial surface. The assembly of these actin tails provides force that propels the organisms through the cell cytoplasm to the cell periphery or into adjacent cells. Each of these organisms utilizes preexisting mammalian pathways of actin rearrangement to induce its own actin-based motility. Particularly remarkable is that while all of these microbes use the same or overlapping pathways, each intercepts the pathway at a different step. In addition, the microbial molecules involved are each distinctly different from the others. Taken together, these observations suggest that each of these microbes separately and convergently evolved a mechanism to utilize the cellular actin assembly machinery. The current understanding of the molecular mechanisms of microbial actin-based motility is the subject of this review. [Abstract/Link to Full Text]

Recent Articles in Applied and Environmental Microbiology

Kienesberger S, Gorkiewicz G, Joainig MM, Scheicher SR, Leitner E, Zechner EL
Development of experimental genetic tools for Campylobacter fetus.
Appl Environ Microbiol. 2007 Jul;73(14):4619-30.
Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus. [Abstract/Link to Full Text]

Lami R, Cottrell MT, Ras J, Ulloa O, Obernosterer I, Claustre H, Kirchman DL, Lebaron P
High abundances of aerobic anoxygenic photosynthetic bacteria in the South Pacific Ocean.
Appl Environ Microbiol. 2007 Jul;73(13):4198-205.
Little is known about the abundance, distribution, and ecology of aerobic anoxygenic phototrophic (AAP) bacteria, particularly in oligotrophic environments, which represent 60% of the ocean. We investigated the abundance of AAP bacteria across the South Pacific Ocean, including the center of the gyre, the most oligotrophic water body of the world ocean. AAP bacteria, Prochlorococcus, and total prokaryotic abundances, as well as bacteriochlorophyll a (BChl a) and divinyl-chlorophyll a concentrations, were measured at several depths in the photic zone along a gradient of oligotrophic conditions. The abundances of AAP bacteria and Prochlorococcus were high, together accounting for up to 58% of the total prokaryotic community. The abundance of AAP bacteria alone was up to 1.94 x 10(5) cells ml(-1) and as high as 24% of the overall community. These measurements were consistent with the high BChl a concentrations (up to 3.32 x 10(-3) microg liter(-1)) found at all stations. However, the BChl a content per AAP bacterial cell was low, suggesting that AAP bacteria are mostly heterotrophic organisms. Interestingly, the biovolume and therefore biomass of AAP bacteria was on average twofold higher than that of other prokaryotic cells. This study demonstrates that AAP bacteria can be abundant in various oligotrophic conditions, including the most oligotrophic regime of the world ocean, and can account for a large part of the bacterioplanktonic carbon stock. [Abstract/Link to Full Text]

Narita S, Kageyama D, Nomura M, Fukatsu T
Unexpected mechanism of symbiont-induced reversal of insect sex: feminizing Wolbachia continuously acts on the butterfly Eurema hecabe during larval development.
Appl Environ Microbiol. 2007 Jul;73(13):4332-41.
When the butterfly Eurema hecabe is infected with two different strains (wHecCI2 and wHecFem2) of the bacterial endosymbiont Wolbachia, genetic males are transformed into functional females, resulting in production of all-female broods. In an attempt to understand how and when the Wolbachia endosymbiont feminizes genetically male insects, larval insects were fed an antibiotic-containing diet beginning at different developmental stages until pupation. When the adult insects emerged, strikingly, many of them exhibited sexually intermediate traits in their wings, reproductive organs, and genitalia. The expression of intersexual phenotypes was strong in the insects treated from first instar, moderate in the insects treated from third instar, and weak in the insects treated from fourth instar. The insects treated from early larval instar grew and pupated normally but frequently failed to emerge and died in the pupal case. The dead insects in the pupal case contained lower densities of the feminizing Wolbachia endosymbiont than the successfully emerged insects, although none of them were completely cured of the symbiont infection. These results suggest the following: (i) the antibiotic treatment suppressed the population of feminizing Wolbachia endosymbionts; (ii) the suppression probably resulted in attenuated feminizing activity of the symbiont, leading to expression of intersexual host traits; (iii) many of the insects suffered pupal mortality, possibly due to either intersexual defects or Wolbachia-mediated addiction; and hence (iv) the feminizing Wolbachia endosymbiont continuously acts on the host insects during larval development for expression of female phenotypes under a male genotype. Our finding may prompt reconsideration of the notion that Wolbachia-induced reproductive manipulations are already complete before the early embryonic stage and provide insights into the mechanism underlying the symbiont-induced reversal of insect sex. [Abstract/Link to Full Text]

Chen J, Yu Z, Michel FC, Wittum T, Morrison M
Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin B in livestock manure and manure management systems.
Appl Environ Microbiol. 2007 Jul;73(14):4407-16.
Erythromycin and tylosin are commonly used in animal production, and such use is perceived to contribute to the overall antimicrobial resistance (AR) reservoirs. Quantitative measurements of this type of AR reservoir in microbial communities are required to understand AR ecology (e.g., emergence, persistence, and dissemination). We report here the development, validation, and use of six real-time PCR assays for quantifying six classes of erm genes (classes A through C, F, T, and X) that encode the major mechanism of resistance to macrolides-lincosamides-streptogramin B (MLS(B)). These real-time PCR assays were validated and used in quantifying the six erm classes in five types of samples, including those from bovine manure, swine manure, compost of swine manure, swine waste lagoons, and an Ekokan upflow biofilter system treating hog house effluents. The bovine manure samples were found to contain much smaller reservoirs of each of the six erm classes than the swine manure samples. Compared to the swine manure samples, the composted swine manure samples had substantially reduced erm gene abundances (by up to 7.3 logs), whereas the lagoon or the biofilter samples had similar erm gene abundances. These preliminary results suggest that the methods of manure storage and treatment probably have a substantial impact on the persistence and decline of MLS(B) resistance originating from food animals, thus likely affecting the dissemination of such resistance genes into the environment. The abundances of these erm genes appeared to be positively correlated with those of the tet genes determined previously among these samples. These real-time PCR assays provide a rapid, quantitative, and cultivation-independent measurement of six major classes of erm genes, which should be useful for ecological studies of AR. [Abstract/Link to Full Text]

Parada V, Sintes E, van Aken HM, Weinbauer MG, Herndl GJ
Viral abundance, decay, and diversity in the meso- and bathypelagic waters of the north atlantic.
Appl Environ Microbiol. 2007 Jul;73(14):4429-38.
To elucidate the potential importance of deep-water viruses in controlling the meso- and bathypelagic picoplankton community, the abundance, decay rate, and diversity of the virioplankton community were determined in the meso- and bathypelagic water masses of the eastern part of the subtropical North Atlantic. Viral abundance averaged 1.4 x 10(6) ml(-1) at around 100 m of depth and decreased only by a factor of 2 at 3,000 to 4,000 m of depth. In contrast, picoplankton abundance decreased by 1 order of magnitude to the Lower Deep Water (LDW; 3,500- to 5,000-m depth). The virus-to-picoplankton ratio increased from 9 at about 100 m of depth to 110 in the LDW. Mean viral decay rates were 3.5 x 10(-3) h(-1) between 900 m and 2,750 m and 1.1 x 10(-3) h(-1) at 4,000 m of depth, corresponding to viral turnover times of 11 and 39 days, respectively. Pulsed-field gel electrophoresis fingerprints obtained from the viral community between 2,400 m and 4,000 m of depth revealed a maximum of only four bands from 4,000 m of depth. Based on the high viral abundance and the low picoplankton production determined via leucine incorporation, we conclude that the viral production calculated from the viral decay is insufficient to maintain the high viral abundance in the deep North Atlantic. Rather, we propose that substantial allochthonous viral input or lysogenic or pseudolysogenic production is required to maintain the high viral abundance detected in the meso- and bathypelagic North Atlantic. Consequently, deep-water prokaryotes are apparently far less controlled in their abundance and taxon richness by lytic prokaryotic phages than the high viral abundance and the virus-to-picoplankton ratio would suggest. [Abstract/Link to Full Text]

Medellin-Pe�a MJ, Wang H, Johnson R, Anand S, Griffiths MW
Probiotics affect virulence-related gene expression in Escherichia coli O157:H7.
Appl Environ Microbiol. 2007 Jul;73(13):4259-67.
The attachment of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) to host intestinal epithelial cells is essential for the development of hemorrhagic colitis and hemolytic-uremic syndrome in humans. Genes involved in attachment are carried within a pathogenicity island named the locus of enterocyte effacement (LEE), known to be directly activated by quorum sensing (QS). In the present study, we investigated autoinducer-2 (AI-2) production and the expression of several virulence-related genes in EHEC O157 grown in the absence and presence of a Lactobacillus acidophilus-secreted molecule(s). Transcription of important EHEC O157 virulence-related genes was studied by constructing promoter-reporter fusions and reverse transcriptase PCR. Shiga toxin (Stx) production was assayed by an enzyme immunoassay. When EHEC O157 was grown in the presence of chromatographically selected fractions of L. acidophilus La-5 cell-free spent medium, we observed a significant reduction of both extracellular AI-2 concentration and the expression of important virulence-related genes, although no significant difference in Stx production was observed. We show here that L. acidophilus La-5 secretes a molecule(s) that either acts as a QS signal inhibitor or directly interacts with bacterial transcriptional regulators, controlling the transcription of EHEC O157 genes involved in colonization. [Abstract/Link to Full Text]

Rademaker JL, Vissers MM, Te Giffel MC
Effective heat inactivation of Mycobacterium avium subsp. paratuberculosis in raw milk contaminated with naturally infected feces.
Appl Environ Microbiol. 2007 Jul;73(13):4185-90.
The effectiveness of high-temperature, short holding time (HTST) pasteurization and homogenization with respect to inactivation of Mycobacterium avium subsp. paratuberculosis was evaluated quantitatively. This allowed a detailed determination of inactivation kinetics. High concentrations of feces from cows with clinical symptoms of Johne's disease were used to contaminate raw milk in order to realistically mimic possible incidents most closely. Final M. avium subsp. paratuberculosis concentrations varying from 10(2) to 3.5 x 10(5) cells per ml raw milk were used. Heat treatments including industrial HTST were simulated on a pilot scale with 22 different time-temperature combinations, including 60 to 90 degrees C at holding (mean residence) times of 6 to 15 s. Following 72 degrees C and a holding time of 6 s, 70 degrees C for 10 and 15 s, or under more stringent conditions, no viable M. avium subsp. paratuberculosis cells were recovered, resulting in >4.2- to >7.1-fold reductions, depending on the original inoculum concentrations. Inactivation kinetic modeling of 69 quantitative data points yielded an E(a) of 305,635 J/mol and an lnk(0) of 107.2, corresponding to a D value of 1.2 s at 72 degrees C and a Z value of 7.7 degrees C. Homogenization did not significantly affect the inactivation. The conclusion can be drawn that HTST pasteurization conditions equal to 15 s at > or =72 degrees C result in a more-than-sevenfold reduction of M. avium subsp. paratuberculosis. [Abstract/Link to Full Text]

J�nsch A, Korakli M, Vogel RF, G�nzle MG
Glutathione reductase from Lactobacillus sanfranciscensis DSM20451T: contribution to oxygen tolerance and thiol exchange reactions in wheat sourdoughs.
Appl Environ Microbiol. 2007 Jul;73(14):4469-76.
The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451(T) on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451(T)DeltagshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451(T)DeltagshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 microM to 10.5 microM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451(T)DeltagshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 microM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451(T)DeltagshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat. [Abstract/Link to Full Text]

Felnagle EA, Rondon MR, Berti AD, Crosby HA, Thomas MG
Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin.
Appl Environ Microbiol. 2007 Jul;73(13):4162-70.
Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance. [Abstract/Link to Full Text]

Verwaal R, Wang J, Meijnen JP, Visser H, Sandmann G, van den Berg JA, van Ooyen AJ
High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous.
Appl Environ Microbiol. 2007 Jul;73(13):4342-50.
To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae. [Abstract/Link to Full Text]

Kikuchi Y, Hosokawa T, Fukatsu T
Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation.
Appl Environ Microbiol. 2007 Jul;73(13):4308-16.
The broad-headed bug Riptortus clavatus (Heteroptera: Alydidae) possesses a number of crypts at a posterior midgut region, which house a dense population of a bacterial symbiont belonging to the genus Burkholderia. Although the symbiont is highly prevalent (95 to 100%) in the host populations, the symbiont phylogeny did not reflect the host systematics at all. In order to understand the mechanisms underlying the promiscuous host-symbiont relationship despite the specific and prevalent association, we investigated the transmission mode and the fitness effects of the Burkholderia symbiont in R. clavatus. Inspection of eggs and a series of rearing experiments revealed that the symbiont is not vertically transmitted but is environmentally acquired by nymphal insects. The Burkholderia symbiont was present in the soil of the insect habitat, and a culture strain of the symbiont was successfully isolated from the insect midgut. Rearing experiments by using sterilized soybean bottles demonstrated that the cultured symbiont is able to establish a normal and efficient infection in the host insect, and the symbiont infection significantly improves the host fitness. These results indicated that R. clavatus postnatally acquires symbiont of a beneficial nature from the environment every generation, uncovering a previously unknown pathway through which a highly specific insect-microbe association is maintained. We suggest that the stinkbug-Burkholderia relationship may be regarded as an insect analogue of the well-known symbioses between plants and soil-associated microbes, such as legume-Rhizobium and alder-Frankia relationships, and we discuss the evolutionary relevance of the mutualistic but promiscuous insect-microbe association. [Abstract/Link to Full Text]

Wright AD, Auckland CH, Lynn DH
Molecular diversity of methanogens in feedlot cattle from Ontario and Prince Edward Island, Canada.
Appl Environ Microbiol. 2007 Jul;73(13):4206-10.
The molecular diversity of rumen methanogens in feedlot cattle and the composition of the methanogen populations in these animals from two geographic locations were investigated using 16S rRNA gene libraries prepared from pooled PCR products from 10 animals in Ontario (127 clones) and 10 animals from Prince Edward Island (114 clones). A total of 241 clones were examined, with Methanobrevibacter ruminantium accounting for more than one-third (85 clones) of the clones identified. From these 241 clones, 23 different 16S rRNA phylotypes were identified. Feedlot cattle from Ontario, which were fed a corn-based diet, revealed 11 phylotypes (38 clones) not found in feedlot cattle from Prince Edward Island, whereas the Prince Edward Island cattle, which were fed potato by-products as a finishing diet, had 7 phylotypes (42 clones) not found in cattle from Ontario. Five sequences, representing the remaining 161 clones (67% of the clones), were common in both herds. Of the 23 different sequences, 10 sequences (136 clones) were 89.8 to 100% similar to those from cultivated methanogens belonging to the orders Methanobacteriales, Methanomicrobiales, and Methanosarcinales, and the remaining 13 sequences (105 clones) were 74.1 to 75.8% similar to those from Thermoplasma volcanium and Thermoplasma acidophilum. Overall, nine possible new species were identified from the two clone libraries, including two new species belonging to the order Methanobacteriales and a new genus/species within the order Methanosarcinales. From the present survey, it is difficult to conclude whether the geographical isolation between these two herds or differences between the two finishing diets directly influenced community structure in the rumen. Further studies are warranted to properly assess the differences between these two finishing diets. [Abstract/Link to Full Text]

Ruas-Madiedo P, Moreno JA, Salazar N, Delgado S, Mayo B, Margolles A, de Los Reyes-Gavil�n CG
Screening of exopolysaccharide-producing Lactobacillus and Bifidobacterium strains isolated from the human intestinal microbiota.
Appl Environ Microbiol. 2007 Jul;73(13):4385-8.
Using phenotypic approaches, we have detected that 17% of human intestinal Lactobacillus and Bifidobacterium strains could be exopolysaccharide (EPS) producers. However, PCR techniques showed that only 7% harbored genes related to the synthesis of heteropolysaccharides. This is the first work to screen the human intestinal ecosystem for the detection of EPS-producing strains. [Abstract/Link to Full Text]

Park GW, Boston DM, Kase JA, Sampson MN, Sobsey MD
Evaluation of liquid- and fog-based application of Sterilox hypochlorous acid solution for surface inactivation of human norovirus.
Appl Environ Microbiol. 2007 Jul;73(14):4463-8.
Noroviruses (NVs) are the most frequent cause of outbreaks of gastroenteritis in common settings, with surface-mediated transfer via contact with fecally contaminated surfaces implicated in exposure. NVs are environmentally stable and persistent and have a low infectious dose. Several disinfectants have been evaluated for efficacy to control viruses on surfaces, but the toxicity and potential damage to treated materials limits their applicability. Sterilox hypochlorous acid (HOCl) solution (HAS) has shown broad-spectrum antimicrobial activity while being suitable for general use. The objectives of this study were to evaluate the efficacy of HAS to reduce NV both in aqueous suspensions and on inanimate carriers. HOCl was further tested as a fog to decontaminate large spaces. HOCl effectiveness was evaluated using nonculturable human NV measured by reverse transcriptase PCR (RT-PCR) and two surrogate viruses, coliphage MS2 and murine NV, that were detected by both infectivity and RT-PCR. Exposing virus-contaminated carriers of ceramic tile (porous) and stainless steel (nonporous) to 20 to 200 ppm of HOCl solution resulted in > or = 99.9% (> or = 3 log10) reductions of both infectivity and RNA titers of tested viruses within 10 min of exposure time. HOCl fogged in a confined space reduced the infectivity and RNA titers of NV, murine NV, and MS2 on these carriers by at least 99.9% (3 log10), regardless of carrier location and orientation. We conclude that HOCl solution as a liquid or fog is likely to be effective in disinfecting common settings to reduce NV exposures and thereby control virus spread via fomites. [Abstract/Link to Full Text]

Love DC, Sobsey MD
Simple and rapid F+ coliphage culture, latex agglutination, and typing assay to detect and source track fecal contamination.
Appl Environ Microbiol. 2007 Jul;73(13):4110-8.
Simple, rapid, and reliable fecal indicator tests are needed to better monitor and manage ambient waters and treated waters and wastes. Antibody-coated polymeric bead agglutination assays can fulfill these needs and are inexpensive and portable for nonlaboratory settings, and their reagents can be stored at ambient temperatures for months. The goal of this study was to develop, optimize, and validate a rapid microbial water quality monitoring assay using F+ coliphage culture, latex agglutination, and typing (CLAT) to detect F+ coliphage groups with antibody-coated particles. Rapid (180 min) F+ coliphage culture gave comparable results to those with the 16- to 24-h culture time used in EPA method 1601 and was amenable to CLAT assay detection. CLAT was performed on a cardboard card by mixing a drop of coliphage enrichment culture with a drop of antibody-coated polymeric beads as the detection reagent. Visual agglutination or clumping of positive samples occurred in <60 seconds. The CLAT assay had sensitivities of 96.4% (185/192 samples) and 98.2% (161/164 samples) and specificities of 100% (34/34 samples) and 97.7% (129/132 samples) for F+ RNA and DNA coliphages, respectively. CLAT successfully classified F+ RNA coliphages into serogroups typically obtained from human (groups II and III) and animal (groups I and IV) fecal sources, in similar proportions to those obtained with a nucleic acid hybridization assay. This novel group-specific antibody-based particle agglutination technique for rapid and simple detection and grouping of F+ coliphages provides a new and improved tool for monitoring the microbiological quality of drinking, recreational, shellfishing, and other waters. [Abstract/Link to Full Text]

Hill VR, Kahler AM, Jothikumar N, Johnson TB, Hahn D, Cromeans TL
Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples.
Appl Environ Microbiol. 2007 Jul;73(13):4218-25.
Ultrafiltration (UF) is increasingly being recognized as a potentially effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. Because of their very small pore sizes, UF membranes are capable of simultaneously concentrating viruses, bacteria, and parasites based on size exclusion. In this study, a UF-based water sampling procedure was used to simultaneously recover representatives of these three microbial classes seeded into 100-liter samples of tap water collected from eight cities covering six hydrologic areas of the United States. The UF-based procedure included hollow-fiber UF as the primary step for concentrating microbes and then used membrane filtration for bacterial culture assays, immunomagnetic separation for parasite recovery and quantification, and centrifugal UF for secondary concentration of viruses. Water samples were tested for nine water quality parameters to investigate whether water quality data correlated with measured recovery efficiencies and molecular detection levels. Average total method recovery efficiencies were 71, 97, 120, 110, and 91% for phiX174 bacteriophage, MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts, respectively. Real-time PCR and reverse transcription-PCR (RT-PCR) for seeded microbes and controls indicated that tap water quality could affect the analytical performance of molecular amplification assays, although no specific water quality parameter was found to correlate with reduced PCR or RT-PCR performance. [Abstract/Link to Full Text]

Thompson FL, Gomez-Gil B, Vasconcelos AT, Sawabe T
Multilocus sequence analysis reveals that Vibrio harveyi and V. campbellii are distinct species.
Appl Environ Microbiol. 2007 Jul;73(13):4279-85.
Identification and classification of Vibrio species have relied upon band pattern methods (e.g., amplified fragment length polymorphism) and DNA-DNA hybridization. However, data generated by these methods cannot be used to build an online electronic taxonomy. In order to overcome these limitations, we developed the first standard multilocus sequence scheme focused on the ubiquitous and pathogenic Vibrio harveyi species group (i.e., V. harveyi, V. campbellii, V. rotiferianus, and a new as yet unnamed species). We examined a collection of 104 isolates from different geographical regions and hosts using segments of seven housekeeping genes. These two species formed separated clusters on the basis of topA, pyrH, ftsZ, and mreB gene sequences. The phylogenetic picture obtained by the other three loci, i.e., gyrB, recA, and gapA, was more complex though. V. campbellii appeared nested within V. harveyi in the recA trees, whereas V. harveyi formed a tight nested cluster within V. campbellii by gapA. The gyrB gene had no taxonomic resolution and grouped the two species together. The fuzziness observed in these three genes seems not be related to recombination but to low divergence due to the accumulation of only a few substitutions. In spite of this, the concatenated sequences provided evidence that the two species form two separated clusters. These clusters did not arise by recombination but by accumulation of point mutations. V. harveyi and V. campbellii isolates can be readily identified through the open database resource developed in this study (http://www.taxvibrio.lncc.br/). We argue that the species should be defined by evolutionary criteria. Strains of the same species will share at least 95% concatenated sequence similarity using the seven loci, and, most importantly, cospecific strains will form cohesive readily recognizable phylogenetic clades. [Abstract/Link to Full Text]

Hatamoto M, Imachi H, Yashiro Y, Ohashi A, Harada H
Diversity of anaerobic microorganisms involved in long-chain fatty acid degradation in methanogenic sludges as revealed by RNA-based stable isotope probing.
Appl Environ Microbiol. 2007 Jul;73(13):4119-27.
Long-chain fatty acid (LCFA) degradation is a key step in methanogenic treatment of wastes/wastewaters containing high concentrations of lipids. However, despite the importance of LCFA-degrading bacteria, their natural diversity is little explored due to the limited availability of isolate information and the lack of appropriate molecular markers. We therefore investigated these microbes by using RNA-based stable isotope probing. We incubated four methanogenic sludges (mesophilic sludges MP and MBF and thermophilic sludges TP and JET) with (13)C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect (13)C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in (13)C-labeled and unlabeled rRNA fractions. The bacterial populations in the (13)C-labeled rRNA fractions were identified by cloning and sequencing of reverse-transcribed 16S rRNA. Diverse phylogenetic bacterial sequences were retrieved, including those of members of the family Syntrophaceae, clone cluster MST belonging to the class Deltaproteobacteria, Clostridium clusters III and IV, phylum Bacteroidetes, phylum Spirochaetes, and family Syntrophomonadaceae. Although Syntrophomonadaceae species are considered to be the major fatty acid-degrading syntrophic microorganisms under methanogenic conditions, they were detected in only two of the clone libraries. These results suggest that phylogenetically diverse bacterial groups were active in situ in the degradation of LCFA under methanogenic conditions. [Abstract/Link to Full Text]

H�ppener-Ogawa S, Leveau JH, Smant W, van Veen JA, de Boer W
Specific detection and real-time PCR quantification of potentially mycophagous bacteria belonging to the genus Collimonas in different soil ecosystems.
Appl Environ Microbiol. 2007 Jul;73(13):4191-7.
The bacterial genus Collimonas has the remarkable characteristic that it grows at the expense of living fungal hyphae under laboratory conditions. Here, we report the first field inventory of the occurrence and abundance of Collimonas in soils (n = 45) with naturally different fungal densities, which was performed in order to test the null hypothesis that there is a relationship between the presence of Collimonas and fungal biomass. Estimates of fungal densities were based on ergosterol measurements. Each soil was also characterized in terms of its physical and chemical properties and vegetation and management types. Culturable Collimonas was identified in plate-spread soil samples by its ability to clear colloidal chitin, in combination with a Collimonas-specific restriction fragment length polymorphism analysis of 16S rRNA PCR amplified from individual colonies. Using this approach, we found culturable collimonads only in (semi)natural grasslands. A real-time PCR assay for the specific quantification of Collimonas 16S rRNA in total soil DNA was developed. Collimonas was detectable in 80% of the soil samples, with densities up to 10(5) cells g(-1) (dry weight) soil. The numbers of Collimonas cells per gram of soil were consistently lowest in fungus-poor arable soils and, surprisingly, also in fungus-rich organic layers of forest soils. When all soils were included, no significant correlation was observed between the number of Collimonas cells and ergosterol-based soil fungal biomass. Based on this result, we rejected our null hypothesis, and possible explanations for this were addressed. [Abstract/Link to Full Text]

Passoth V, Blomqvist J, Schn�rer J
Dekkera bruxellensis and Lactobacillus vini form a stable ethanol-producing consortium in a commercial alcohol production process.
Appl Environ Microbiol. 2007 Jul;73(13):4354-6.
The ethanol production process of a Swedish alcohol production plant was dominated by Dekkera bruxellensis and Lactobacillus vini, with a high number of lactic acid bacteria. The product quality, process productivity, and stability were high; thus, D. bruxellensis and L. vini can be regarded as commercial ethanol production organisms. [Abstract/Link to Full Text]

Ruecker NJ, Braithwaite SL, Topp E, Edge T, Lapen DR, Wilkes G, Robertson W, Medeiros D, Sensen CW, Neumann NF
Tracking host sources of Cryptosporidium spp. in raw water for improved health risk assessment.
Appl Environ Microbiol. 2007 Jun;73(12):3945-57.
Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium. [Abstract/Link to Full Text]

Hayes M, Stanton C, Slattery H, O'Sullivan O, Hill C, Fitzgerald GF, Ross RP
Casein fermentate of Lactobacillus animalis DPC6134 contains a range of novel propeptide angiotensin-converting enzyme inhibitors.
Appl Environ Microbiol. 2007 Jul;73(14):4658-67.
This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (+/-15%) and had a 50% inhibitory concentration (IC50) of 0.8 mg protein/ml compared to captopril, which had an IC50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (+/-15), 83.71 (+/-19), and 42.36 (+/-11), respectively, where ACE inhibition was determined with 80 microl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from alpha-, beta-, and kappa-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to beta-casein f(73-89); peptide IGSENSEKTTMP, corresponding to alpha(s1)-casein f(201212); peptide SQSKVLPVPQ, corresponding to beta-casein f(166-175); peptide MPFPKYPVEP, corresponding to beta-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to beta-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC50 values in the range of 92 microM to 790 microM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract. [Abstract/Link to Full Text]

Hou S, Burton EA, Simon KA, Blodgett D, Luk YY, Ren D
Inhibition of Escherichia coli biofilm formation by self-assembled monolayers of functional alkanethiols on gold.
Appl Environ Microbiol. 2007 Jul;73(13):4300-7.
Bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. To further understand bacterium-surface interactions and to develop efficient control strategies, self-assembled monolayers (SAMs) of alkanethiols presenting different functional groups on gold films were analyzed to determine their resistance to biofilm formation. Escherichia coli was labeled with green florescence protein, and its biofilm formation on SAM-modified surfaces was monitored by confocal laser scanning microscopy. The three-dimensional structures of biofilms were analyzed with the COMSTAT software to obtain information about biofilm thickness and surface coverage. SAMs presenting methyl, L-gulonamide (a sugar alcohol tethered with an amide bond), and tri(ethylene glycol) (TEG) groups were tested. Among these, the TEG-terminated SAM was the most resistant to E. coli biofilm formation; e.g., it repressed biofilm formation by E. coli DH5alpha by 99.5% +/- 0.1% for 1 day compared to the biofilm formation on a bare gold surface. When surfaces were patterned with regions consisting of methyl-terminated SAMs surrounded by TEG-terminated SAMs, E. coli formed biofilms only on methyl-terminated patterns. Addition of TEG as a free molecule to growth medium at concentrations of 0.1 and 1.0% also inhibited biofilm formation, while TEG at concentrations up to 1.5% did not have any noticeable effects on cell growth. The results of this study suggest that the reduction in biofilm formation on surfaces modified with TEG-terminated SAMs is a result of multiple factors, including the solvent structure at the interface, the chemorepellent nature of TEG, and the inhibitory effect of TEG on cell motility. [Abstract/Link to Full Text]

Quir�s C, Herrero M, Garc�a LA, D�az M
Application of flow cytometry to segregated kinetic modeling based on the physiological states of microorganisms.
Appl Environ Microbiol. 2007 Jun;73(12):3993-4000.
Flow cytometry (FC) has been introduced to characterize and to assess the physiological states of microorganisms in conjunction with the classical plate-counting method. To show the applicability of the technique, in particular for the development of kinetic models, pure culture fermentation experiments were followed over time, using both prokaryotic (Lactobacillus hilgardii) and eukaryotic (Saccharomyces cerevisiae) microorganisms growing in standard culture media (MRS and YPD). The differences observed between the active and viable cells determined by FC and CFU, respectively, allowed us to determine that a large number of cells were in a viable but nonculturable (VBNC) state, which resulted in a subpopulation much larger than the damaged-cell (double-stained) subpopulation. Finally, the determination of the evolution of viable, the VBNC, and the dead cells allowed us to develop a segregated kinetic model to describe the yeast and the bacteria population dynamics and glucose consumption in batch cultures. This model, more complete than that which is traditionally used, based only on viable cell measurements, describes better the behavior and the functionality of the cultures, giving a deeper knowledge in real time about the status and the course of the bioprocesses. [Abstract/Link to Full Text]

Graczyk TK, Sunderland D, Tamang L, Shields TM, Lucy FE, Breysse PN
Quantitative evaluation of the impact of bather density on levels of human-virulent microsporidian spores in recreational water.
Appl Environ Microbiol. 2007 Jul;73(13):4095-9.
Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively. Enterocytozoon bieneusi spores were detected in 59% of weekend (n = 27) and 30% of weekday (n = 33) samples, and Encephalitozoon intestinalis spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples; P values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with P values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with Enterocytozoon bieneusi spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores. [Abstract/Link to Full Text]

Kothary MH, McCardell BA, Frazar CD, Deer D, Tall BD
Characterization of the zinc-containing metalloprotease encoded by zpx and development of a species-specific detection method for Enterobacter sakazakii.
Appl Environ Microbiol. 2007 Jul;73(13):4142-51.
Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused "rounding" of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37 degrees C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135 E. sakazakii strains possessed the metalloprotease gene, zpx, and 25 non-E. sakazakii strains did not. The cloned zpx gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases. [Abstract/Link to Full Text]

Boczek LA, Rice EW, Johnston B, Johnson JR
Occurrence of antibiotic-resistant uropathogenic Escherichia coli clonal group A in wastewater effluents.
Appl Environ Microbiol. 2007 Jul;73(13):4180-4.
Isolates of Escherichia coli belonging to clonal group A (CGA), a recently described disseminated cause of drug-resistant urinary tract infections in humans, were present in four of seven sewage effluents collected from geographically dispersed areas of the United States. All 15 CGA isolates (1% of the 1,484 isolates analyzed) exhibited resistance to trimethoprim-sulfamethoxazole (TMP-SMZ), accounting for 19.5% of the 77 TMP-SMZ-resistant isolates. Antimicrobial resistance patterns, virulence traits, O:H serotypes, and phylogenetic groupings were compared for CGA and selected non-CGA isolates. The CGA isolates exhibited a wider diversity of resistance profiles and somatic antigens than that found in most previous characterizations of this clonal group. This is the first report of recovery from outside a human host of E. coli CGA isolates with virulence factor and antibiotic resistance profiles typical of CGA isolates from a human source. The occurrence of "human-type" CGA in wastewater effluents demonstrates a potential mode for the dissemination of this clonal group in the environment, with possible secondary transmission to new human or animal hosts. [Abstract/Link to Full Text]

Graczyk TK, Sunderland D, Rule AM, da Silva AJ, Moura IN, Tamang L, Girouard AS, Schwab KJ, Breysse PN
Urban feral pigeons (Columba livia) as a source for air- and waterborne contamination with Enterocytozoon bieneusi spores.
Appl Environ Microbiol. 2007 Jul;73(13):4357-8.
This study demonstrated that a person with 30 min of occupational or nonoccupational exposure to urban feral pigeons, such as exposure through the cleaning of surfaces contaminated with pigeon excrement, could inhale approximately 3.5 x 10(3) Enterocytozoon bieneusi spores and that 1.3 x 10(3) spores could be inhaled by a nearby person. [Abstract/Link to Full Text]

Schlesinger DJ, Shoemaker NB, Salyers AA
Possible origins of CTnBST, a conjugative transposon found recently in a human colonic Bacteroides strain.
Appl Environ Microbiol. 2007 Jul;73(13):4226-33.
A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species. [Abstract/Link to Full Text]

Chuang AS, Mattes TE
Identification of polypeptides expressed in response to vinyl chloride, ethene, and epoxyethane in Nocardioides sp. strain JS614 by using peptide mass fingerprinting.
Appl Environ Microbiol. 2007 Jul;73(13):4368-72.
Enzymes expressed in response to vinyl chloride, ethene, and epoxyethane by Nocardioides sp. strain JS614 were identified by using a peptide mass fingerprinting (PMF) approach. PMF provided insight concerning vinyl chloride biodegradation in strain JS614 and extends the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry as a tool to enhance characterization of biodegradation pathways. [Abstract/Link to Full Text]

Lee J, Bansal T, Jayaraman A, Bentley WE, Wood TK
Enterohemorrhagic Escherichia coli biofilms are inhibited by 7-hydroxyindole and stimulated by isatin.
Appl Environ Microbiol. 2007 Jul;73(13):4100-9.
Since indole is present at up to 500 microM in the stationary phase and is an interspecies biofilm signal (J. Lee, A. Jayaraman, and T. K. Wood, BMC Microbiol. 7:42, 2007), we investigated hydroxyindoles as biofilm signals and found them also to be nontoxic interspecies biofilm signals for enterohemorrhagic Escherichia coli O157:H7 (EHEC), E. coli K-12, and Pseudomonas aeruginosa. The genetic basis of EHEC biofilm formation was also explored, and notably, virulence genes in biofilm cells were repressed compared to those in planktonic cells. In Luria-Bertani medium (LB) on polystyrene with quiescent conditions, 7-hydroxyindole decreased EHEC biofilm formation 27-fold and decreased K-12 biofilm formation 8-fold without affecting the growth of planktonic cells. 5-Hydroxyindole also decreased biofilm formation 11-fold for EHEC and 6-fold for K-12. In contrast, isatin (indole-2,3-dione) increased biofilm formation fourfold for EHEC, while it had no effect for K-12. When continuous-flow chambers were used, confocal microscopy revealed that EHEC biofilm formation was reduced 6-fold by indole and 10-fold by 7-hydroxyindole in LB. Whole-transcriptome analysis revealed that isatin represses indole synthesis by repressing tnaABC 7- to 37-fold in EHEC, and extracellular indole levels were found to be 20-fold lower. Furthermore, isatin repressed the AI-2 transporters lsrABCDFGKR, while significantly inducing the flagellar genes flgABCDEFGHIJK and fliAEFGILMNOPQ (which led to a 50% increase in motility). 7-Hydroxyindole induces the biofilm inhibitor/stress regulator ycfR and represses cysADIJPU/fliC (which led to a 50% reduction in motility) and purBCDEFHKLMNRT. Isogenic mutants showed that 7-hydroxyindole inhibits E. coli biofilm through cysteine metabolism. 7-Hydroxyindole (500 microM) also stimulates P. aeruginosa PAO1 biofilm formation twofold; therefore, hydroxyindoles are interspecies bacterial signals, and 7-hydroxyindole is a potent EHEC biofilm inhibitor. [Abstract/Link to Full Text]

Recent Articles in BMC Immunology

Su DM, Manley NR
Stage-specific changes in fetal thymocyte proliferation during the CD4-8- to CD4+8+ transition in wild type, Rag1-/-, and Hoxa3,Pax1 mutant mice.
BMC Immunol. 2002 Sep 19;312.
BACKGROUND: The function of the thymic microenvironment is to promote thymocyte maturation, in part via regulation of thymocyte proliferation and cell death. Defects in fetal thymic epithelial cell (TEC) development and function, and therefore in the formation of a functional microenvironment, can be caused either directly by TEC differentiation defects or indirectly by defective thymocyte maturation. In this paper we studied fetal thymocyte proliferation during the early transition from the CD3-4-8- (triple negative, TN) to CD4+8+ (double positive, DP) stages. We compared wild type mice with Rag1-/- mice and with Hoxa3+/-Pax1-/- compound mutant mice, which have blocks at different stages of thymocyte development. RESULTS: Wild type fetal and adult thymus showed stage-specific differences in the proliferation profiles of developing thymocytes, with fetal stages showing generally higher levels of proliferation. The proliferation profile of fetal thymocytes from Rag1-/- mutants also had stage-specific increases in proliferation compared to wild type fetal thymocytes, in contrast to the lower proliferation previously reported for thymocytes from adult Rag1-/- mutants. We have previously shown that Hoxa3+/-Pax1-/- mice have abnormal fetal TEC development, resulting in increased apoptosis at the TN to DP transition and decreased DP cell numbers. Fetal thymocytes from Hoxa3+/-Pax1-/- compound mutants had increased proliferation, but fewer proliferating cells, at the DP stage. We also observed a decrease in the level of the cytokines IL-7 and SCF produced by Hoxa3+/-Pax1-/-TECs. CONCLUSION: Our results indicate complex and stage-specific effects of abnormal TEC development on thymocyte proliferation. [Abstract/Link to Full Text]

Hofmann N, Lachnit N, Streppel M, Witter B, Neiss WF, Guntinas-Lichius O, Angelov DN
Increased expression of ICAM-1, VCAM-1, MCP-1, and MIP-1 alpha by spinal perivascular macrophages during experimental allergic encephalomyelitis in rats.
BMC Immunol. 2002 Aug 26;311.
BACKGROUND: T-cells extravasation and CNS parenchyma infiltration during autoimmune neurodegenerative disease can be evoked by local antigen presenting cells. Studying the chemoattracting potential of spinal perivascular macrophages (SPM) during experimental allergic encephalomyelitis (EAE), we observed numerous infiltrates of densely-packed mononuclear cells. Apart from the poor spatial and optical resolution, no differentiation between the resident SPM (mabs ED1+, ED2+) and the just recruited monocytes/macrophages (mab ED1+) was possible. RESULTS: This is why we labeled SPM by injections of different fluoresecent dyes into the lateral cerebral ventricle before induction of active EAE. Within an additional experimental set EAE was induced by an intraperitoneal injection of T-cells specifically sensitized to myelin basic protein (MBP) and engineered to express the green fluorescent protein (GFP). In both experiments we observed a strong activation of SPM (mabs OX6+, SILK6+, CD40+, CD80+, CD86+) which was accompanied by a consistently increased expression of ICAM-1, VCAM-1, and the chemokines MCP-1 and MIP-1alpha. CONCLUSION: These observations indicate that SPM play a role in promoting lymphocyte extravasation. [Abstract/Link to Full Text]

Allen CE, Mak CH, Wu LC
The kappa B transcriptional enhancer motif and signal sequences of V(D)J recombination are targets for the zinc finger protein HIVEP3/KRC: a site selection amplification binding study.
BMC Immunol. 2002 Aug 22;310.
BACKGROUND: The ZAS family is composed of proteins that regulate transcription via specific gene regulatory elements. The amino-DNA binding domain (ZAS-N) and the carboxyl-DNA binding domain (ZAS-C) of a representative family member, named kappaB DNA binding and recognition component (KRC), were expressed as fusion proteins and their target DNA sequences were elucidated by site selection amplification binding assays, followed by cloning and DNA sequencing. The fusion proteins-selected DNA sequences were analyzed by the MEME and MAST computer programs to obtain consensus motifs and DNA elements bound by the ZAS domains. RESULTS: Both fusion proteins selected sequences that were similar to the kappaB motif or the canonical elements of the V(D)J recombination signal sequences (RSS) from a pool of degenerate oligonucleotides. Specifically, the ZAS-N domain selected sequences similar to the canonical RSS nonamer, while ZAS-C domain selected sequences similar to the canonical RSS heptamer. In addition, both KRC fusion proteins selected oligonucleoties with sequences identical to heptamer and nonamer sequences within endogenous RSS. CONCLUSIONS: The RSS are cis-acting DNA motifs which are essential for V(D)J recombination of antigen receptor genes. Due to its specific binding affinity for RSS and kappaB-like transcription enhancer motifs, we hypothesize that KRC may be involved in the regulation of V(D)J recombination. [Abstract/Link to Full Text]

Nirmala R, Narayanan PR
Flow cytometry--a rapid tool to correlate functional activities of human peripheral blood lymphocytes with their corresponding phenotypes after in vitro stimulation.
BMC Immunol. 2002 Aug 7;39.
BACKGROUND: While dealing with mixed in vitro lymphocyte cultures one is faced with the problem of relative contributions of different populations to the activity being studied. This is especially true in the controversy relating to the contributions of lymphocyte sub-populations to the Lymphokine Activated Killer (LAK) phenomenon. Flowcytometry can be used to highlight relative contributions of lymphocyte subpopulations towards LAK activity without resorting to difficult purification strategies. We set up long-term in vitro lymphocyte cultures, stimulated them with cytokines IL-2/IL-12, recorded their phenotypic changes and cytotoxic activity against U-937 tumor targets. RESULTS: The results indicated that natural killer cells (NK) constituted the predominant proliferating cell population in the cytokine stimulatedcultures. Flowcytometric evidence revealed that CD56+ T cells contributed little to LAK activity against U937 target cells as compared to cells with NK phenotype which were predominantly responsible for spontaneous killing of the tumor targets. The two cytokines, IL-2 and IL-12, had an additive effect on cell proliferation and spontaneous cytotoxicity. CONCLUSION: Flowcytometry can be used to rapidly delineate phenotypic changes in immune cells after stimulation and simultaneously correlate them with corresponding functional activity. This approach may find application as a initial screening tool for studying different types of cells in mixed cultures and their respective activities under stimulatory / inhibitory conditions. [Abstract/Link to Full Text]

Dutra WO, Correa-Oliveira R, Dunne D, Cecchini LF, Fraga L, Roberts M, Soares-Silveira AM, Webster M, Yssel H, Gollob KJ
Polarized Th2 like cells, in the absence of Th0 cells, are responsible for lymphocyte produced IL-4 in high IgE-producer schistosomiasis patients.
BMC Immunol. 2002 Jul 6;38.
BACKGROUND: Human resistance to re-infection with S. mansoni is correlated with high levels of anti-soluble adult worm antigens (SWAP) IgE. Although it has been shown that IL-4 and IL-5 are crucial in establishing IgE responses in vitro, the active in vivo production of these cytokines by T cells, and the degree of polarization of Th2 vs. Th0 in human schistosomiasis is not known. To address this question, we determined the frequency of IL-4 and IFN-gamma or IL-5 and IL-2 producing lymphocytes from schistosomiasis patients with high or low levels of IgE anti-SWAP. RESULTS: Our analysis showed that high and low IgE-producers responded equally to schistosomiasis antigens as determined by proliferation. Moreover, patients from both groups displayed similar percentages of circulating lymphocytes. However, high IgE-producers had an increased percentage of activated CD4+ T cells as compared to the low IgE-producers. Moreover, intracellular cytokine analysis, after short-term stimulation with anti-CD3/CD28 mAbs, showed that IgE high-producers display an increase in the percentage of T lymphocytes expressing IL-4 and IL-5 as compared to IgE low-responders. A coordinate control of the frequency of IL-4 and IL-5 producing lymphocytes in IgE high, but not IgE low-responders, was observed. CONCLUSIONS: High IgE phenotype human schistosomiasis patients exhibit a coordinate regulation of IL-4 and IL-5 producing cells and the lymphocyte derived IL-4 comes from true polarized Th2 like cells, in the absence of measurable Th0 cells as measured by co-production of IL-4 and IFN-gamma. [Abstract/Link to Full Text]

Loke P, Nair MG, Parkinson J, Guiliano D, Blaxter M, Allen JE
IL-4 dependent alternatively-activated macrophages have a distinctive in vivo gene expression phenotype.
BMC Immunol. 2002 Jul 4;37.
BACKGROUND: "Alternatively-activated" macrophages are found in Th2-mediated inflammatory settings such as nematode infection and allergic pulmonary inflammation. Due in part to a lack of markers, these cells have not been well characterized in vivo and their function remains unknown. RESULTS: We have used murine macrophages elicited by nematode infection (NeM(phi)) as a source of in vivo derived alternatively activated macrophages. Using three distinct yet complementary molecular approaches we have established a gene expression profile of alternatively activated macrophages and identified macrophage genes that are regulated in vivo by IL-4. First, genes abundantly expressed were identified by an expressed sequence tag strategy. Second, an array of 1176 known mouse genes was screened for differential expression between NeM(phi) from wild type or IL-4 deficient mice. Third, a subtractive library was screened to identify novel IL-4 dependent macrophage genes. Differential expression was confirmed by real time RT-PCR analysis. CONCLUSIONS: Our data demonstrate that alternatively activated macrophages generated in vivo have a gene expression profile distinct from any macrophage population described to date. Several of the genes we identified, including those most abundantly expressed, have not previously been associated with macrophages and thus this study provides unique new information regarding the phenotype of macrophages found in Th2-mediated, chronic inflammatory settings. Our data also provide additional in vivo evidence for parallels between the inflammatory processes involved in nematode infection and allergy. [Abstract/Link to Full Text]

Iribarren P, Correa SG, Sodero N, Riera CM
Activation of macrophages by silicones: phenotype and production of oxidant metabolites.
BMC Immunol. 2002 Jul 1;36.
BACKGROUND: The effect of silicones on the immune function is not fully characterized. In clinical and experimental studies, immune alterations associated with silicone gel seem to be related to macrophage activation. In this work we examined in vivo, phenotypic and functional changes on peritoneal macrophages early (24 h or 48 h) and late (45 days) after the intraperitoneal (i.p.) injection of dimethylpolysiloxane (DMPS) (silicone). We studied the expression of adhesion and co-stimulatory molecules and both the spontaneous and the stimulated production of reactive oxygen intermediates and nitric oxide (NO). RESULTS: The results presented here demonstrate that the fluid compound DMPS induced a persistent cell recruitment at the site of the injection. Besides, cell activation was still evident 45 days after the silicone injection: activated macrophages exhibited an increased expression of adhesion (CD54 and CD44) and co-stimulatory molecules (CD86) and an enhanced production of oxidant metabolites and NO. CONCLUSIONS: Silicones induced a persistent recruitment of leukocytes at the site of the injection and macrophage activation was still evident 45 days after the injection. [Abstract/Link to Full Text]

Sayama K, Diehn M, Matsuda K, Lunderius C, Tsai M, Tam SY, Botstein D, Brown PO, Galli SJ
Transcriptional response of human mast cells stimulated via the Fc(epsilon)RI and identification of mast cells as a source of IL-11.
BMC Immunol. 2002 Jun 12;35.
BACKGROUND: In asthma and other allergic disorders, the activation of mast cells by IgE and antigen induces the cells to release histamine and other mediators of inflammation, as well as to produce certain cytokines and chemokines. To search for new mast cell products, we used complementary DNA microarrays to analyze gene expression in human umbilical cord blood-derived mast cells stimulated via the high-affinity IgE receptor (Fc(epsilon)RI). RESULTS: One to two hours after Fc(epsilon)RI-dependent stimulation, more than 2,400 genes (about half of which are of unknown function) exhibited 2-200 fold changes in expression. The transcriptional program included changes in the expression of IL-11 and at least 30 other cytokines and chemokines. Human mast cells secreted 130-529 pg of IL-11/106 cells by 6 h after stimulation with anti-IgE. CONCLUSION: Our initial analysis of the transcriptional program induced in in vitro-derived human mast cells stimulated via the Fc(epsilon)RI has identified many products that heretofore have not been associated with this cell type, but which may significantly influence mast cell function in IgE-associated host responses. We also have demonstrated that mast cells stimulated via the Fc(epsilon)RI can secrete IL-11. Based on the previously reported biological effects of IL-11, our results suggest that production of IL-11 may represent one link between IgE-dependent mast cell activation in subjects with allergic asthma and the development of a spectrum of structural changes in the airways of these individuals; such changes, collectively termed "airway remodeling," can constitute an important long term consequence of asthma. [Abstract/Link to Full Text]

Hurez V, Dzialo-Hatton R, Oliver J, Matthews RJ, Weaver CT
Efficient adenovirus-mediated gene transfer into primary T cells and thymocytes in a new coxsackie/adenovirus receptor transgenic model.
BMC Immunol. 2002 May 2;34.
BACKGROUND: Gene transfer studies in primary T cells have suffered from the limitations of conventional viral transduction or transfection techniques. Replication-defective adenoviral vectors are an attractive alternative for gene delivery. However, naive lymphocytes are not readily susceptible to infection with adenoviruses due to insufficient expression of the coxsackie/adenovirus receptor. RESULTS: To render T cells susceptible to adenoviral gene transfer, we have developed three new murine transgenic lines in which expression of the human coxsackie/adenovirus receptor (hCAR) with a truncated cytoplasmic domain (hCAR(Delta)cyt) is limited to thymocytes and lymphocytes under direction of a human CD2 mini-gene. hCAR(Delta)cyt.CD2 transgenic mice were crossed with DO11.10 T cell receptor transgenic mice (DO11.hCAR(Delta)cyt) to allow developmental studies in a defined, clonal T cell population. Expression of hCAR(Delta)cyt enabled adenoviral transduction of resting primary CD4+ T cells, differentiated effector T cells and thymocytes from DO11.hCAR(Delta)cyt with high efficiency. Expression of hCAR(Delta)cyt transgene did not perturb T cell development in these mice and adenoviral transduction of DO11.hCAR(Delta)cyt T cells did not alter their activation status, functional responses or differentiative potential. Adoptive transfer of the transduced T cells into normal recipients did not modify their physiologic localization. CONCLUSION: The DO11.hCAR(Delta)cyt transgenic model thus allows efficient gene transfer in primary T cell populations and will be valuable for novel studies of T cell activation and differentiation. [Abstract/Link to Full Text]

Ferret PJ, Soum E, Negre O, Fradelizi D
Auto-protective redox buffering systems in stimulated macrophages.
BMC Immunol. 2002 Mar 12;33.
BACKGROUND: Macrophages, upon encounter with micro-organisms or stimulated by cytokines, produce various effector molecules aimed at destroying the foreign agents and protecting the organism. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are front line molecules exerting strong cytotoxic activities against micro-organisms and many cells, including macrophages themselves. Using cells of the murine macrophage cell line (RAW 264.7) stimulated in vitro with lipopolysaccharide (LPS) and/or interferon (IFN-gamma), which induce strong endogenous NO production, we examined by which mechanisms a fraction of activated macrophages protect themselves from nitrosative stress and manage to escape destruction? RESULTS: We observed that survivors (10-50% depending on the experiments) had acquired a resistant phenotype being capable to survive when further exposed in vitro to an apoptosis inducing dose of the NO donor compound DETA-NO. These cells expressed an increased steady-state levels of Mn SOD, CuZn SOD and catalase mRNA (130-200%), together with an increased activity of the corresponding enzymes. Intracellular concentration of glutathione was also increased (x 3.5 fold at 6 hours, still maintained x 5.2 fold at 48 hours). Neither mRNA for glutathione peroxydase, gamma-glutamylcysteine synthase and glutathione reductase, nor thioredoxine and thioredoxine reductase, were significantly modified. Additional experiments in which RAW 264.7 cells were stimulated with LPS and/or IFN-gamma in the presence of relatively specific inhibitors of both Mn and Cu/Zn SOD, aminotriazol (ATZ) catalase inhibitor and buthionine sulfoximine (BSO) glutathione inhibitor, showed that inhibiting LPS-induced up-regulation of intracellular redox buffering systems also prevented acquisition of the resistant phenotype. CONCLUSIONS: Our data suggest a direct causal relationship between survival of a fraction of macrophages and a up-regulation of key sets of auto-protective intracellular redox buffering systems, occurring simultaneously with modulation of expression of apoptotic molecules of the Bcl2-Bcl-XL/Bax-Bad family. [Abstract/Link to Full Text]

Mucha JM, Stickler MM, Poulose AJ, Ganshaw G, Saldajeno M, Collier K, Huang MT, Harding FA
Enhanced immunogenicity of a functional enzyme by T cell epitope modification.
BMC Immunol. 2002;32.
BACKGROUND: T helper epitopes are necessary for the induction of high titers of antigen-specific IgG antibodies. We are interested in the epitope modification of intact proteins as a method to enhance their immunogenicity for the generation of recombinant protein-based vaccines. RESULTS: Hartley strain guinea pig T cell epitopes were mapped for two related bacterial proteases. Two T cell epitopes were found in one of the proteases, while a comparatively reduced immunogenicity protease had no detectable T cell epitopes. A T cell epitope sequence homologous to the immunogenic protease was created in the less immunogenic protease by changing a single amino acid. Proliferative responses to the whole protein parent enzyme were two-fold higher in splenocyte cultures from variant-immunized animals. We found that the single amino acid change in the variant resulted in a protein immunogen that induced higher titers of antigen-specific IgG antibody at low doses and at early time points during the immunization protocol. The serum from parent- and variant-immunized guinea pigs cross-reacted at both the protein and the peptide level. Finally, animals primed to the variant but boosted with the parent enzyme had higher levels of antigen-specific IgG than animals immunized with the parent enzyme alone. CONCLUSIONS: With a single amino acid change we have introduced a T cell epitope into a comparatively low-immunogenic enzyme and have increased its immunogenicity while retaining the enzyme's original proteolytic function. The ability to immunomodulate proteins while leaving their function intact has important implication for the development of recombinant vaccines and protein-based therapeutics. [Abstract/Link to Full Text]

Martinez-Valdez H, Madrid-Marina V, Cohen A
Phorbol esters and cAMP differentially regulate the expression of CD4 and CD8 in human thymocytes.
BMC Immunol. 2002;31.
BACKGROUND: Intrathymic development and selection of the T lymphocyte repertoire is restricted by the interactions of the T cell antigen receptor and CD4 or CD8 co-receptors with self major histocompatibility complex molecules. Positive or negative selection depends on a tight regulatory control of CD4 and CD8 expression. Determining the intracellular signals that differentially regulate the expression of CD4 and CD8 is important to understand the mechanisms that are implicated in selection of single positive CD4+CD8- or CD4-CD8+. RESULTS: The present study shows that stimulation of human thymocytes by phorbol esters or cAMP result in a differential regulation of CD4 and CD8 expression, both at the mRNA and cell surface glycoprotein level. CONCLUSIONS: The differential regulation of CD4 and CD8 gene expression suggests that the selective activation of protein kinase C (PKC) and cAMP-dependent protein kinases (PKA) may be required for the selection of single positive CD4+CD8- and CD4-CD8+ cells during Intrathymic differentiation. [Abstract/Link to Full Text]

Eylar EH, Lefranc CE, Yamamura Y, B�ez I, Col�n-Martinez SL, Rodriguez N, Breithaupt TB
HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8+ CD28- T subset.
BMC Immunol. 2001;210.
BACKGROUND: T cells from HIV+ and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8+ CD28- T cells. In order to compare cytokine production from T cells from these two states, CD4+ and CD8+ T cells from HIV+ aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8+ T cell subsets CD28+ and CD28- from the HIV+ and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls. RESULTS: Flow cytometric analysis indicated that CD8+ T cells from both HIV+ and aged donors showed an increase of approximately 2-3 fold over controls in percentage of cells producing inflammatory cytokines IFN-gamma and TNF-alpha. Similar analysis also revealed that the production of interleukins-4,6 and 10, production was very low (1-2% of cells) and unchanged in these cells. Quantitative PCR also showed a substantial increase (4-5 fold) in IFN-gamma and TNF-alpha mRNA from HIV+ and aged CD8+ T cells, as did ELISA for secreted IFN-gamma and TNF-alpha (2.3-4 fold). Flow cytometric analysis showed that the CD8+ CD28- T cell subset accounts for approximately 80-86% of the IFN-gamma and TNF-alpha production from the CD8+ subset in the aged and HIV+ states. The CD4+ T cell, while not significantly changed in the HIV+ or aged states in terms of IFN-gamma production, showed a small but significant increase in TNF-alpha production in both states. CONCLUSIONS: Our data appear compatible with physiologic conditions existing in HIV+ and aged individuals, i.e. elevated serum levels and elevated CD8+ T cell production of IFN-gamma and TNF-alpha. Thus, the capacity for increased production of cytokines IFN-gamma and TNF-alpha in the aged individual by the dominant CD8+ CD28- subset may have a profound influence on the clinical state by aggravating inflammatory pathologies such as rheumatoid arthritis, and possibly Alzheimer's disease and Crohn's disease. In AIDS, these cytokines may contribute to wasting and cachexia. We theorize that the predominant phenotypic change to the cytotoxic CD8+ CD28- T cell subsets in both the HIV+ and the aged states may reflect a natural "endpoint" in CD8+ T cell differentiation induced after a lifetime of immune activity (toward viruses, etc) in the aged, and after a massive accelerated response to HIV in the HIV-positive individual. [Abstract/Link to Full Text]

Hume DA, Underhill DM, Sweet MJ, Ozinsky AO, Liew FY, Aderem A
Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state.
BMC Immunol. 2001;211.
BACKGROUND: Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. RESULTS: RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1beta promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. CONCLUSIONS: These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge. [Abstract/Link to Full Text]

Brown DC, Larson RS
Improvements to parallel plate flow chambers to reduce reagent and cellular requirements.
BMC Immunol. 2001;29.
BACKGROUND: The parallel plate flow chamber has become a mainstay for examination of leukocytes under physiologic flow conditions. Several design modifications have occurred over the years, yet a comparison of these different designs has not been performed. In addition, the reagent requirements of many designs prohibit the study of rare leukocyte populations and require large amounts of reagents. RESULTS: In this study, we evaluate modifications to a newer parallel plate flow chamber design in comparison to the original parallel plate flow chamber described by Lawrence et al. We show that modifications in the chamber size, internal tubing diameters, injection valves, and a recirculation design may dramatically reduce the cellular and reagent requirements without altering measurements. CONCLUSIONS: These modifications are simple and easily implemented so that study of rare leukocyte subsets using scarce or expensive reagents can occur. [Abstract/Link to Full Text]

Liu SZ, Jin SZ, Liu XD, Sun YM
Role of CD28/B7 costimulation and IL-12/IL-10 interaction in the radiation-induced immune changes.
BMC Immunol. 2001;28.
BACKGROUND: The present paper aims at studying the role of B7/CD28 interaction and related cytokine production in the immunological changes after exposure to different doses of ionizing radiation. RESULTS: The stimulatory effect of low dose radiation (LDR) on the proliferative response of lymphocytes to Con A was found to require the presence of APCs. The addition of APCs obtained from both low- and high-dose-irradiated mice to splenic lymphocytes separated from low-dose-irradiated mice caused stimulation of lymphocyte proliferation. B7-1/2 expression on APCs was up-regulated after both low and high doses of radiation. There was up-regulation of CD28 expression on splenic and thymic lymphocytes after LDR and its suppression after high dose radiation (HDR), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression showed changes in the opposite direction. IL-12 secretion by macrophages was stimulated after both low and high doses of radiation, but IL-10 synthesis by splenocytes was suppressed by low dose radiation and up-regulated by high dose radiation. CONCLUSION: The status of CD28/CTLA-4 expression on T lymphocytes in the presence of up-regulated B7 expression on APCs determined the outcome of the immune changes in response to radiation, i.e., up-regulation of CD28 after LDR resulted in immunoenhancement, and up-regulation of CTLA-4 associated with down-regulation of CD28 after HDR led to immunosuppression. Both low and high doses of radiation up-regulated B7-1/2 expression on APCs. After LDR, the stimulated proliferative effect of increased IL-12 secretion by APCs, reinforced by the suppressed secretion of IL-10, further strengthened the intracellular signaling induced by B7-CD28 interaction. [Abstract/Link to Full Text]

Dom�nguez-Gerpe L, Rey-M�ndez M
Alterations induced by chronic stress in lymphocyte subsets of blood and primary and secondary immune organs of mice.
BMC Immunol. 2001;27.
BACKGROUND: The immune system is particularly sensitive to stress. Although acute stress generally has positive effects, chronic stress typically provokes immunosuppression. The elucidation of the mechanisms involved in immunosuppression are of interest for the design of therapeutic approaches to avoid the appearance of stress disorders. This study aimed to investigate chronic stress-induced alterations on lymphocyte subset distribution and percentages. The experiments were performed with C57BL/6 mice subjected to chronic immobilization stress. RESULTS: Stress caused a marked increase in apoptosis inside the thymus, and a reduction in the total number of thymocytes. Furthermore, the proportion of immature thymocytes declined significantly suggesting that the increased apoptosis mainly affected cells of immature phenotype. In blood, the total number of lymphocytes diminished but not all lymphocyte populations showed the same tendency: while the relative proportion of B cells declined slightly, the relative proportion of circulating CD3+ cells, and particularly some T cell subsets showing an immature phenotype (CD3+PNA+), increased under stress. The spleen and lymph nodes show a marked reduction in cellularity, but the relative proportion of T cells increased, while no change or only a slight reduction was observed in the relative proportion of B cells. Similarly, the relative proportion of T cells increased in bone marrow. CONCLUSIONS: Detailed data on the alterations of lymphoid cell subsets occurring under immobilization stress, both in the bloodstream and in different lymphoid tissues, are obtained. In general, T cells are more affected than B cells and, in particular, a marked increase in the percentage of a subset of circulating PNA+CD3+ T cells is observed. [Abstract/Link to Full Text]

Nunez R, Filgueira L
Flow cytometric assessment of the reactivity of a panel of monoclonal antibodies (mAb) against two populations of human dendritic cells (DC).
BMC Immunol. 2001;26.
BACKGROUND: The identification of antigens on human DC has been a very difficult and elusive task because of the lack of appropriate reagents. Therefore, we evaluated by flow cytometry a panel of mAb that recognize antigens on human DC, aiming to determine the kinetics of DC antigen expression at 7, 14, 21 and 28 days in (i) Dermal DC like cells (Mo-DC) and (ii) Langerhans cell like DC (Mo-LC). In addition we aimed to identify markers for DC subpopulations. RESULTS: It was found at day 7, that mAb BG6, HP-F1, BU10, RFD-1, CMRF-44 recognized <20% of Mo-DC. In contrast, 7H5, ZM3.8, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-DC. Moreover, 7H5, ZM3.8, CMRF-56, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 showed increased MFI reactivity against Mo-DC. mAb BG6, BU10 and CMRF-44 recognized <20% Mo-LC while RFD-1 reacted with 21% of Mo-LC. In contrast, HP-F1 showed 87% of Mo-LC positive. Also, 7H5, ZM3.8, RFD-7, MR15-2, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-LC. The increase in % of positive cells was paralleled by MFI increases.At day 14, fourteen mAb recognized >50% of the Mo-DC, while five recognized 20-50% of Mo-DC. BG6 reacted with 7% of the Mo-DC. Nineteen mAb recognized >48% of Mo-LC while BG6 had negative reactivity.At day 21 and 28, all mAb reacted with >20% of Mo-DC and yielded a significant MFI with Mo-DC. Also nineteen mAb yielded significant MFI with Mo-LC while RFD-7 did not. CONCLUSIONS: The immunophenotyping assays demonstrated differences between the two DC populations as well as variations in the reactivity of the mAb at diverse time points, suggesting the existence of subpopulations within the Mo-DC and Mo-LC. [Abstract/Link to Full Text]

Stevens J, Joly E, Trowsdale J, Butcher GW
Peptide binding characteristics of the non-classical class Ib MHC molecule HLA-E assessed by a recombinant random peptide approach.
BMC Immunol. 2001;25.
BACKGROUND: Increasing evidence suggests that the effect of HLA-E on Natural Killer (NK) cell activity can be affected by the nature of the peptides bound to this non-classical, MHC class Ib molecule. However, its reduced cell surface expression, and until recently, the lack of specific monoclonal antibodies hinder studying the peptide-binding specificity HLA-E. RESULTS: An in vitro refolding system was used to assess binding of recombinant HLA-E to either specific peptides or a nonamer random peptide library. Peptides eluted from HLA-E molecules refolded around the nonamer library were then used to determine a binding motif for HLA-E. Hydrophobic and non-charged amino acids were found to predominate along the peptide motif, with a leucine anchor at P9, but surprisingly there was no methionine preference at P2, as suggested by previous studies. CONCLUSIONS: Compared to the results obtained with rat classical class Ia MHC molecules, RT1-A1c and RT1-Au, HLA-E appears to refold around a random peptide library to reduced but detectable levels, suggesting that this molecule's specificity is tight but probably not as exquisite as has been previously suggested. This, and a previous report that it can associate with synthetic peptides carrying a viral sequence, suggests that HLA-E, similar to its mouse counterpart (Qa-1b), could possibly bind peptides different from MHC class I leader peptides and present them to T lymphocytes. [Abstract/Link to Full Text]

Tomlinson MG, Woods DB, McMahon M, Wahl MI, Witte ON, Kurosaki T, Bolen JB, Johnston JA
A conditional form of Bruton's tyrosine kinase is sufficient to activate multiple downstream signaling pathways via PLC Gamma 2 in B cells.
BMC Immunol. 2001;24.
BACKGROUND: Bruton's tyrosine kinase (Btk) is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-gamma2 (PLCgamma2) and calcium mobilization. However it is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCgamma2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCgamma2-deficient DT40 B cell line. RESULTS: Activation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCgamma2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCgamma2, mobilization of intracellular calcium, activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, and apoptosis. In DT40 B cells deficient for PLCgamma2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis. CONCLUSIONS: These data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCgamma2 in B cells. While it is not known whether activated Btk:ER precisely mimics activated Btk, this conditional system will likely facilitate the dissection of the role of Btk and its family members in a variety of biological processes in many different cell types. [Abstract/Link to Full Text]

Chamorro M, Czar MJ, Debnath J, Cheng G, Lenardo MJ, Varmus HE, Schwartzberg PL
Requirements for activation and RAFT localization of the T-lymphocyte kinase Rlk/Txk.
BMC Immunol. 2001;23.
BACKGROUND: The Tec family kinases are implicated in signaling from lymphocyte antigen receptors and are activated following phosphorylation by Src kinases. For most Tec kinases, this activation requires an interaction between their pleckstrin homology (PH) domains and the products of phosphoinositide 3-Kinase, which localizes Tec kinases to membrane RAFTs. Rlk/Txk is a Tec related kinase expressed in T cells that lacks a pleckstrin homology domain, having instead a palmitoylated cysteine-string motif. To evaluate Rlk's function in T cell receptor signaling cascades, we examined the requirements for Rlk localization and activation by Src family kinases. RESULTS: We demonstrate that Rlk is also associated with RAFTs, despite its lack of a pleckstrin homology domain. Rlk RAFT association requires the cysteine-string motif and is independent of PI3 Kinase activity. We further demonstrate that Rlk can be phosphorylated and activated by Src kinases, leading to a decrease in its half-life. A specific tyrosine in the activation loop of Rlk, Y420, is required for phosphorylation and activation, as well as for decreased stability, but is not required for lipid RAFT association. Mutation of this tyrosine also prevents increased tyrosine phosphorylation of Rlk after stimulation of the T cell receptor, suggesting that Rlk is phosphorylated by Src family kinases in response to T cell receptor engagement. CONCLUSIONS: Like the other related Tec kinases, Rlk is associated with lipid RAFTs and can be phosphorylated and activated by Src family kinases, supporting a role for Rlk in signaling downstream of Src kinases in T cell activation. [Abstract/Link to Full Text]

Vidal-Rubio B, Sanchez-Carril M, Oliver-Morales J, Gonz�lez-Femandez A, Gamb�n-Deza F
Changes in human lymphocyte subpopulations in tonsils and regional lymph nodes of human head and neck squamous carcinoma compared to control lymph nodes.
BMC Immunol. 2001;22.
BACKGROUND: Lymphoid tissues constitute basic structures where specific immune responses take place. This leads to the development of germinal centres (GCs), migration of cells and the generation of memory cells. Here, we have compared human tumour reactive lymph nodes and tonsils with control lymph nodes. RESULTS: The study by flow cytometry shows that in control lymph nodes the majority of cells were naive T-lymphocytes (CD45RA+/CD7+). In reactive nodes, although the percentage of CD45RO+ T cells remains constant, there is an increase in the number of B-lymphocytes, and a reduction in naive T cells. The percentage of cells expressing CD69 was similar in reactive nodes and in controls. In both cases, we have found two populations of B cells of either CD69- or CD69dull. Two populations of T cells, which are either negative for CD69 or express it in bright levels (CD69bright), were also found.The analysis of tissue sections by confocal microscopy revealed differences between control, tonsils and tumor reactive lymph nodes. In control lymph nodes, CD19 B cells are surrounded by a unique layer of CD69bright/CD45RO+ T cells. GCs from tonsils and from tumour reactive nodes are mainly constituted by CD19 B cells and have four distinct layers. The central zone is composed of CD69- B cells surrounded by CD69bright/CD45RO+ T cells. The mantle region has basically CD69dull B-lymphocytes and, finally, there is an outer zone with CD69-/CD45RO+ T cells. CONCLUSIONS: Human secondary lymphoid organs react with an increase in the proportion of B lymphocytes and a decrease in the number of CD45RA+ T cells (naive). In tonsils, this is due to chronic pathogen stimulation, whereas in lymph nodes draining head and neck carcinomas the reaction is prompted by surrounded tumors. During this process, secondary lymphoid organs develop secondary follicles with a special organization of T and B cells in consecutive layers, that are described here by confocal microscopy. This pattern of cellular distribution may suggest a model of cell migration into the secondary lymphoid follicles. [Abstract/Link to Full Text]

Torreilles J, Romestand B
In vitro production of peroxynitrite by haemocytes from marine bivalves: C-ELISA determination of 3-nitrotyrosine level in plasma proteins from Mytilus galloprovincialis and Crassostrea gigas.
BMC Immunol. 2001;21.
BACKGROUND: Peroxynitrite is increasingly proposed as a contributor to defence system in marine bivalve. It can be formed by combination of superoxide and nitric oxide, and can react with tyrosine residues of proteins giving rise to 3-nitrotyrosine. RESULTS: The present article describes a competitive ELISA for the measurement of 3-nitrotyrosine contents of plasma proteins from marine bivalves by means of a monoclonal anti 3-nitrotyrosine antibody mouse IgG. CONCLUSIONS: This assay is sensitive enough to determine the amounts of 3-nitrotyrosine in plasma proteins from one animal only.Using the C-ELISA, we have shown that the phagocytosis of zymosan particles increased the 3-nitrotyrosine levels of plasma proteins from mussel M. galloprovincialis and oyster C. gigas 5.8 and 7.5 times respectively. [Abstract/Link to Full Text]

Kim LJ, Ferguson HA, Seto AG, Goodrich JA
Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp.
BMC Immunol. 2000;11.
BACKGROUND: NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. RESULTS: We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. CONCLUSIONS: We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins. [Abstract/Link to Full Text]

Recent Articles in BMC Infectious Diseases

Echchannaoui H, Leib SL, Neumann U, Landmann RM
Adjuvant TACE inhibitor treatment improves the outcome of TLR2-/- mice with experimental pneumococcal meningitis.
BMC Infect Dis. 2007;725.
BACKGROUND: Streptococcus (S.) pneumoniae meningitis has a high lethality despite antibiotic treatment. Inflammation is a major pathogenetic factor, which is unresponsive to antibiotics. Therefore adjunctive therapies with antiinflammatory compounds have been developed. TNF484 is a TNF-alpha converting enzyme (TACE) inhibitor and has been found efficacious in experimental meningitis. Toll-like receptor 2 (TLR2) contributes to host response in pneumococcal meningitis by enhancing bacterial clearing and downmodulating inflammation. In this study, TNF484 was applied in mice, which lacked TLR2 and exhibited a strong meningeal inflammation. METHODS: 103 CFU S. pneumoniae serotype 3 was inoculated subarachnoidally into C57BL/6 wild type (wt) mice or TLR2-/-, CD14-/- and CD14-/-/TLR2-/- mice. Severity of disease and survival was followed over 9 days. Response to antibiotics (80 mg/kg ceftriaxone i.p. for 5 days) and/or TACE inhibitor treatment (1 mg/kg s.c. twice daily for 4 days) was evaluated. Animals were sacrificed after 12, 24, and 48 h for analysis of bacterial load in cerebrospinal fluid (CSF) and brain and for TNF and leukocyte measurements in CSF. RESULTS: TLR2-/- mice were significantly sicker than the other mouse strains 24 h after infection. All knockout mice showed higher disease severity after 48 h and died earlier than wt mice. TNF release into CSF was significantly more elevated in TLR2-/- than in the other strains after 24 h. Brain bacterial numbers were significantly higher in all knockout than wt mice after 24 h. Modulation of outcome by antibiotic and TACE inhibitor treatment was evaluated. With antibiotic therapy all wt, CD14-/- and TLR2-/-/CD14-/- mice, but only 79% of TLR2-/- mice, were rescued. TACE inhibitor treatment alone did not rescue, but prolonged survival in wt mice, and in TLR2-/- and CD14-/- mice to the values observed in untreated wt mice. By combined antibiotic and TACE inhibitor treatment 95% of TLR2-/- mice were rescued. CONCLUSION: During pneumococcal meningitis strong inflammation in TLR2-deficiency was associated with incomplete responsiveness to antibiotics and complete response to combined antibiotic and TACE inhibitor treatment. TACE inhibitor treatment offers a promising adjuvant therapeutic strategy in pneumococcal meningitis. [Abstract/Link to Full Text]

de Bruin MA, Riley LW
Does vancomycin prescribing intervention affect vancomycin-resistant enterococcus infection and colonization in hospitals? A systematic review.
BMC Infect Dis. 2007;724.
BACKGROUND: Vancomycin resistant enterococcus (VRE) is a major cause of nosocomial infections in the United States and may be associated with greater morbidity, mortality, and healthcare costs than vancomycin-susceptible enterococcus. Current guidelines for the control of VRE include prudent use of vancomycin. While vancomycin exposure appears to be a risk factor for VRE acquisition in individual patients, the effect of vancomycin usage at the population level is not known. We conducted a systematic review to determine the impact of reducing vancomycin use through prescribing interventions on the prevalence and incidence of VRE colonization and infection in hospitals within the United States. METHODS: To identify relevant studies, we searched three electronic databases, and hand searched selected journals. Thirteen studies from 12 articles met our inclusion criteria. Data were extracted and summarized for study setting, design, patient characteristics, types of intervention(s), and outcome measures. The relative risk, 95% confidence interval, and p-value associated with change in VRE acquisition pre- and post-vancomycin prescription interventions were calculated and compared. Heterogeneity in study results was formally explored by stratified analysis. RESULTS: No randomized clinical trials on this topic were found. Each of the 13 included studies used a quasi-experimental design of low hierarchy. Seven of the 13 studies reported statistically significant reductions in VRE acquisition following interventions, three studies reported no significant change, and three studies reported increases in VRE acquisition, one of which reported statistical significance. Results ranged from a reduction of 82.5% to an increase of 475%. Studies of specific wards, which included sicker patients, were more likely to report positive results than studies of an entire hospital including general inpatients (Fisher's exact test 0.029). The type of intervention, endemicity status, type of study design, and the duration of intervention were not found to significantly modify the results. Among the six studies that implemented vancomycin reduction strategies as the sole intervention, two of six (33%) found a significant reduction in VRE colonization and/or infection. In contrast, among studies implementing additional VRE control measures, five of seven (71%) reported a significant reduction. CONCLUSION: It was not possible to conclusively determine a potential role for vancomycin usage reductions in controlling VRE colonization and infection in hospitals in the United States. The effectiveness of such interventions and their sustainability remains poorly defined because of the heterogeneity and quality of studies. Future research using high-quality study designs and implementing vancomycin as the sole intervention are needed to answer this question. [Abstract/Link to Full Text]

Vicidomini S, Cancrini G, Gabrielli S, Naspetti R, Bartoloni A
Muscular cystic hydatidosis: case report.
BMC Infect Dis. 2007;723.
BACKGROUND: Hydatidosis is a zoonosis caused by Echinococcus granulosus, and ingesting eggs released through the faeces from infected dogs infects humans. The location of the hydatid cysts is mostly hepatic and/or pulmonary, whereas musculoskeletal hydatidosis is very rare. CASE PRESENTATION: We report an unusual case of primary muscular hydatidosis in proximity of the big adductor in a young Sicilian man. The patient, 34 years old, was admitted to the Department of Infectious and Tropical Diseases for ultrasonographic detection, with successive confirmation by magnetic resonance imaging, of an ovular mass (13 x 8 cm) in the big adductor of the left thigh, cyst-like, and containing several small cystic formations. Serological tests for hydatidosis gave negative results. A second drawing of blood was done 10 days after the first one and showed an increase in the antibody titer for hydatidosis. The patient was submitted to surgical excision of the lesion with perioperatory prophylaxis with albendazole. The histopathological examination of the bioptic material was not diriment in the diagnosis, therefore further tests were performed: additional serological tests for hydatidosis for the evaluation of IgE and IgG serotype (Western Blot and REAST), and molecular analysis of the excised material. These more specific serological tests gave positive results for hydatidosis, and the sequencing of the polymerase chain reaction products from the cyst evidenced E. granulosus DNA, genotype G1. Any post-surgery complications was observed during 6 following months. CONCLUSION: Cystic hydatidosis should always be considered in the differential diagnosis of any cystic mass, regardless of its location, also in epidemiological contests less suggestive of the disease. The diagnosis should be achieved by taking into consideration the clinical aspects, the epidemiology of the disease, the imaging and immunological tests but, as demonstrated in this case, without neglecting the numerous possibilities offered by new serological devices and modern day molecular biology techniques. [Abstract/Link to Full Text]

Setiati TE, Mairuhu AT, Koraka P, Supriatna M, Mac Gillavry MR, Brandjes DP, Osterhaus AD, van der Meer JW, van Gorp EC, Soemantri A
Dengue disease severity in Indonesian children: an evaluation of the World Health Organization classification system.
BMC Infect Dis. 2007;722.
BACKGROUND: Dengue disease severity is usually classified using criteria set up by the World Health Organization (WHO). We aimed to assess the diagnostic accuracy of the WHO classification system and modifications to this system, and evaluated their potential practical usefulness. METHODS: Patients, admitted consecutively to the hospital with severe dengue, were classified using the WHO classification system and modifications to this system. Treating physicians were asked to classify patients immediately after discharge. We calculated the sensitivity of the various classification systems for the detection of shock and the agreement between the various classification systems and the treating physician's classification. RESULTS: Of 152 patients with confirmed dengue, sixty-six (43%) had evidence of circulatory failure. The WHO classification system had a sensitivity of 86% (95%CI 76-94) for the detection of patients with shock. All modifications to the WHO classification system had a higher sensitivity than the WHO classification system (sensitivity ranging from 88% to 99%). The WHO classification system was in only modest agreement with the intuitive classification by treating physicians whereas several modified classification systems were in good agreement. CONCLUSION: The use of the WHO classification system to classify dengue disease severity is to be questioned, because it is not accurate in correctly classifying dengue disease severity and it lacks sufficient agreement with clinical practice. [Abstract/Link to Full Text]

Mettler J, Simcock M, Sendi P, Widmer AF, Bingisser R, Battegay M, Fluckiger U, Bassetti S
Empirical use of antibiotics and adjustment of empirical antibiotic therapies in a university hospital: a prospective observational study.
BMC Infect Dis. 2007;721.
BACKGROUND: Several strategies to optimise the use of antibiotics have been developed. Most of these interventions can be classified as educational or restrictive. Restrictive measures are considered to be more effective, but the enforcement of these measures may be difficult and lead to conflicts with prescribers. Any intervention should be aimed at targets with the highest impact on antibiotic prescribing. The aim of the present study was to assess the adequacy of empirical and adjusted antibiotic therapies in a Swiss university hospital where no antibiotic use restrictions are enforced, and to identify risk factors for inadequate treatment and targets for intervention. METHODS: A prospective observational study was performed during 9 months. All patients admitted through the emergency department who received an antibiotic therapy within 24 hours of admission were included. Data on demographic characteristics, diagnoses, comorbidities, systemic inflammatory response syndrome (SIRS) parameters, microbiological tests, and administered antibiotics were collected prospectively. Antibiotic therapy was considered adequate if spectrum, dose, application modus, and duration of therapy were appropriate according to local recommendations or published guidelines. RESULTS: 2943 admitted patients were evaluated. Of these, 572 (19.4%) received antibiotics within 24 hours and 539 (94%) were analysed in detail. Empirical antibiotic therapy was inadequate in 121 patients (22%). Initial therapy was adjusted in 168 patients (31%). This adjusted antibiotic therapy was inadequate in 46 patients (27%). The main reason for inadequacy was the use of antibiotics with unnecessarily broad spectrum (24% of inadequate empirical, and 52% of inadequate adjusted therapies). In 26% of patients with inadequate adjusted therapy, antibiotics used were either ineffective against isolated pathogenic bacteria or antibiotic therapy was continued despite negative results of microbiological investigations. CONCLUSION: The rate of inadequate antibiotic therapies was similar to the rates reported from other institutions despite the absence of a restrictive antibiotic policy. Surprisingly, adjusted antibiotic therapies were more frequently inappropriate than empirical therapies. Interventions aiming at improving antibiotic prescribing should focus on both initial empirical therapy and streamlining and adjustment of therapy once microbiological results become available. [Abstract/Link to Full Text]

Constantin de Magny G, Gu�gan JF, Petit M, Cazelles B
Regional-scale climate-variability synchrony of cholera epidemics in West Africa.
BMC Infect Dis. 2007;720.
BACKGROUND: The relationship between cholera and climate was explored in Africa, the continent with the most reported cases, by analyzing monthly 20-year cholera time series for five coastal adjoining West African countries: C�te d'Ivoire, Ghana, Togo, Benin and Nigeria. METHODS: We used wavelet analyses and derived methods because these are useful mathematical tools to provide information on the evolution of the periodic component over time and allow quantification of non-stationary associations between time series. RESULTS: The temporal variability of cholera incidence exhibits an interannual component, and a significant synchrony in cholera epidemics is highlighted at the end of the 1980's. This observed synchrony across countries, even if transient through time, is also coherent with both the local variability of rainfall and the global climate variability quantified by the Indian Oscillation Index. CONCLUSION: Results of this study suggest that large and regional scale climate variability influence both the temporal dynamics and the spatial synchrony of cholera epidemics in human populations in the Gulf of Guinea, as has been described for two other tropical regions of the world, western South America and Bangladesh. [Abstract/Link to Full Text]

Leung KH, Yip SP, Wong WS, Yiu LS, Chan KK, Lai WM, Chow EY, Lin CK, Yam WC, Chan KS
Sex- and age-dependent association of SLC11A1 polymorphisms with tuberculosis in Chinese: a case control study.
BMC Infect Dis. 2007;719.
BACKGROUND: Host genetic factors are important determinants in tuberculosis (TB). The SLC11A1 (or NRAMP1) gene has been studied extensively for genetic association with TB, but with inconsistent findings. In addition, no study has yet looked into the effect of sex and age on the relationship between SLC11A1 polymorphisms and TB. METHODS: A case-control study was conducted. In total, 278 pulmonary TB patients and 282 sex- and age-matched controls without TB were recruited. All subjects were ethnic Chinese. On the basis of linkage disequilibrium pattern, three genetic markers from SLC11A1 and one from the nearby IL8RB locus were selected and examined for association with TB susceptibility. These markers were genotyped using single strand conformation polymorphism analysis or fragment analysis of amplified products. RESULTS: Statistically significant differences in allele (P = 0.0165, OR = 1.51) and genotype (P = 0.0163, OR = 1.59) frequencies of the linked markers SLC6a/b (classically called D543N and 3'UTR) of the SLC11A1 locus were found between patients and controls. With stratification by sex, positive associations were identified in the female group for both allele (P = 0.0049, OR = 2.54) and genotype (P = 0.0075, OR = 2.74) frequencies. With stratification by age, positive associations were demonstrated in the young age group (age < or =65 years) for both allele (P = 0.0047, OR = 2.52) and genotype (P = 0.0031, OR = 2.92) frequencies. All positive findings remained significant even after correction for multiple comparisons. No significant differences were noted in either the male group or the older age group. No significant differences were found for the other markers (one SLC11A1 marker and one IL8RB marker) either. CONCLUSION: This study confirmed the association between SLC11A1 and TB susceptibility and demonstrated for the first time that the association was restricted to females and the young age group. [Abstract/Link to Full Text]

Lau JT, Kim JH, Tsui HY, Griffiths S
Anticipated and current preventive behaviors in response to an anticipated human-to-human H5N1 epidemic in the Hong Kong Chinese general population.
BMC Infect Dis. 2007;718.
BACKGROUND: The prevalence of self-reported preventive behaviors in response to an anticipated local human-to-human H5N1 transmission outbreak and factors associated with such behaviors have not been examined. METHODS: A random, anonymous, cross-sectional telephone survey of 503 Hong Kong Chinese adults. RESULTS: The public in Hong Kong is likely to adopt self-protective behaviors (e.g., wearing face mask in public venues (73.8%), increasing the frequency of handwashing (86.7%)) and behaviors that protect others (e.g., wearing face masks when experiencing influenza-like illness (ILI, 92.4%), immediately seeking medical consultation (94.2%), making declarations when crossing the border with ILI (87.1%), complying to quarantine policies (88.3%)). Multivariate analyses indicated that factors related to age, full-time employment, perceived susceptibility, perceived efficacy of preventive measures, perceived higher fatality as compared to SARS, perceived chance of a major local outbreak, and being worried about self/family members contracting the virus were significantly associated with the inclination to adopt self-protective measures. Similar analyses showed that education level, variables related to perceived efficacy, perceived major local outbreak and such were significantly associated with various behaviors directed towards protecting others. CONCLUSION: In the event of a human-to-human H5N1 outbreak, the public in Hong Kong is likely to adopt preventive measures that may help contain the spread of the virus in the community. [Abstract/Link to Full Text]

Eichner M, Schwehm M, Duerr HP, Brockmann SO
The influenza pandemic preparedness planning tool InfluSim.
BMC Infect Dis. 2007;717.
BACKGROUND: Planning public health responses against pandemic influenza relies on predictive models by which the impact of different intervention strategies can be evaluated. Research has to date rather focused on producing predictions for certain localities or under specific conditions, than on designing a publicly available planning tool which can be applied by public health administrations. Here, we provide such a tool which is reproducible by an explicitly formulated structure and designed to operate with an optimal combination of the competing requirements of precision, realism and generality. RESULTS: InfluSim is a deterministic compartment model based on a system of over 1,000 differential equations which extend the classic SEIR model by clinical and demographic parameters relevant for pandemic preparedness planning. It allows for producing time courses and cumulative numbers of influenza cases, outpatient visits, applied antiviral treatment doses, hospitalizations, deaths and work days lost due to sickness, all of which may be associated with economic aspects. The software is programmed in Java, operates platform independent and can be executed on regular desktop computers. CONCLUSION: InfluSim is an online available software http://www.influsim.info which efficiently assists public health planners in designing optimal interventions against pandemic influenza. It can reproduce the infection dynamics of pandemic influenza like complex computer simulations while offering at the same time reproducibility, higher computational performance and better operability. [Abstract/Link to Full Text]

Nagelkerke NJ, Moses S, de Vlas SJ, Bailey RC
Modelling the public health impact of male circumcision for HIV prevention in high prevalence areas in Africa.
BMC Infect Dis. 2007;716.
BACKGROUND: Recent clinical trials in Africa, in combination with several observational epidemiological studies, have provided evidence that male circumcision can reduce HIV female-to-male transmission risk by 60% or more. However, the public health impact of large-scale male circumcision programs for HIV prevention is unclear. METHODS: Two mathematical models were examined to explore this issue: a random mixing model and a compartmental model that distinguishes risk groups associated with sex work. In the compartmental model, two scenarios were developed, one calculating HIV transmission and prevalence in a context similar to the country of Botswana, and one similar to Nyanza Province, in western Kenya. RESULTS: In both models, male circumcision programs resulted in large and sustained declines in HIV prevalence over time among both men and women. Men benefited somewhat more than women, but prevalence among women was also reduced substantially. With 80% male circumcision uptake, the reductions in prevalence ranged from 45% to 67% in the two "countries", and with 50% uptake, from 25% to 41%. It would take over a decade for the intervention to reach its full effect. CONCLUSION: Large-scale uptake of male circumcision services in African countries with high HIV prevalence, and where male circumcision is not now routinely practised, could lead to substantial reductions in HIV transmission and prevalence over time among both men and women. [Abstract/Link to Full Text]

Rocha PN, Rehem AP, Santana JF, Castro N, Muniz AL, Salgado K, Rocha H, Carvalho EM
The cause of urinary symptoms among Human T Lymphotropic Virus Type I (HLTV-I) infected patients: a cross sectional study.
BMC Infect Dis. 2007;715.
BACKGROUND: HTLV-I infected patients often complain of urinary symptomatology. Epidemiological studies have suggested that these individuals have a higher prevalence and incidence of urinary tract infection (UTI) than seronegative controls. However, the diagnosis of UTI in these studies relied only on patient information and did not require confirmation by urine culture. The purpose of this study was to investigate the role of urinary tract infection (UTI) as the cause of urinary symptoms in HTLV-I infected patients. METHODS: In this cross sectional study we interviewed, and cultured urine from, 157 HTLV-I seropositive individuals followed regularly at a specialized clinic. All patients were evaluated by a neurologist and classified according to the Expanded Disability Status Scale (EDSS). Urodynamic studies were performed at the discretion of the treating physician. RESULTS: Sixty-four patients complained of at least one active urinary symptom but UTI was confirmed by a positive urine culture in only 12 of these patients (19%); the majority of symptomatic patients (81%) had negative urine cultures. To investigate the mechanism behind the urinary complaints in symptomatic individuals with negative urine cultures, we reviewed the results of urodynamic studies performed in 21 of these patients. Most of them (90.5%) had abnormal findings. The predominant abnormalities were detrusor sphincter hyperreflexia and dyssynergia, findings consistent with HTLV-I-induced neurogenic bladder. On a multivariate logistic regression, an abnormal EDSS score was the strongest predictor of urinary symptomatology (OR 9.87, 95% CI 3.465 to 28.116, P < 0.0001). CONCLUSION: Urinary symptomatology suggestive of UTI is highly prevalent among HTLV-I seropositive individuals but true UTI is responsible for the minority of cases. We posit that the main cause of urinary symptoms in this population is neurogenic bladder. Our data imply that HLTV-I infected patients with urinary symptomatology should not be empirically treated for UTI but rather undergo urine culture; if a UTI is excluded, further investigation with urodynamic studies should be considered. [Abstract/Link to Full Text]

Manosuthi W, Athichathanabadi C, Uttayamakul S, Phoorisri T, Sungkanuparph S
Plasma nevirapine levels, adverse events and efficacy of antiretroviral therapy among HIV-infected patients concurrently receiving nevirapine-based antiretroviral therapy and fluconazole.
BMC Infect Dis. 2007;714.
BACKGROUND: The clinical data of plasma NVP level, safety and efficacy of antiretroviral therapy (ART) for the concurrent use of nevirapine (NVP)-based ART and fluconazole (FLU) is scanty. METHODS: A retrospective study was conducted in patients who were initiated NVP-based ART between October 2004 and November 2005. The objectives were to compare NVP levels, adverse events, and 36-week efficacy of NVP-based ART between patients who did not receive FLU (group A) and those who received FLU 200 mg/day or 400 mg/day (group B). RESULTS: There were 122 patients with mean +/- SD age of 36 +/- 9 years; 81 in group A and 41 in group B. Median (IQR) baseline CD4 cell count was 29 (8-79) cell/mm3 in group A and 19 (8-33) cell/mm3 in group B (P = 0.102). Baseline characteristics between the two groups were similar. Mean +/- SD NVP levels were 6.5 +/- 3.0 mg/L in group A and 11.4 +/- 6.1 mg/L in group B(P < 0.001). One (2.4%) patient in group B developed clinical hepatitis (P = 0.336). Six (7.4%) patients in group A developed NVP-related skin rashes (P = 0.096). There were no differences in term of 36-week antiviral efficacy between the two groups (P > 0.05). CONCLUSION: Co-administration of NVP and daily dosage of FLU (200 mg/day and 400 mg/day) results in markedly increased trough plasma NVP level when compared to the administration of NVP alone. The concurrent use of NVP and FLU in very advanced HIV-infected patients is well-tolerated. The immunological and virological responses are favorable. [Abstract/Link to Full Text]

Huttunen R, Laine J, Lumio J, Vuento R, Syrj�nen J
Obesity and smoking are factors associated with poor prognosis in patients with bacteraemia.
BMC Infect Dis. 2007;713.
BACKGROUND: Bacteraemia is still a major cause of case fatality in all age groups. Our aim was to identify the major underlying conditions constituting risk factors for case fatality in bacteraemia patients. METHODS: The study involved 149 patients (79 male and 70 female) with bacteraemia caused by Staphylococcus aureus (S. aureus) (41 patients), Streptococcus pneumoniae (Str. pneumoniae) (42 patients), beta-hemolytic streptococcae (beta-hml str.) (23 patients) and Eschericia coli (E. coli) (43 patients). Underlying diseases, alcohol and tobacco consumption and body mass index (BMI) were registered. Laboratory findings and clinical data were registered on admission and 6 consecutive days and on day 10-14. Case fatality was studied within 30 days after positive blood culture. Associations between underlying conditions and case fatality were studied in univariate analysis and in a multivariate model. RESULTS: Nineteen patients (12.8%) died of bacteraemia. We found obesity (p = 0.002, RR 9.8; 95% CI 2.3 to 41.3), smoking (p < 0.001, RR 16.9; 95% CI 2.1 to 133.5), alcohol abuse (p = 0.008, RR 3.9; 95% CI 1.3 to 11.28), COPD (p = 0.01, RR 8.4; 95% CI 1.9 to 37.1) and rheumatoid arthritis (p = 0.045, RR 5.9; 95% CI 1.2 to 28.8) to be significantly associated with case fatality in bacteraemia in univariate model. The median BMI was significantly higher among those who died compared to survivors (33 vs. 26, p = 0.003). Obesity and smoking also remained independent risk factors for case fatality when their effect was studied together in a multivariate model adjusted with the effect of alcohol abuse, age (continuos variable), sex and causative organism. CONCLUSION: Our results indicate that obesity and smoking are prominent risk factors for case fatality in bacteraemic patients. Identification of risk factors underlying fatal outcome in bacteraemia may allow targeting of preventive efforts to individuals likely to derive greatest potential benefit. [Abstract/Link to Full Text]

Louw A, Tikly M
Purulent pericarditis due to co-infection with Streptococcus pneumoniae and Mycobacterium tuberculosis in a patient with features of advanced HIV infection.
BMC Infect Dis. 2007;712.
BACKGROUND: Both Mycobacterium tuberculosis and Streptococcus pneumoniae are common pathogens in patients with HIV infection. CASE PRESENTATION: We present an unusual case of purulent pericarditis resulting in cardiac tamponade due to infection with both organisms. We highlight the re-emergence of pneumococcal pericarditis in the HIV era and describe the pitfalls and challenges in the diagnosis of this condition. CONCLUSION: Clinicians working in HIV endemic areas need to consider dual infection with these organisms in patients who respond inadequately to either antibiotics or anti-tuberculous therapy alone. [Abstract/Link to Full Text]

M�ller B, Harbarth S, Stolz D, Bingisser R, Mueller C, Leuppi J, Nusbaumer C, Tamm M, Christ-Crain M
Diagnostic and prognostic accuracy of clinical and laboratory parameters in community-acquired pneumonia.
BMC Infect Dis. 2007;710.
BACKGROUND: Community-acquired pneumonia (CAP) is the most frequent infection-related cause of death. The reference standard to diagnose CAP is a new infiltrate on chest radiograph in the presence of recently acquired respiratory signs and symptoms. This study aims to evaluate the diagnostic and prognostic accuracy of clinical signs and symptoms and laboratory biomarkers for CAP. METHODS: 545 patients with suspected lower respiratory tract infection, admitted to the emergency department of a university hospital were included in a pre-planned post-hoc analysis of two controlled intervention trials. Baseline assessment included history, clinical examination, radiography and measurements of procalcitonin (PCT), highly sensitive C-reactive protein (hsCRP) and leukocyte count. RESULTS: Of the 545 patients, 373 had CAP, 132 other respiratory tract infections, and 40 other final diagnoses. The AUC of a clinical model including standard clinical signs and symptoms (i.e. fever, cough, sputum production, abnormal chest auscultation and dyspnea) to diagnose CAP was 0.79 [95% CI, 0.75-0.83]. This AUC was significantly improved by including PCT and hsCRP (0.92 [0.89-0.94]; p < 0.001). PCT had a higher diagnostic accuracy (AUC, 0.88 [0.84-0.93]) in differentiating CAP from other diagnoses, as compared to hsCRP (AUC, 0.76 [0.69-0.83]; p < 0.001) and total leukocyte count (AUC, 0.69 [0.62-0.77]; p < 0.001). To predict bacteremia, PCT had a higher AUC (0.85 [0.80-0.91]) as compared to hsCRP (p = 0.01), leukocyte count (p = 0.002) and elevated body temperature (p < 0.001). PCT, in contrast to hsCRP and leukocyte count, increased with increasing severity of CAP, as assessed by the pneumonia severity index (p < 0.001). CONCLUSION: PCT, and to a lesser degree hsCRP, improve the accuracy of currently recommended approaches for the diagnosis of CAP, thereby complementing clinical signs and symptoms. PCT is useful in the severity assessment of CAP. [Abstract/Link to Full Text]

Murdoch DM, McDonald JR
Mycobacterium avium-intracellulare cellulitis occurring with septic arthritis after joint injection: a case report.
BMC Infect Dis. 2007;79.
BACKGROUND: Cellulitis caused by Mycobacterium avium-intracellulare has rarely been described. Mycobacterium avium-intracellulare is a rare cause of septic arthritis after intra-articular injection, though the causative role of injection is difficult to ascertain in such cases. CASE PRESENTATION: A 57-year-old with rheumatoid arthritis treated with prednisone and azathioprine developed bilateral painful degenerative shoulder arthritis. After corticosteroid injections into both acromioclavicular joints, he developed bilateral cellulitis centered over the injection sites. Skin biopsy showed non-caseating granulomas, and culture grew Mycobacterium avium-intracellulare. Joint aspiration also revealed Mycobacterium avium-intracellulare infection. CONCLUSION: Although rare, skin and joint infections caused by Mycobacterium avium-intracellulare should be considered in any immunocompromised host, particularly after intra-articular injection. Stains for acid-fast bacilli may be negative in pathologic samples even in the presence of infection; cultures of tissue specimens should always be obtained. [Abstract/Link to Full Text]

Bhatti Z, Berenson CS
Adult systemic cat scratch disease associated with therapy for hepatitis C.
BMC Infect Dis. 2007;78.
BACKGROUND: We describe the first case of systemic cat scratch disease in a patient receiving peginterferon alpha-2a and ribavirin for treatment of hepatitis C. Cases of adult systemic CSD are extremely infrequent and immunomodulatory treatment for hepatitis C has been associated with aberrant host responses to common pathogens. CASE PRESENTATION: A 52 year old man being treated for hepatitis C presented with diffuse lymphadenopathy, weight loss, fevers and splenic lesions. Symptoms were initially confused with adverse effects of his regimen, delaying recognition of his infection. Diagnostic investigation, including histopathology, microbiology and serologic parameters, confirmed that his illness was due to disseminated cat scratch disease with Bartonella henselae. CONCLUSION: Disseminated CSD is exceptionally rare in adults. We describe the first case of disseminated cat scratch disease associated with peginterferon alpha and ribavirin to alert clinicians of the need to be aware of unusual manifestations of common infections in this population. [Abstract/Link to Full Text]

Giudice A, Camada I, Leopoldo PT, Pereira JM, Riley LW, Wilson ME, Ho JL, de Jesus AR, Carvalho EM, Almeida RP
Resistance of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis to nitric oxide correlates with disease severity in Tegumentary Leishmaniasis.
BMC Infect Dis. 2007;77.
BACKGROUND: Nitric oxide (NO*) plays a pivotal role as a leishmanicidal agent in mouse macrophages. NO* resistant Escherichia coli and Mycobacterium tuberculosis have been associated with a severe outcome of these diseases. METHODS: In this study we evaluated the in vitro toxicity of nitric oxide for the promastigote stages of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis parasites, and the infectivity of the amastigote stage for human macrophages. Parasites were isolated from patients with cutaneous, mucosal or disseminated leishmaniasis, and NO* resistance was correlated with clinical presentation. RESULTS: Seventeen isolates of L. (L.) amazonensis or L. (V.) braziliensis promastigotes were killed by up to 8 mM of more of NaNO2 (pH 5.0) and therefore were defined as nitric oxide-susceptible. In contrast, eleven isolates that survived exposure to 16 mM NaNO2 were defined as nitric oxide-resistant. Patients infected with nitric oxide-resistant Leishmania had significantly larger lesions than patients infected with nitric oxide-susceptible isolates. Furthermore, nitric oxide-resistant L. (L.) amazonensis and L. (V.) braziliensis multiplied significantly better in human macrophages than nitric oxide-susceptible isolates. CONCLUSION: These data suggest that nitric oxide-resistance of Leishmania isolates confers a survival benefit for the parasites inside the macrophage, and possibly exacerbates the clinical course of human leishmaniasis. [Abstract/Link to Full Text]

Saeed M, Zaidi SZ, Naeem A, Masroor M, Sharif S, Shaukat S, Angez M, Khan A
Epidemiology and clinical findings associated with enteroviral acute flaccid paralysis in Pakistan.
BMC Infect Dis. 2007;76.
BACKGROUND: Enteroviruses are among the most common viruses infecting humans worldwide and they are associated with diverse clinical syndromes. Acute flaccid paralysis (AFP) is a clinical manifestation of enteroviral neuropathy, transverse myelitis, Guillian-Barre Syndrome, Traumatic neuritis and many other nervous system disorders. The objective of this study was to understand the role of Non-Polio Enteroviruses (NPEV) towards this crippling disorder. METHODS: Stool specimens of 1775 children, aged less than 15 years, suffering from acute flaccid paralysis were collected after informed consent within 14 days of onset of symptoms during January 2003 to September 2003. The specimens were inoculated on RD and L20B cells using conventional tube cell culture while micro-neutralization test was used to identify the non-polio enterovirus (NPEV) serotypes. Detailed clinical information and 60-days follow-up reports were analyzed for NPEV-associated AFP cases. RESULTS: NPEV were isolated from 474 samples. The male to female ratio was 1.4:1. The isolation of NPEV decreased significantly with the increase in age. Cases associated with fever at the onset of NPEV-associated AFP were found to be 62%. The paralysis was found asymmetrical in 67% cases, the progression of paralysis to peak within 4 days was found in 72% cases and residual paralysis after 60 days of paralysis onset was observed in 39% cases associated with NPEV. A clinical diagnosis of Guillian-Barre syndrome was made in 32% cases. On Microneutralization assay, echo-6 (13%) and coxsackievirus B (13%) were the most commonly isolated serotypes of NPEV along with E-7, E-13, E-11, E-4 and E-30. The isolates (n = 181) found untypable by the antiserum pools were confirmed as NPEV by PCR using Pan-Enterovirus primers. CONCLUSION: The present study suggests that NPEV are a dominant cause of AFP and different serotypes of NPEV are randomly distributed in Pakistan. The untypable isolates need further characterization and analysis in order to determine their association with clinical presentation of a case. [Abstract/Link to Full Text]

Kampf G, Steinmann J, Rabenau H
Suitability of vaccinia virus and bovine viral diarrhea virus (BVDV) for determining activities of three commonly-used alcohol-based hand rubs against enveloped viruses.
BMC Infect Dis. 2007;75.
BACKGROUND: A procedure for including activity against enveloped viruses in the post-contamination treatment of hands has been recommended, but so far no European standard is available to implement it. In 2004, the German Robert Koch-Institute (RKI) and the German Association for the Control of Virus Disease (DVV) suggested that vaccinia virus and bovine viral diarrhea virus (BVDV) should be used as test viruses in a quantitative suspension test to determine the activity of a disinfectant against all enveloped viruses. METHODS: We have studied the activities of three commonly-used alcohol-based hand rubs (hand rub A, based on 45% propan-2-ol, 30% propan-1-ol and 0.2% mecetronium etilsulfate; hand rub B, based on 80% ethanol; hand rub C, based on 95% ethanol) against vaccinia virus and BVDV, and in addition against four other clinically relevant enveloped viruses: herpes simplex virus (HSV) types 1 and 2, and human and avian influenza A virus. The hand rubs were challenged with different organic loads at exposure time of 15, 30 and 60 s. According to the guidelines of both BGA/RKI and DVV, and EN 14476:2005, the reduction of infectivity of each test virus was measured on appropriate cell lines using a quantitative suspension test. RESULTS: All three alcohol-based hand rubs reduced the infectivity of vaccinia virus and BVDV by > or = 4 log10-steps within 15 s, irrespective of the type of organic load. Similar reductions of infectivity were seen against the other four enveloped viruses within 15 s in the presence of different types of organic load. CONCLUSION: Commonly used alcohol-based hand rubs with a total alcohol concentration > or = 75% can be assumed to be active against clinically relevant enveloped viruses if they effectively reduce the infectivities of vaccinia virus and BVDV in a quantitative suspension test. [Abstract/Link to Full Text]

Belmares J, Detterline S, Pak JB, Parada JP
Corynebacterium endocarditis species-specific risk factors and outcomes.
BMC Infect Dis. 2007;74.
BACKGROUND: Corynebacterium species are recognized as uncommon agents of endocarditis, but little is known regarding species-specific risk factors and outcomes in Corynebacterium endocarditis. METHODS: Case report and Medline search of English language journals for cases of Corynebacterium endocarditis. Inclusion criteria required that cases be identified as endocarditis, having persistent Corynebacterium bacteremia, murmurs described by the authors as identifying the affected valve, or vegetations found by echocardiography or in surgical or autopsy specimens. Cases also required patient-specific information on risk factors and outcomes (age, gender, prior prosthetic valve, other prior nosocomial risk factors (infected valve, involvement of native versus prosthetic valve, need for valve replacement, and death) to be included in the analysis. Publications of Corynebacterium endocarditis which reported aggregate data were excluded. Univariate analysis was conducted with chi-square and t-tests, as appropriate, with p = 0.05 considered significant. RESULTS: 129 cases of Corynebacterium endocarditis involving nine species met inclusion criteria. Corynebacterium endocarditis typically infects the left heart of adult males and nearly one third of patients have underlying valvular disease. One quarter of patients required valve replacement and one half of patients died. Toxigenic C. diphtheriae is associated with pediatric infections (p < 0.001). Only C. amycolatum has a predilection for women (p = 0.024), while C. pseudodiphtheriticum infections are most frequent in men (p = 0.023). C. striatum, C. jeikeium and C. hemolyticum are associated with nosocomial risk factors (p < 0.001, 0.028, and 0.024, respectively). No species was found to have a predilection for any particular heart valve. C. pseudodiphtheriticum is associated with a previous prosthetic valve replacement (p = 0.004). C. jeikeium infections are more likely to require valve replacement (p = 0.026). Infections involving toxigenic C. diphtheriae and C. pseudodiphtheriticum are associated with decreased survival (p = 0.001 and 0.032, respectively). CONCLUSION: We report the first analysis of species-specific risk factors and outcomes in Corynebacterium endocarditis. In addition to species-specific associations with age, gender, prior valvular diseases, and other nosocomial risk factors, we found differences in rates of need for valve replacement and death. This review highlights the seriousness of these infections, as up to 28% of patients required valve replacement and 43.5% died. [Abstract/Link to Full Text]

Reithinger R, Coleman PG
Treating cutaneous leishmaniasis patients in Kabul, Afghanistan: cost-effectiveness of an operational program in a complex emergency setting.
BMC Infect Dis. 2007;73.
BACKGROUND: Although Kabul city, Afghanistan, is currently the worldwide largest focus of cutaneous leishmaniasis (CL) with an estimated 67,500 cases, donor interest in CL has been comparatively poor because the disease is non-fatal. Since 1998 HealthNet TPO (HNTPO) has implemented leishmaniasis diagnosis and treatment services in Kabul and in 2003 alone 16,390 were treated patients in six health clinics in and around the city. The aim of our study was to calculate the cost-effectiveness for the implemented treatment regimen of CL patients attending HNTPO clinics in the Afghan complex emergency setting. METHODS: Using clinical and cost data from the on-going operational HNTPO program in Kabul, published and unpublished sources, and discussions with researchers, we developed models that included probabilistic sensitivity analysis to calculate ranges for the cost per disability adjusted life year (DALY) averted for implemented CL treatment regimen. We calculated the cost-effectiveness of intralesional and intramuscular administration of the pentavalent antimonial drug sodium stibogluconate, HNTPO's current CL 'standard treatment'. RESULTS: The cost of the standard treatment was calculated to be 27 US dollars (95% C.I. 20-36) per patient treated and cured. The cost per DALY averted per patient cured with the standard treatment was estimated to be approximately 1,200 US dollars (761-1,827). CONCLUSION: According to WHO-CHOICE criteria, treatment of CL in Kabul, Afghanistan, is not a cost-effective health intervention. The rationale for treating CL patients in Afghanistan and elsewhere is discussed. [Abstract/Link to Full Text]

Hill PC, Onyeama CO, Ikumapayi UN, Secka O, Ameyaw S, Simmonds N, Donkor SA, Howie SR, Tapgun M, Corrah T, Adegbola RA
Bacteraemia in patients admitted to an urban hospital in West Africa.
BMC Infect Dis. 2007;72.
BACKGROUND: Few studies on bacteraemia in Africa have been published. We aimed to prospectively identify the causative organisms of bacteraemia in The Gambia and their relation to clinical diagnoses, outcome and antimicrobial susceptibility. METHODS: Between November 2003 and February 2005 we studied those admitted to the Medical Research Council hospital who were suspected of having bacteraemia. We documented clinical features, outcome, pathogens identified and their susceptibility patterns, and searched for factors associated with bacteraemia. RESULTS: 871 patients were admitted and had a blood culture taken. The median age was 2 years (range 2 months to 80 years) and 36 of 119 tested were HIV positive; 54.5% were male. 297 (34%) had a positive result and 93 (10.7% overall) were considered a genuine pathogen. Those with bacteraemia were more likely to die in hospital (OR 2.79; 1.17-6.65, p = 0.017) and to have a high white cell count (WCC; OR 1.81;95% CI 1.09-3.02; p = 0.022). Three organisms accounted for 73% of bacteraemias: Streptococcus pneumoniae (45.2%), Staphylococcus aureus (18.3%) and Escherichia coli (9.7%) while non-typhoidal salmonellae (NTS) accounted for 8.6%. Antimicrobial susceptibility of S. pneumoniae was very high to penicillin (97.5%); high resistance was found to co-trimoxazole. S. aureus was generally highly susceptible to cloxacillin, gentamicin and chloramphenicol. E. coli and NTS were all susceptible to ciprofloxacin and mostly susceptible to gentamicin. Thirteen (33%) S. pneumoniae isolates were of serotypes contained in a 7-valent pneumococcal conjugate vaccine and 20 (51.3%) were of the same serogroup. CONCLUSION: In The Gambia, those with bacteraemia are more likely than those without to die in hospital and to have a raised peripheral blood WCC. S. pneumoniae is the most common organism isolated. Introduction of a pneumococcal conjugate vaccine can be expected to lead to a reduction in disease incidence. [Abstract/Link to Full Text]

Yang ML, Chen YH, Chen TC, Lin WR, Lin CY, Lu PL
Case report: infective endocarditis caused by Brevundimonas vesicularis.
BMC Infect Dis. 2006;6179.
BACKGROUND: There are few reports in the literature of invasive infection caused by Brevundimonas vesicularis in patients without immunosuppression or other predisposing factors. The choice of antimicrobial therapy for bacteremia caused by the pathogen requires more case experience to be determined. CASE PRESENTATION: The case of a 40-year-old previously healthy man with subacute endocarditis proposed to be contributed from an occult dental abscess is described. The infection was found to be caused by B. vesicularis on blood culture results. The patient recovered without sequelae after treatment with ceftriaxone followed by subsequent ciprofloxacin therapy owing to an allergic reaction to ceftriaxone and treatment failure with ampicillin/sulbactam. CONCLUSION: To our knowledge, this is the first report of B. vesicularis as a cause of infective endocarditis. According to an overview of the literature and our experience, we suggest that third-generation cephalosporins, piperacillin/tazobactam, and ciprofloxacin are effective in treating invasive B. vesicularis infections, while the efficacy of ampicillin-sulbactam needs further evaluation. [Abstract/Link to Full Text]

Alvarado-Esquivel C, Alanis-Qui�ones OP, Arreola-Valenzuela MA, Rodr�guez-Briones A, Piedra-Nevarez LJ, Duran-Morales E, Estrada-Mart�nez S, Mart�nez-Garc�a SA, Liesenfeld O
Seroepidemiology of Toxoplasma gondii infection in psychiatric inpatients in a northern Mexican city.
BMC Infect Dis. 2006;6178.
BACKGROUND: Patients with psychiatric disorders were found to show a high seroprevalence of Toxoplasma gondii infection. There is scarce information about the epidemiology of T. gondii infection in psychiatric patients in Mexico. Therefore, we sought to determine the prevalence of T. gondii infection and associated socio-demographic, clinical and behavioural characteristics in a population of psychiatric patients in Durango City, Mexico. Seroprevalence in patients was compared with that obtained in a control population. METHODS: One hundred and thirty seven inpatients of a public psychiatric hospital and 180 controls were examined for the presence of IgG and IgM antibodies against T. gondii by enzyme-linked immunoassay (Diagnostic Automation Inc., Calabasas, CA, USA). The control population consisted of blood donors of a public blood bank and elderly persons attending a senior center in the same city. Age in controls (42 years +/- 20.2) was comparable with that of the psychiatric patients (43.7 years +/-13.8) (p = 0.42). Socio-demographic, clinical and behavioral characteristics from the patients were also obtained. RESULTS: Anti-T. gondii IgG antibodies indicating latent infection with T. gondii was found in 25 (18.2%) of 137 psychiatric inpatients and 16 (8.9%) of 180 controls (p = 0.02). Ten (26.3%) of 38 schizophrenic patients had latent infection and this prevalence was also significantly higher than that observed in controls (p = 0.005). Prevalence of anti-T. gondii IgM antibodies was comparable among patients and controls (4.4% vs 2.2%, respectively, p = 0.22). Multivariate analysis showed that T. gondii infection in inpatients was positively associated with sexual promiscuity (adjusted OR = 15.8; 95% CI: 3.8-64.8), unwashed raw fruit consumption (adjusted OR = 5.19; 95% CI: 2.3-11.3), and a history of surgery (adjusted OR = 6.5; 95% CI: 2.6-16), and negatively associated with lamb meat consumption (adjusted OR = 0.26; 95% CI: 0.10-0.63). CONCLUSION: In the present study, psychiatric inpatients in Durango, Mexico, in general and schizophrenia inpatients in particular had a significantly higher prevalence of T. gondii infection than the control group. Results suggest that unwashed raw fruit consumption might be the most important route of T. gondii transmission in our psychiatric inpatients while lamb meat consumption the less important. Additional studies will have to elucidate the causative relation between infection with T. gondii and psychiatric disorders. [Abstract/Link to Full Text]

Pittalis S, Nicastri E, Spinazzola F, Ghirga P, De Marco M, Paglia MG, Narciso P
Leishmania infantum leishmaniasis in corticosteroid--treated patients.
BMC Infect Dis. 2006;6177.
BACKGROUND: The number of leishmaniasis cases associated with immunosuppression has increased regularly over the past 20 years. Immunosuppression related to HIV infection, immunosuppressive treatment, organ transplantation, and neoplastic diseases increases the risk for Leishmania-infected people to develop visceral illness. CASE PRESENTATION: Three cases of Leishmania infantum leishmaniasis in corticosteroid (CS)-treated patients are reported: an isolated lingual leishmaniasis in a farmer treated with CS for asthma, a severe visceral leishmaniasis associated with cutaneous lesions in a woman with myasthenia gravis, and a visceral involvement after cutaneous leishmaniasis in a man receiving CS. CONCLUSION: Physicians should recognise CS-treated patients as a population likely to be immune-suppressed. In immunodeficiency conditions, unusual forms of leishmaniasis can develop and foster the risk of a diagnostic delay and of poor response to therapy. [Abstract/Link to Full Text]

Lu CY, Lauderdale TL, Fang YH, Wang CY, Ho YH, Hung CL, Chang LY, Lee CY, Huang LM
Disease burden and related medical costs of rotavirus infections in Taiwan.
BMC Infect Dis. 2006;6176.
BACKGROUND: The disease burden and associated medical costs of rotavirus infections in inpatient and outpatient sectors in Taiwan were examined in anticipation of the availability of new rotavirus vaccines. METHODS: The yearly national case number and medical costs for all for inpatients and outpatients with acute gastroenteritis (AGE) were extracted from the Bureau of National Health Insurance database in Taiwan according to ICD-9-CM codes. A retrospective study was also performed using records of children with AGE seen at three hospitals in Taiwan in 2001 to identify laboratory confirmed rotavirus infection cases. The annual incidence and related medical costs of AGE due to rotavirus infection were then estimated. RESULTS: Children <5 years old comprised 83.6% of inpatient and 62.0% of outpatient pediatric AGE cases in Taiwan in 2001. Rotavirus was the most common agent detected among AGE patients in this age group in the three hospitals, and was detected in 32.9% (221/672) of inpatient and 24% (23/96) of outpatient stool specimens tested for microbial etiologies. An estimated 277,400 to 624,892 cases of rotavirus infections sought medical care in Taiwan in 2001, equaling one in 2 to 5 children <5 years old required medical care due to rotavirus infection. The incidence of hospitalization due to rotavirus infections was 1,528-1,997/100,000 for children <5 years old. The total associated medical costs due to rotavirus infection were estimated at US $10-16 millions in Taiwan in 2001. Although the per-capita medical cost of rotavirus infection was lower in Taiwan than in the United States or Hong Kong, the personal economic burden was similar among the three places when normalized for gross national incomes per capita. CONCLUSION: Infections caused by rotavirus constitute an important human and economic burden among young children in Taiwan. A safe and effective vaccine is urgently needed. [Abstract/Link to Full Text]

Fjaerli HO, Bukholm G, Krog A, Skjaeret C, Holden M, Nakstad B
Whole blood gene expression in infants with respiratory syncytial virus bronchiolitis.
BMC Infect Dis. 2006;6175.
BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of viral bronchiolitis in infants worldwide, and environmental, viral and host factors are all of importance for disease susceptibility and severity. To study the systemic host response to this disease we used the microarray technology to measure mRNA gene expression levels in whole blood of five male infants hospitalised with acute RSV, subtype B, bronchiolitis versus five one year old male controls exposed to RSV during infancy without bronchiolitis. The gene expression levels were further evaluated in a new experiment using quantitative real-time polymerase chain reaction (QRT-PCR) both in the five infants selected for microarray and in 13 other infants hospitalised with the same disease. RESULTS: Among the 30 genes most differentially expressed by microarray nearly 50% were involved in immunological processes. We found the highly upregulated interferon, alpha-inducible protein 27 (IFI27) and the highly downregulated gene Charcot-Leyden crystal protein (CLC) to be the two most differentially expressed genes in the microarray study. When performing QRT-PCR on these genes IFI27 was upregulated in all but one infant, and CLC was downregulated in all 18 infants, and similar to that given by microarray. CONCLUSION: The gene IFI27 is upregulated and the gene CLC is downregulated in whole blood of infants hospitalised with RSV, subtype B, bronchiolitis and is not reported before. More studies are needed to elucidate the specificity of these gene expressions in association with host response to this virus in bronchiolitis of moderate severity. [Abstract/Link to Full Text]

Srinivasa Rao AS, Chen MH, Pham BZ, Tricco AC, Gilca V, Duval B, Krahn MD, Bauch CT
Cohort effects in dynamic models and their impact on vaccination programmes: an example from hepatitis A.
BMC Infect Dis. 2006;6174.
BACKGROUND: Infection rates for many infectious diseases have declined over the past century. This has created a cohort effect, whereby older individuals experienced a higher infection rate in their past than younger individuals do now. As a result, age-stratified seroprevalence profiles often differ from what would be expected from constant infection rates. METHODS: Here, we account for the cohort effect by fitting an age-structured compartmental model with declining transmission rates to Hepatitis A seroprevalence data for Canadian-born individuals. We compare the predicted impact of universal vaccination with and without including the cohort effect in the dynamic model. RESULTS: We find that Hepatitis A transmissibility has declined by a factor of 2.8 since the early twentieth century. When the cohort effect is not included in the model, incidence and mortality both with and without vaccination are significantly over-predicted. Incidence (respectively mortality) over a 20 year period of universal vaccination is 34% (respectively 90%) higher than if the cohort effect is included. The percentage reduction in incidence and mortality due to vaccination are also over-predicted when the cohort effect is not included. Similar effects are likely for many other infectious diseases where infection rates have declined significantly over past decades and where immunity is lifelong. CONCLUSION: Failure to account for cohort effects has implications for interpreting seroprevalence data and predicting the impact of vaccination programmes with dynamic models. Cohort effects should be included in dynamic modelling studies whenever applicable. [Abstract/Link to Full Text]

Yu DT, Seger DL, Peterson JF, Kumar RN, Bates DW
Fluconazole for empiric antifungal therapy in cancer patients with fever and neutropenia.
BMC Infect Dis. 2006;6173.
BACKGROUND: Several clinical trials have demonstrated the efficacy of fluconazole as empiric antifungal therapy in cancer patients with fever and neutropenia. Our objective was to assess the frequency and resource utilization associated with treatment failure in cancer patients given empiric fluconazole antifungal therapy in routine inpatient care. METHODS: We performed a retrospective cohort study of cancer patients treated with oral or intravenous fluconazole between 7/97 and 6/01 in a tertiary care hospital. The final study cohort included cancer patients with neutropenia (an absolute neutrophil count below 500 cells/mm3) and fever (a temperature above 38 degrees C or 100.4 degrees F), who were receiving at least 96 hours of parenteral antibacterial therapy prior to initiating fluconazole. Patients' responses to empiric therapy were assessed by reviewing patient charts. RESULTS: Among 103 cancer admissions with fever and neutropenia, treatment failure after initiating empiric fluconazole antifungal therapy occurred in 41% (95% confidence interval (CI) 31%-50%) of admissions. Patients with a diagnosis of hematological malignancy had increased risk of treatment failure (OR = 4.6, 95% CI 1.5-14.8). When treatment failure occurred the mean adjusted increases in length of stay and total costs were 7.4 days (95% CI 3.3-11.5) and $18,925 (95% CI 3,289-34,563), respectively. CONCLUSION: Treatment failure occurred in more than one-third of neutropenic cancer patients on fluconazole as empiric antifungal treatment for fever in routine clinical treatment. The increase in costs when treatment failure occurs is substantial. [Abstract/Link to Full Text]

Recent Articles in BMC Microbiology

Qin Z, Zhang J, Xu B, Chen L, Wu Y, Yang X, Shen X, Molin S, Danchin A, Jiang H, Qu D
Structure-based discovery of inhibitors of the YycG histidine kinase: new chemical leads to combat Staphylococcus epidermidis infections.
BMC Microbiol. 2006;696.
BACKGROUND: Coagulase-negative Staphylococcus epidermidis has become a major frequent cause of infections in relation to the use of implanted medical devices. The pathogenicity of S. epidermidis has been attributed to its capacity to form biofilms on surfaces of medical devices, which greatly increases its resistance to many conventional antibiotics and often results in chronic infection. It has an urgent need to design novel antibiotics against staphylococci infections, especially those can kill cells embedded in biofilm. RESULTS: In this report, a series of novel inhibitors of the histidine kinase (HK) YycG protein of S. epidermidis were discovered first using structure-based virtual screening (SBVS) from a small molecular lead-compound library, followed by experimental validation. Of the 76 candidates derived by SBVS targeting of the homolog model of the YycG HATPase_c domain of S. epidermidis, seven compounds displayed significant activity in inhibiting S. epidermidis growth. Furthermore, five of them displayed bactericidal effects on both planktonic and biofilm cells of S. epidermidis. Except for one, the compounds were found to bind to the YycG protein and to inhibit its auto-phosphorylation in vitro, indicating that they are potential inhibitors of the YycG/YycF two-component system (TCS), which is essential in S. epidermidis. Importantly, all these compounds did not affect the stability of mammalian cells nor hemolytic activities at the concentrations used in our study. CONCLUSION: These novel inhibitors of YycG histidine kinase thus are of potential value as leads for developing new antibiotics against infecting staphylococci. The structure-based virtual screening (SBVS) technology can be widely used in screening potential inhibitors of other bacterial TCSs, since it is more rapid and efficacious than traditional screening technology. [Abstract/Link to Full Text]

Slack AT, Symonds ML, Dohnt MF, Smythe LD
Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene.
BMC Microbiol. 2006;695.
BACKGROUND: Leptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing. RESULTS: A conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results. CONCLUSION: This research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification. [Abstract/Link to Full Text]

Iversen C, Waddington M, Farmer JJ, Forsythe SJ
The biochemical differentiation of Enterobacter sakazakii genotypes.
BMC Microbiol. 2006;694.
BACKGROUND: Enterobacter sakazakii is an emergent pathogen that has been associated with neonatal infections through contaminated powdered infant milk formula. The species was defined by Farmer et al. (1980) who described 15 biogroups according to the biochemical characterization of 57 strains. This present study compares genotypes (DNA cluster groups based on partial 16S rDNA sequence analysis) with the biochemical traits for 189 E. sakazakii strains. RESULTS: Analysis of partial 16S rDNA sequences gave 4 well defined phylogenetic groups. Cluster group 1 was composed of the majority of strains (170/189) and included Biogroups 1-5, 7-9, 11, 13 and 14. Cluster 3 corresponded with Biogroup 15 and cluster 4 with Biogroups 6, 10 and 12. Cluster group 2 comprised a new Biogroup 16. For the isolates in this study, the four DNA cluster groups can be distinguished using the inositol, dulcitol and indole tests. CONCLUSION: This study demonstrates an agreement between genotyping (partial 16S rDNA) and biotyping and describes a new biogroup of E. sakazakii. [Abstract/Link to Full Text]

Rohde BH, Quadri LE
Functional characterization of a three-component regulatory system involved in quorum sensing-based regulation of peptide antibiotic production in Carnobacterium maltaromaticum.
BMC Microbiol. 2006;693.
BACKGROUND: Quorum sensing is a form of cell-to-cell communication that allows bacteria to control a wide range of physiological processes in a population density-dependent manner. Production of peptide antibiotics is one of the processes regulated by quorum sensing in several species of Gram-positive bacteria, including strains of Carnobacterium maltaromaticum. This bacterium and its peptide antibiotics are of interest due to their potential applications in food preservation. The molecular bases of the quorum sensing phenomenon controlling peptide antibiotic production in C. maltaromaticum remain poorly understood. The present study was aimed at gaining a deeper insight into the molecular mechanism involved in quorum sensing-mediated regulation of peptide antibiotic (bacteriocin) production by C. maltaromaticum. We report the functional analyses of the CS (autoinducer)-CbnK (histidine protein kinase)-CbnR (response regulator) three-component regulatory system and the three regulated promoters involved in peptide antibiotic production in C. maltaromaticum LV17B. RESULTS: CS-CbnK-CbnR system-dependent activation of carnobacterial promoters was demonstrated in both homologous and heterologous hosts using a two-plasmid system with a beta-glucuronidase (GusA) reporter read-out. The results of our analyses support a model in which the CbnK-CbnR two-component signal transduction system is necessary and sufficient to transduce the signal of the peptide autoinducer CS into the activation of the promoters that drive the expression of the genes required for production of the carnobacterial peptide antibiotics and the immunity proteins that protect the producer bacterium. CONCLUSIONS: The CS-CbnK-CbnR triad forms a three-component regulatory system by which production of peptide antibiotics by C. maltaromaticum LV17B is controlled in a population density-dependent (or cell proximity-dependent) manner. This regulatory mechanism would permit the bacterial population to synchronize the production of peptide antibiotics and immunity proteins. Such a population-wide action would afford a substantial peptide antibiotic production burst that could increase the ability of the bacterium to inhibit susceptible bacterial competitors. Finally, our CS-CbnK-CbnR-based two-plasmid expression system represents a suitable genetic tool for undertaking structure-function relationship analyses to map the amino acid residues in the components of the CS-CbnK-CbnR system that are required for biological activity. This plasmid system also has potential as a starting point for developing alternative vectors for controlled gene expression in C. maltaromaticum, Lactococcus lactis, and related lactic acid bacteria. [Abstract/Link to Full Text]

Tuominen-Gustafsson H, Penttinen M, Hyt�nen J, Viljanen MK
Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils.
BMC Microbiol. 2006;692.
BACKGROUND: Species of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi) cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis) which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry. RESULTS: In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii), or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain. CONCLUSION: These results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The results also indicate a clear difference in the association with neutrophils between virulent and non-virulent borrelial strains. [Abstract/Link to Full Text]

Njuguna JT, Nassar M, Hoerauf A, Kaiser AE
Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax.
BMC Microbiol. 2006;691.
BACKGROUND: Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites. RESULTS: We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs) sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098) after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot.The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa) in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7), suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins. CONCLUSION: We identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains. [Abstract/Link to Full Text]

Aristimu�o L, Armengol R, Cebollada A, Espa�a M, Guilarte A, Lafoz C, Lezcano MA, Revillo MJ, Mart�n C, Ram�rez C, Rastogi N, Rojas J, de Salas AV, Sola C, Samper S
Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela.
BMC Microbiol. 2006;690.
BACKGROUND: Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD). RESULTS: Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised. Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis. CONCLUSION: This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies. [Abstract/Link to Full Text]

Hayes ET, Wilks JC, Sanfilippo P, Yohannes E, Tate DP, Jones BD, Radmacher MD, BonDurant SS, Slonczewski JL
Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition in Escherichia coli K-12.
BMC Microbiol. 2006;689.
BACKGROUND: In Escherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated. RESULTS: The pH dependence of gene expression was analyzed in oxygen-limited cultures of E. coli K-12 strain W3110. E. coli K-12 strain W3110 was cultured in closed tubes containing LBK broth buffered at pH 5.7, pH 7.0, and pH 8.5. Affymetrix array hybridization revealed pH-dependent expression of 1,384 genes and 610 intergenic regions. A core group of 251 genes showed pH responses similar to those in a previous study of cultures grown with aeration. The highly acid-induced gene yagU was shown to be required for extreme-acid resistance (survival at pH 2). Acid also up-regulated fimbriae (fimAC), periplasmic chaperones (hdeAB), cyclopropane fatty acid synthase (cfa), and the "constitutive" Na+/H+ antiporter (nhaB). Base up-regulated core genes for maltodextrin transport (lamB, mal), ATP synthase (atp), and DNA repair (recA, mutL). Other genes showed opposite pH responses with or without aeration, for example ETS components (cyo,nuo, sdh) and hydrogenases (hya, hyb, hyc, hyf, hyp). A hypF strain lacking all hydrogenase activity showed loss of extreme-acid resistance. Under oxygen limitation only, acid down-regulated ribosome synthesis (rpl,rpm, rps). Acid up-regulated the catabolism of sugar derivatives whose fermentation minimized acid production (gnd, gnt, srl), and also a cluster of 13 genes in the gadA region. Acid up-regulated drug transporters (mdtEF, mdtL), but down-regulated penicillin-binding proteins (dacACD, mreBC). Intergenic regions containing regulatory sRNAs were up-regulated by acid (ryeA, csrB, gadY, rybC). CONCLUSION: pH regulates a core set of genes independently of oxygen, including yagU, fimbriae, periplasmic chaperones, and nhaB. Under oxygen limitation, however, pH regulation is reversed for genes encoding electron transport components and hydrogenases. Extreme-acid resistance requires yagU and hydrogenase production. Ribosome synthesis is down-regulated at low pH under oxygen limitation, possibly due to the restricted energy yield of catabolism. Under oxygen limitation, pH regulates metabolism and transport so as to maximize alternative catabolic options while minimizing acidification or alkalinization of the cytoplasm. [Abstract/Link to Full Text]

Zhang CY, Wei JF, He SH
Adaptive evolution of the spike gene of SARS coronavirus: changes in positively selected sites in different epidemic groups.
BMC Microbiol. 2006;688.
BACKGROUND: It is believed that animal-to-human transmission of severe acute respiratory syndrome (SARS) coronavirus (CoV) is the cause of the SARS outbreak worldwide. The spike (S) protein is one of the best characterized proteins of SARS-CoV, which plays a key role in SARS-CoV overcoming species barrier and accomplishing interspecies transmission from animals to humans, suggesting that it may be the major target of selective pressure. However, the process of adaptive evolution of S protein and the exact positively selected sites associated with this process remain unknown. RESULTS: By investigating the adaptive evolution of S protein, we identified twelve amino acid sites (75, 239, 244, 311, 479, 609, 613, 743, 765, 778, 1148, and 1163) in the S protein under positive selective pressure. Based on phylogenetic tree and epidemiological investigation, SARS outbreak was divided into three epidemic groups: 02-04 interspecies, 03-early-mid, and 03-late epidemic groups in the present study. Positive selection was detected in the first two groups, which represent the course of SARS-CoV interspecies transmission and of viral adaptation to human host, respectively. In contrast, purifying selection was detected in 03-late group. These indicate that S protein experiences variable positive selective pressures before reaching stabilization. A total of 25 sites in 02-04 interspecies epidemic group and 16 sites in 03-early-mid epidemic group were identified under positive selection. The identified sites were different between these two groups except for site 239, which suggests that positively selected sites are changeable between groups. Moreover, it was showed that a larger proportion (24%) of positively selected sites was located in receptor-binding domain (RBD) than in heptad repeat (HR)1-HR2 region in 02-04 interspecies epidemic group (p = 0.0208), and a greater percentage (25%) of these sites occurred in HR1-HR2 region than in RBD in 03-early-mid epidemic group (p = 0.0721). These suggest that functionally different domains of S protein may not experience same positive selection in each epidemic group. In addition, three specific replacements (F360S, T487S and L665S) were only found between 03-human SARS-CoVs and strains from 02-04 interspecies epidemic group, which reveals that selective sweep may also force the evolution of S genes before the jump of SARS-CoVs into human hosts. Since certain residues at these positively selected sites are associated with receptor recognition and/or membrane fusion, they are likely to be the crucial residues for animal-to-human transmission of SARS-CoVs, and subsequent adaptation to human hosts. CONCLUSION: The variation of positive selective pressures and positively selected sites are likely to contribute to the adaptive evolution of S protein from animals to humans. [Abstract/Link to Full Text]

Herthnek D, B�lske G
New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis.
BMC Microbiol. 2006;687.
BACKGROUND: Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. RESULTS: Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. CONCLUSION: We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used. [Abstract/Link to Full Text]

Andersen JB, Roldgaard BB, Lindner AB, Christensen BB, Licht TR
Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes.
BMC Microbiol. 2006;686.
BACKGROUND: Existing virulence models are often difficult to apply for quantitative comparison of invasion potentials of Listeria monocytogenes. Well-to-well variation between cell-line based in vitro assays is practically unavoidable, and variation between individual animals is the cause of large deviations in the observed capacity for infection when animal models are used.One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains. RESULTS: A fluorescent marker system, allowing visualization and identification of single L. monocytogenes cells as well as colonies in a non-destructive manner, was developed. Five different fluorescent labels are available, and allowed simultaneous visual discrimination between three differently labelled strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations.The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible. CONCLUSION: The constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between differently labelled bacteria after internalization in these cells. [Abstract/Link to Full Text]

Mercado-Curiel RF, Esquinca-Avil�s HA, Tovar R, D�az-Badillo A, Camacho-Nuez M, Mu�oz Mde L
The four serotypes of dengue recognize the same putative receptors in Aedes aegypti midgut and Ae. albopictus cells.
BMC Microbiol. 2006;685.
ABSTRACT: BACKGROUND: Dengue viruses (DENV) attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. Several primary and low affinity co-receptors for this flavivirus have been identified. However, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection and in designing control strategies. RESULTS: (1) Overlay protein binding assay showed two proteins with molecular masses of 80 and 67 kDa (R80 and R67). (2) Specific antibodies against these two proteins inhibited cell binding and infection. (3) Both proteins were bound by all four serotypes of dengue virus. (4) R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5) In addition, a protein with molecular mass of 57 kDa was purified by affinity chromatography from the midgut extracts. (6) R80 and R67 from radiolabeled surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against Ae. aegypti midgut. CONCLUSION: Our results strongly suggest that R67 and R80 are receptors for the four serotypes of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells. [Abstract/Link to Full Text]

Halling SM, Jensen AE
Intrinsic and selected resistance to antibiotics binding the ribosome: analyses of Brucella 23S rrn, L4, L22, EF-Tu1, EF-Tu2, efflux and phylogenetic implications.
BMC Microbiol. 2006;684.
BACKGROUND: Brucella spp. are highly similar, having identical 16S RNA. However, they have important phenotypic differences such as differential susceptibility to antibiotics binding the ribosome. Neither the differential susceptibility nor its basis has been rigorously studied. Differences found among other conserved ribosomal loci could further define the relationships among the classical Brucella spp. RESULTS: Minimum inhibitory concentration (MIC) values of Brucella reference strains and three marine isolates to antibiotics binding the ribosome ranged from 0.032 to >256 microg/ml for the macrolides erythromycin, clarithromycin, and azithromycin and 2 to >256 microg/ml for the lincosamide, clindamycin. Though sequence polymorphisms were identified among ribosome associated loci 23S rrn, rplV, tuf-1 and tuf-2 but not rplD, they did not correlate with antibiotic resistance phenotypes. When spontaneous erythromycin resistant (eryR) mutants were examined, mutation of the peptidyl transferase center (A2058G Ec) correlated with increased resistance to both erythromycin and clindamycin. Brucella efflux was examined as an alternative antibiotic resistance mechanism by use of the inhibitor L-phenylalanine-L-arginine beta-naphthylamide (PAbetaN). Erythromycin MIC values of reference and all eryR strains, except the B. suis eryR mutants, were lowered variably by PAbetaN. A phylogenetic tree based on concatenated ribosomal associated loci supported separate evolutionary paths for B. abortus, B. melitensis, and B. suis/B. canis, clustering marine Brucella and B. neotomae with B. melitensis. Though Brucella ovis was clustered with B. abortus, the bootstrap value was low. CONCLUSION: Polymorphisms among ribosomal loci from the reference Brucella do not correlate with their highly differential susceptibility to erythromycin. Efflux plays an important role in Brucella sensitivity to erythromycin. Polymorphisms identified among ribosome associated loci construct a robust phylogenetic tree supporting classical Brucella spp. designations. [Abstract/Link to Full Text]

Johnson G, Millar MR, Matthews S, Skyrme M, Marsh P, Barringer E, O'Hara S, Wilks M
Evaluation of BacLite Rapid MRSA, a rapid culture based screening test for the detection of ciprofloxacin and methicillin resistant S. aureus (MRSA) from screening swabs.
BMC Microbiol. 2006;683.
BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide. The need for accurate and rapid screening methods to detect MRSA carriers has been clearly established. The performance of a novel assay, BacLite Rapid MRSA (Acolyte Biomedica, UK) for the rapid detection (5 h) and identification of hospital associated ciprofloxacin resistant strains of MRSA directly from nasal swab specimens was compared to that obtained by culture on Mannitol salt agar containing Oxacillin (MSAO) after 48 h incubation. RESULTS: A total of 1382 nasal screening swabs were tested by multiple operators. The BacLite Rapid MRSA test detected 142 out of the 157 confirmed MRSA that were detected on MSAO giving a diagnostic sensitivity of 90.4, diagnostic specificity of 95.7% and a negative predictive value of 98.7%. Of the 15 false negatives obtained by the BacLite Rapid MRSA test, seven grew small amounts (< 10 colonies of MRSA) on the MSAO culture plate and five isolates were ciprofloxacin sensitive. However there were 13 confirmed BacLite MRSA positive samples, which were negative by the direct culture method, probably due to overgrowth on the MSAO plate. There were 53 false positive results obtained by the BacLite Rapid MRSA test at 5 h and 115 cases where MRSA colonies were tentatively identified on the MSAO plate when read at 48 h, and which subsequently proved not to be MRSA. CONCLUSION: The BacLite MRSA test is easy to use and provides a similar level of sensitivity to conventional culture for the detection of nasal carriage of MRSA with the advantage that the results are obtained much more rapidly. [Abstract/Link to Full Text]

Nygaard TK, Liu M, McClure MJ, Lei B
Identification and characterization of the heme-binding proteins SeShp and SeHtsA of Streptococcus equi subspecies equi.
BMC Microbiol. 2006;682.
BACKGROUND: Heme is a preferred iron source of bacterial pathogens. Streptococcus equi subspecies equi is a bacterial pathogen that causes strangles in horses. Whether S. equi has a heme acquisition transporter is unknown. RESULTS: An S. equi genome database was blasted with the heme binding proteins Shp and HtsA of Streptococcus pyogenes, and found that S. equi has the homologue of Shp (designated SeShp) and HtsA (designated SeHtsA). Tag-free recombinant SeShp and SeHtsA and 6xHis-tagged SeHtsA (SeHtsAHis) were prepared and characterized. Purified holoSeShp and holoSeHtsA bind Fe(II)-protoporphyrin IX (heme) and Fe(III)-protoporphyrin IX (hemin) in a 1:1 stoichiometry, respectively, and are designated hemoSeShp and hemiSeHtsA. HemiSeShp and hemiSeHtsAHis can be reconstituted from apoSeShp and apoSeHtsAHis and hemin. HemoSeShp is stable in air and can be oxidized to hemiSeShp by ferricyanide. HemiSeHtsA can be reduced into hemoSeHtsA, which autoxidizes readily. HemoSeShp rapidly transfers its heme to apoSeHtsAHis. In addition, hemoSeShp can also transfer its heme to apoHtsA, and hemoShp is able to donate heme to apoSeHtsAHis. CONCLUSION: The primary structures, optical properties, oxidative stability, and in vitro heme transfer reaction of SeShp and SeHtsA are very similar to those of S. pyogenes Shp and HtsA. The data suggest that the putative cell surface protein SeShp and lipoprotein SeHtsA are part of the machinery to acquire heme in S. equi. The results also imply that the structure, function, and functional mechanism of the heme acquisition machinery are conserved in S. equi and S. pyogenes. [Abstract/Link to Full Text]

Michalski CW, Di Mola FF, K�mmel K, Wendt M, K�ninger JS, Giese T, Giese NA, Friess H
Human inflammatory bowel disease does not associate with Lawsonia intracellularis infection.
BMC Microbiol. 2006;681.
BACKGROUND: There is increasing evidence that bacterial infection of the intestinal mucosa may contribute to the pathogenesis of inflammatory bowel diseases (IBD). In pigs, an obligate intracellular bacterium, Lawsonia intracellularis (LI), was shown to cause proliferative enteropathy (PE) of which some forms display histological and clinical similarities to human IBD. Since LI-similar Desulfovibrio spp. may infect human cells, we hypothesized that LI might be associated with the development of human IBD. RESULTS: In human intestinal tissue samples, PCR using LLG, 50SL27, LSA and strictly LI-specific 16SII primers, yielded either no amplicons or products with weak homology to human genomic sequences. Sequencing of these amplicons revealed no specificity for LI. However, amplification of DNA with less specific 16SI primers resulted in products bearing homology to certain Streptococcus species. These 16SI-amplified products were present in healthy and diseased specimens, without obvious prevalence. CONCLUSION: LI is not associated with the pathogenesis of UC or CD. Whether an immunologic response to commensal bacteria such as streptococci may contribute to the chronic inflammatory condition in IBD, remained to be determined. [Abstract/Link to Full Text]

Harr B, Schl�tterer C
Gene expression analysis indicates extensive genotype-specific crosstalk between the conjugative F-plasmid and the E. coli chromosome.
BMC Microbiol. 2006;680.
BACKGROUND: Plasmids are an important component of the bacterial genome, but the crosstalk between genes encoded on the chromosome and on the plasmid is still poorly understood. RESULTS: We performed a large-scale survey for genes on the E. coli chromosome that are affected by the presence of the conjugative F-plasmid (crosstalk). The expression pattern of about 4% (107 genes) of the genes encoded by the chromosome was affected by the presence of the F-plasmid. Comparing two different Escherichia coli strains, MG1655 and DH5alpha, we found a strong host genotype-specific crosstalk of the host chromosome with the F-plasmid. About 88% of the genes affected by the presence of the F-plasmid showed a significant plasmid by host genotype interaction, i.e. the presence of the F-plasmid resulted in a different gene expression in the two host genotypes. Less than 12% of the genes showed an additive effect of gene expression, i.e. host genotype independent crosstalk between plasmid and host chromosome. CONCLUSION: We propose that epistatic effects also contribute to the maintenance of F-plasmids in natural populations. [Abstract/Link to Full Text]

Sala�n L, Saunders NJ
Population-associated differences between the phase variable LPS biosynthetic genes of Helicobacter pylori.
BMC Microbiol. 2006;679.
BACKGROUND: Population structures are normally determined using genes under minimal functional selection. In this study we have assessed genes that are not always essential, show differences in alleles between strains, and are involved in the directly host-selectable phenotype of LPS biosynthesis. RESULTS: Eight complete LPS biosynthesis genes, seven of which are associated with phase variation in some or all strains of Helicobacter pylori, have been sequenced and their divergence analyzed. The differences observed indicate that recombination within these genes largely reflects exchange between strains within the population lineages previously determined on the basis of MLST using housekeeping genes. This indicates that the differences that are used for MLST are likely to broadly associate with genes under functional selection, and differences in strain behaviour. However, instances of exchange between the subpopulations were identified, including the hpAfrica2 subpopulation. Further, there were other differences in gene complements and the chromosomal location of genes indicative of greater diversity within the population than is revealed by the available genome sequences and comparative genome hybridization studies. CONCLUSION: These results indicate that the described population structure based upon MLST is broadly a good basis for studying the biology of H. pylori, but that individual alleles may not follow these associations. As a consequence, when working in unsequenced strains, it is necessary to carefully check the presence, sequence, and distribution of any individual gene of interest. [Abstract/Link to Full Text]

Hasan Z, Ashraf M, Tayyebi A, Hussain R
M. leprae inhibits apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 gene expression.
BMC Microbiol. 2006;678.
BACKGROUND: Virulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG. RESULTS: M. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1-4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA. CONCLUSION: This study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression. [Abstract/Link to Full Text]

M�rquez M, Iturriaga EA, Quesada-Moraga E, Santiago-Alvarez C, Monte E, Hermosa R
Detection of potentially valuable polymorphisms in four group I intron insertion sites at the 3'-end of the LSU rDNA genes in biocontrol isolates of Metarhizium anisopliae.
BMC Microbiol. 2006;677.
BACKGROUND: The entomopathogenic anamorphic fungus Metarhizum anisopliae is currently used as a biocontrol agent (BCA) of insects. In the present work, we analyzed the sequence data obtained from group I introns in the large subunit (LSU) of rDNA genes with a view to determining the genetic diversity present in an autochthonous collection of twenty-six M. anisopliae isolates selected as BCAs. RESULTS: DNA fragments corresponding to the 3'-end of the nuclear LSU rDNA genes of 26 M. anisopliae isolates were amplified by PCR. The amplicon sizes ranged from 0.8 to 3.4-kb. Four intron insertion sites, according to Escherichia coli J01695 numbering, were detected--Ec1921, Ec2066, Ec2449 and Ec2563--after sequencing and analysis of the PCR products. The presence/absence of introns allowed the 26 isolates to be distributed into seven genotypes. Nine of the isolates tested showed no introns, 4 had only one, 3 two, and 10 displayed three introns. The most frequent insertion sites were Ec1921 and Ec2449. Of the 26 isolates, 11 showed insertions at Ec2563 and a 1754-bp sequence was observed in ten of them. The most-parsimonious (MP) tree obtained from parsimony analysis of the introns revealed a main set containing four-groups that corresponded to the four insertion sites. CONCLUSION: Four insertion sites of group I introns in the LSU rDNA genes allowed the establishment of seven genotypes among the twenty-six biocontrol isolates of M. anisopliae. Intron insertions at the Ec2563 site were observed for first time in this species. [Abstract/Link to Full Text]

Eldholm V, Matee M, Mfinanga SG, Heun M, Dahle UR
A first insight into the genetic diversity of Mycobacterium tuberculosis in Dar es Salaam, Tanzania, assessed by spoligotyping.
BMC Microbiol. 2006;676.
BACKGROUND: Tanzania has a high tuberculosis incidence, and genotyping studies of Mycobacterium tuberculosis in the country are necessary in order to improve our understanding of the epidemic. Spoligotyping is a potentially powerful genotyping method due to fast generation of genotyping results, high reproducibility and low operation costs. The recently constructed SpolDB4 database and the model-based program 'Spotclust' can be used to assign isolates to families, subfamilies and variants. The results of a study can thus be analyzed in a global context. RESULTS: One hundred forty-seven pulmonary isolates from consecutive tuberculosis patients in Dar es Salaam were spoligotyped. SpolDB4 and 'Spotclust' were used to assign isolates to families, subfamilies and variants. The CAS (37%), LAM (22%) and EAI (17%) families were the most abundant. Despite the dominance of these three families, diversity was high due to variation within M. tuberculosis families. Of the obtained spoligopatterns, 64% were previously unrecorded. CONCLUSION: Spoligotyping is useful to gain an overall understanding of the local TB epidemic. This study demonstrates that the extensive TB epidemic in Dar es Salaam, Tanzania is caused by a few successful M. tuberculosis families, dominated by the CAS family. Import of strains was a minor problem. [Abstract/Link to Full Text]

Torija P, Robles A, Escalante R
Optimization of a large-scale gene disruption protocol in Dictyostelium and analysis of conserved genes of unknown function.
BMC Microbiol. 2006;675.
BACKGROUND: Development of the post-genomic age in Dictyostelium will require the existence of rapid and reliable methods to disrupt genes that would allow the analysis of entire gene families and perhaps the possibility to undertake the complete knock-out analysis of all the protein-coding genes present in Dictyostelium genome. RESULTS: Here we present an optimized protocol based on the previously described construction of gene disruption vectors by in vitro transposition. Our method allows a rapid selection of the construct by a simple PCR approach and subsequent sequencing. Disruption constructs were amplified by PCR and the products were directly transformed in Dictyostelium cells. The selection of homologous recombination events was also performed by PCR. We have constructed 41 disruption vectors to target genes of unknown function, highly conserved between Dictyostelium and human, but absent from the genomes of S. cerevisiae and S. pombe. 28 genes were successfully disrupted. CONCLUSION: This is the first step towards the understanding of the function of these conserved genes and exemplifies the easiness to undertake large-scale disruption analysis in Dictyostelium. [Abstract/Link to Full Text]

Yoke-Fun C, AbuBakar S
Phylogenetic evidence for inter-typic recombination in the emergence of human enterovirus 71 subgenotypes.
BMC Microbiol. 2006;674.
BACKGROUND: Human enterovirus 71 (EV-71) is a common causative agent of hand, foot and mouth disease (HFMD). In recent years, the virus has caused several outbreaks with high numbers of deaths and severe neurological complications. Several new EV-71 subgenotypes were identified from these outbreaks. The mechanisms that contributed to the emergence of these subgenotypes are unknown. RESULTS: Six EV-71 isolates from an outbreak in Malaysia, in 1997, were sequenced completely. These isolates were identified as EV-71 subgenotypes, B3, B4 and C2. A phylogenetic tree that correlated well with the present enterovirus classification scheme was established using these full genome sequences and all other available full genome sequences of EV-71 and human enterovirus A (HEV-A). Using the 5' UTR, P2 and P3 genomic regions, however, isolates of EV-71 subgenotypes B3 and C4 segregated away from other EV-71 subgenotypes into a cluster together with coxsackievirus A16 (CV-A16/G10) and EV-71 subgenotype C2 clustered with CV-A8. Results from the similarity plot analyses supported the clustering of these isolates with other HEV-A. In contrast, at the same genomic regions, a CV-A16 isolate, Tainan5079, clustered with EV-71. This suggests that amongst EV-71 and CV-A16, only the structural genes were conserved. The 3' end of the virus genome varied and consisted of sequences highly similar to various HEV-A viruses. Numerous recombination crossover breakpoints were identified within the non-structural genes of some of these newer EV-71 subgenotypes. CONCLUSION: Phylogenetic evidence obtained from analyses of the full genome sequence supports the possible occurrence of inter-typic recombination involving EV-71 and various HEV-A, including CV-A16, the most common causal agent of HFMD. It is suggested that these recombination events played important roles in the emergence of the various EV-71 subgenotypes. [Abstract/Link to Full Text]

Cunha-Rodrigues M, Portugal S, Febbraio M, Mota MM
Infection by and protective immune responses against Plasmodium berghei ANKA are not affected in macrophage scavenger receptors A deficient mice.
BMC Microbiol. 2006;673.
BACKGROUND: Scavenger receptors (SRs) recognize endogenous molecules modified by pathological processes as well as components of diverse microorganisms. Mice deficient for both SR-AI and II are more susceptible to infections by a variety of bacterial and viral pathogens. RESULTS: Here we show that SR-A deficient mice and wild type mice are equally susceptible to malaria infection both during liver and blood stages. Moreover, like wild type mice, SR-A deficient mice are able to mount a protective immune response against radiation attenuated sporozoites. CONCLUSION: Our results do not reveal a function of SR-A I and II receptors in the Plasmodium berghei ANKA infection, both in the development of CM and parasitemia control. Moreover, these receptors appear not to be required for the establishment of a protective immune response against the malaria liver stages. [Abstract/Link to Full Text]

Erol I, Jeong KC, Baumler DJ, Vykhodets B, Choi SH, Kaspar CW
H-NS controls metabolism and stress tolerance in Escherichia coli O157:H7 that influence mouse passage.
BMC Microbiol. 2006;672.
BACKGROUND: H-NS is a DNA-binding protein with central roles in gene regulation and nucleoid structuring in Escherichia coli. There are over 60 genes that are influenced by H-NS many of which are involved in metabolism. To determine the significance of H-NS-regulated genes in metabolism and stress tolerance, an hns mutant of E. coli O157:H7 was generated (hns::nptI, FRIK47001P) and its growth, metabolism, and gastrointestinal passage compared to the parent strain (43895) and strain FRIK47001P harboring pSC0061 which contains a functional hns and 90-bp upstream of the open-reading frame. RESULTS: The hns mutant grew slower and was non-motile in comparison to the parent strain. Carbon and nitrogen metabolism was significantly altered in the hns mutant, which was incapable of utilizing 42 carbon, and 19 nitrogen sources that the parent strain metabolized. Among the non-metabolized substrates were several amino acids, organic acids, and key metabolic intermediates (i.e., pyruvate) that limit carbon acquisition and energy generation. Growth studies determined that the parent strain grew in LB containing 14 to 15% bile or bile salts, while the hns mutant grew in 6.5 and 9% of these compounds, respectively. Conversely, log-phase cells of the hns mutant were significantly (p < 0.05) more acid tolerant than the parent strain and hns mutant complemented with pSC0061. In mouse passage studies, the parent strain was recovered at a higher frequency (p < 0.01) than the hns mutant regardless of whether log- or stationary-phase phase cells were orally administered. CONCLUSION: These results demonstrate that H-NS is a powerful regulator of carbon and nitrogen metabolism as well as tolerance to bile salts. It is likely that the metabolic impairments and/or the reduced bile tolerance of the E. coli O157:H7 hns mutant decreased its ability to survive passage through mice. Collectively, these results expand the influence of H-NS on carbon and nitrogen metabolism and highlight its role in the ability of O157:H7 strains to respond to changing nutrients and conditions encountered in the environment and its hosts. [Abstract/Link to Full Text]

Yoonim N, Olive C, Pruksachatkunakorn C, Pruksakorn S
Bactericidal activity of M protein conserved region antibodies against group A streptococcal isolates from the Northern Thai population.
BMC Microbiol. 2006;671.
BACKGROUND: Most group A streptococcal (GAS) vaccine strategies have focused on the surface M protein, a major virulence factor of GAS. The amino-terminus of the M protein elicits antibodies, that are both opsonic and protective, but which are type specific. J14, a chimeric peptide that contains 14 amino acids from the M protein conserved C-region at the carboxy-terminus, offers the possibility of a vaccine which will elicit protective opsonic antibodies against multiple different GAS strains. In this study, we searched for J14 and J14-like sequences and the number of their repeats in the C-region of the M protein from GAS strains isolated from the Northern Thai population. Then, we examined the bactericidal activity of J14, J14.1, J14-R1 and J14-R2 antisera against multiple Thai GAS strains. RESULTS: The emm genes of GAS isolates were sequenced and grouped as 14 different J14-types. The most diversity of J14-types was found in the C1-repeat. The J14.1 type was the major sequence in the C2 and C3-repeats. We have shown that antisera raised against the M protein conserved C-repeat region peptides, J14, J14.1, J14-R1 and J14-R2, commonly found in GAS isolates from the Northern Thai population, are able to kill GAS of multiple different emm types derived from an endemic area. The mean percent of bactericidal activities for all J14 and J14-like peptide antisera against GAS isolates were more than 70%. The mean percent of bactericidal activity was highest for J14 antisera followed by J14-R2, J14.1 and J14-R1 antisera. CONCLUSION: Our study demonstrated that antisera raised against the M protein conserved C-repeat region are able to kill multiple different strains of GAS isolated from the Northern Thai population. Therefore, the four conserved "J14" peptides have the potential to be used as GAS vaccine candidates to prevent streptococcal infections in an endemic area. [Abstract/Link to Full Text]

Mahfouz ME, Grayson TH, Dance DA, Gilpin ML
Characterization of the mrgRS locus of the opportunistic pathogen Burkholderia pseudomallei: temperature regulates the expression of a two-component signal transduction system.
BMC Microbiol. 2006;670.
BACKGROUND: Burkholderia pseudomallei is a saprophyte in tropical environments and an opportunistic human pathogen. This versatility requires a sensing mechanism that allows the bacterium to respond rapidly to altered environmental conditions. We characterized a two-component signal transduction locus from B. pseudomallei 204, mrgR and mrgS, encoding products with extensive homology with response regulators and histidine protein kinases of Escherichia coli, Bordetella pertussis, and Vibrio cholerae. RESULTS: The locus was present and expressed in a variety of B. pseudomallei human and environmental isolates but was absent from other Burkholderia species, B. cepacia, B. cocovenenans, B. plantarii, B. thailandensis, B. vandii, and B. vietnamiensis. A 2128 bp sequence, including the full response regulator mrgR, but not the sensor kinase mrgS, was present in the B. mallei genome. Restriction fragment length polymorphism downstream from mrgRS showed two distinct groups were present among B. pseudomallei isolates. Our analysis of the open reading frames in this region of the genome revealed that transposase and bacteriophage activity may help explain this variation. MrgR and MrgS proteins were expressed in B. pseudomallei 204 cultured at different pH, salinity and temperatures and the expression was substantially reduced at 25 degrees C compared with 37 degrees C or 42 degrees C but was mostly unaffected by pH or salinity, although at 25 degrees C and 0.15% NaCl a small increase in MrgR expression was observed at pH 5. MrgR was recognized by antibodies in convalescent sera pooled from melioidosis patients. CONCLUSION: The results suggest that mrgRS regulates an adaptive response to temperature that may be essential for pathogenesis, particularly during the initial phases of infection. B. pseudomallei and B. mallei are very closely related species that differ in their capacity to adapt to changing environmental conditions. Modifications in this region of the genome may assist our understanding of the reasons for this difference. [Abstract/Link to Full Text]

Qin A, Mann BJ
Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2.
BMC Microbiol. 2006;669.
BACKGROUND: Francisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined. METHODS: We constructed a stable transposon mutagenesis library of virulent strain Schu S4 using a derivative of the EZ::TN transposon system. Approximately 2000 mutants were screened for the inability to invade, and replicate in the hepatic carcinoma cell line HepG2. These mutants were also tested for replication within the J774.1 macrophage-like cell line. RESULTS: Eighteen mutants defective in intracellular replication in HepG2 cells were identified. Eight of these mutants were auxotrophs; seven had mutations in nucleotide biosynthesis pathways. The remaining mutants had insertions in genes that were predicted to encode putative transporters, enzymes involved in protein modification and turnover, and hypothetical proteins. A time course of the intracellular growth of a pyrB mutant revealed that this mutant was only able to grow at low levels within HepG2 cells but grew like wild-type bacteria in J774.1 cells. This pyrB mutant was also attenuated in mice. CONCLUSION: This is the first reported large-scale mutagenesis of a type A strain of F. tularensis and the first identification of mutants specifically defective in intracellular growth in a hepatic cell line. We have identified several genes and pathways that are key for the survival and growth of F. tularensis in a hepatic cell line, and a number of novel intracellular growth-defective mutants that have not been previously characterized in other pathogens. Further characterization of these mutants will help provide a better understanding of the pathogenicity of F. tularensis, and may have practical applications as targets for drugs or attenuated vaccines. [Abstract/Link to Full Text]

Gr�ter D, Schmid B, Brandl H
Influence of plant diversity and elevated atmospheric carbon dioxide levels on belowground bacterial diversity.
BMC Microbiol. 2006;668.
BACKGROUND: Changes in aboveground plant species diversity as well as variations of environmental conditions such as exposure of ecosystems to elevated concentrations of atmospheric carbon dioxide may lead to changes in metabolic activity, composition and diversity of belowground microbial communities, both bacterial and fungal. RESULTS: We examined soil samples taken from a biodiversity x CO2 grassland experiment where replicate plots harboring 5, 12, or 31 different plant species had been exposed to ambient or elevated (600 ppm) levels of carbon dioxide for 5 years. Analysis of soil bacterial communities in these plots by temporal temperature gradient gel electrophoresis (TTGE) showed that dominant soil bacterial populations varied only very little between different experimental treatments. These populations seem to be ubiquitous. Likewise, screening of samples on a high-resolution level by terminal restriction fragment length polymorphism (T-RFLP) showed that increased levels of carbon dioxide had no significant influence on both soil bacterial community composition (appearance and frequency of operational taxonomic units, OTUs) and on bacterial richness (total number of different OTUs). In contrast, differences in plant diversity levels had a significant effect on bacterial composition but no influence on bacterial richness. Regarding species level, several bacterial species were found only in specific plots and were related to elevated carbon dioxide or varying plant diversity levels. For example, analysis of T-RFLP showed that the occurrence of Salmonella typhimurium was significantly increased in plots exposed to elevated CO2 (P < 0.05). CONCLUSION: Plant diversity levels are affecting bacterial composition (bacterial types and their frequency of occurrence). Elevated carbon dioxide does not lead to quantitative alteration (bacterial richness), whereas plant diversity is responsible for qualitative changes (bacterial diversity). [Abstract/Link to Full Text]

Serrano I, Melo-Cristino J, Ramirez M
Heterogeneity of pneumococcal phase variants in invasive human infections.
BMC Microbiol. 2006;667.
BACKGROUND: Streptococcus pneumoniae can be carried asymptomatically in the nasopharynx of its human host but can also cause a wide range of infections. A role for pneumococcal phase variants in the different lifestyles of this bacterium has been suggested but no systematic survey of the colony phenotypes of isolates associated with human infections has been undertaken. RESULTS: We report the colony opacity phenotypes of a genetically diverse set of 304 invasive isolates representing 10 serotypes. Over half of the isolates (52%) presented the opaque phenotype whereas transparent variants accounted for only 26% of the total. However, the frequency of recovery of each phase variant was not uniform, while serotypes 1, 4, 12B and 23F presented the opaque phenotype more frequently than expected by chance, serotypes 3 and 14 where less frequently associated with this phenotype. CONCLUSION: The opaque phenotype was the most frequent phenotype found among invasive isolates. An unexpected and equally important finding is the variability of the dominant opacity phenotype found among serotypes. This observation highlights the heterogeneity of opacity phenotypes in invasive isolates and lends further support to the proposal that other factors, in addition to the site of isolation, determine the opacity phenotype of a given isolate. The association between serotype and colonial opacity could help explain epidemiological differences observed among pneumococcal serotypes such as a higher invasive disease potential. [Abstract/Link to Full Text]

Recent Articles in Clinical and Diagnostic Laboratory Immunology

Nardelli DT, Cloute JP, Luk KH, Torrealba J, Warner TF, Callister SM, Schell RF
CD4(+) CD25(+) T cells prevent arthritis associated with Borrelia vaccination and infection.
Clin Diagn Lab Immunol. 2005 Jun;12(6):786-92.
CD4(+) CD25(+) T cells are a population of regulatory T cells associated with control of arthritis in anti-interleukin-17 antibody-treated Borrelia-vaccinated and challenged gamma interferon-deficient mice. Here, we present direct evidence that adoptive transfer of enriched CD4(+) CD25(+) T cells from these mice can prevent the development of arthritis in Borrelia-vaccinated and challenged mice. These findings establish a major role for CD4(+) CD25(+) T cells in the prevention of arthritis in Borrelia-vaccinated and challenged animals. [Abstract/Link to Full Text]

Li F, Stevenson RA, Crabb BS, Studdert MJ, Hartley CA
Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.
Clin Diagn Lab Immunol. 2005 Jun;12(6):778-85.
Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA. [Abstract/Link to Full Text]

Livingstone M, Entrican G, Wattegedera S, Buxton D, McKendrick IJ, Longbottom D
Antibody responses to recombinant protein fragments of the major outer membrane protein and polymorphic outer membrane protein POMP90 in Chlamydophila abortus-infected pregnant sheep.
Clin Diagn Lab Immunol. 2005 Jun;12(6):770-7.
Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans. [Abstract/Link to Full Text]

Levine M, Owen WL, Avery KT
Antibody response to actinomyces antigen and dental caries experience: implications for caries susceptibility.
Clin Diagn Lab Immunol. 2005 Jun;12(6):764-9.
Fluoridated dentifrices reduce dental caries in subjects who perform effective oral hygiene. Actinomyces naeslundii increases in teeth-adherent microbial biofilms (plaques) in these subjects, and a well-characterized serum immunoglobulin G (IgG) antibody response (Actinomyces antibody [A-Ab]) is also increased. Other studies suggest that a serum IgG antibody response to streptococcal d-alanyl poly(glycerophosphate) (S-Ab) may indicate caries experience associated strongly with gingival health and exposure to fluoridated water. The aim of this study was to investigate relationships between A-Ab response, oral hygiene, S-Ab response, and caries experience. Measurements were made of A-Ab and S-Ab concentrations, caries experience (number of decayed, missing, and filled teeth [DMFT], number of teeth surfaces [DMFS], and number of decayed teeth needing treated [DT]), exposure to fluoridated water (Flu), mean clinical pocket depth (PD; in millimeters), and extent of plaque (PL) and gingival bleeding on probing (BOP). A-Ab concentration, the dependent variable in a multiple regression analysis, increased with S-Ab concentration and decreased with PL and DMFT adjusted for Flu (R(2) = 0.51, P < 0.002). Residual associations with age, DMFS, DT, and BOP were not significant. In addition, an elevated A-Ab response, defined from immunoprecipitation and immunoassay measurements, indicated a significant, 30% reduction in DMFT after adjustment for significant age and Flu covariance (analysis of variance with covariance F statistic = 10.6, P < 0.003; S-Ab response and interactions not significant). Thus, an elevated A-Ab response indicates less caries in subjects performing effective oral hygiene using fluoridated dentifrices. Conversely, a low A-Ab response is suggestive of decreased A. naeslundii binding to saliva-coated apatite and greater caries experience, as reported by others. [Abstract/Link to Full Text]

Choi YJ, Lee SH, Park KH, Koh YS, Lee KH, Baik HS, Choi MS, Kim IS, Jang WJ
Evaluation of PCR-based assay for diagnosis of spotted fever group rickettsiosis in human serum samples.
Clin Diagn Lab Immunol. 2005 Jun;12(6):759-63.
A nested PCR assay was developed for the detection of spotted fever group (SFG) rickettsiae in serum samples. The assay was based on specific primers derived from the rickettsial outer membrane protein B gene (rompB) of Rickettsia conorii. An SFG rickettsia-specific signal is obtained from R. akari, R. japonica, R. sibirica, and R. conorii. Other bacterial species tested did not generate any signal, attesting to the specificity of the assay. As few as seven copies of the rompB gene of R. conorii could be detected in 200 microl of serum sample. The assay was evaluated with a panel of sera obtained from patients with acute-phase febrile disease tested by immunofluorescent antibody assay (IFA). The SFG rickettsia-specific DNA fragment was detected in 71 out of 100 sera, which were proven to have immunoglobulin M antibodies against SFG rickettsial antigen by IFA. The results were further confirmed by restriction fragment length polymorphism and sequencing analysis of the DNA fragments. The results indicated that this PCR assay is suitable for the diagnosis of spotted fever group rickettsiosis in Korea. [Abstract/Link to Full Text]

Kashyap RS, Dobos KM, Belisle JT, Purohit HJ, Chandak NH, Taori GM, Daginawala HF
Demonstration of components of antigen 85 complex in cerebrospinal fluid of tuberculous meningitis patients.
Clin Diagn Lab Immunol. 2005 Jun;12(6):752-8.
Tuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. Specifically, there is no generally accepted early confirmative diagnosis protocol available for TBM. Various Mycobacterium tuberculosis antigens are now recognized as potential markers for diagnosis of TBM. However, their presence remains questionable, and many of these antigens are reported in the blood but not in the cerebrospinal fluid (CSF). This study identifies a specific protein marker in CSF which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% (n = 5) of confirmed and 90% (n = 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa band was excised from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Partially purified proteins from CSF samples of TBM were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. The antigen 85 (Ag 85) complex was detected in CSF of TBM patients by indirect ELISA using antibodies against Ag 85 complex. The results of this study showed the 30-kDa protein band contained MTB proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B), both members of the Ag85 complex. This was also confirmed by using immunotechniques such as indirect ELISA and the dot immunobinding assay. Detection of Ag85 complex was observed in CSF of 89% (71 out of 80) of suspected TBM patients that were 30-kDa protein positive. The observed 30-kDa protein in the CSF is comprised of the MTB Ag85 complex. This protein was earlier reported to be present in the blood of patients with extra-central nervous system tuberculosis. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option for the early and confirmatory diagnosis of TBM. [Abstract/Link to Full Text]

Lovrich SD, Jobe DA, Schell RF, Callister SM
Borreliacidal OspC antibodies specific for a highly conserved epitope are immunodominant in human lyme disease and do not occur in mice or hamsters.
Clin Diagn Lab Immunol. 2005 Jun;12(6):746-51.
Humans produce highly specific borreliacidal antibodies against outer surface protein C (OspC) shortly after infection with Borrelia burgdorferi sensu stricto. We previously demonstrated the epitope recognized by immunoglobulin M (IgM) and IgG OspC borreliacidal antibodies was located within the 50 amino acids nearest the carboxy (C) terminus. In this study, we show the immunodominant epitope is located in the highly conserved region within the seven C-terminal amino acids. Six early Lyme disease sera that contained borreliacidal activity and IgM and/or IgG OspC antibodies were chosen randomly and adsorbed with truncated OspC containing the 16 or 7 amino acids nearest the C terminus. Adsorptions with each truncated protein abrogated the borreliacidal activity completely. In addition, only small concentrations of OspC antibodies remained detectable by enzyme-linked immunosorbent assay and Western blotting. Moreover, borreliacidal OspC antibodies were not induced in laboratory mice or hamsters despite heavy infections with B. burgdorferi spirochetes. These findings confirm that borreliacidal antibodies comprise the majority of the IgM and IgG OspC antibody response in human Lyme disease and that the epitope is located in the highly conserved C terminus. In addition, rodent animal models appear to be inappropriate subjects for assessing the effectiveness of the epitope for serodiagnosis or as a human Lyme disease vaccine. [Abstract/Link to Full Text]

Flynn JN, Pistello M, Isola P, Zaccaro L, Del Santo B, Ricci E, Matteucci D, Bendinelli M
Adoptive immunotherapy of feline immunodeficiency virus with autologous ex vivo-stimulated lymphoid cells modulates virus and T-cell subsets in blood.
Clin Diagn Lab Immunol. 2005 Jun;12(6):736-45.
The potential of immunotherapy with autologous virus-specific T cells to affect the course of feline immunodeficiency virus (FIV) infection was explored in a group of specific-pathogen-free cats infected with FIV a minimum of 10 months earlier. Popliteal lymph node cells were stimulated by cocultivation with UV-inactivated autologous fibroblasts infected with recombinant vaccinia viruses expressing either FIV gag or env gene products, followed by expansion in interleukin-2. One or two infusions of both Gag- and Env-stimulated cells resulted in a slow increase in FIV-specific gamma interferon-secreting T cells in the circulation of cats. In the same animals, viral set points fluctuated widely during the first 2 to 3 weeks after adoptive transfer and then returned to pretreatment levels. The preexisting viral quasispecies was also found to be modulated, whereas no novel viral variants were detected. Circulating CD4(+) counts underwent a dramatic decline early after treatment. CD4/CD8 ratios remained instead essentially unchanged and eventually improved in some animals. In contrast, a single infusion of Gag-stimulated cells alone produced no apparent modulations of infection. [Abstract/Link to Full Text]

Waters WR, Palmer MV, Bannantine JP, Greenwald R, Esfandiari J, Andersen P, McNair J, Pollock JM, Lyashchenko KP
Antibody responses in reindeer (Rangifer tarandus) infected with Mycobacterium bovis.
Clin Diagn Lab Immunol. 2005 Jun;12(6):727-35.
Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer. [Abstract/Link to Full Text]

Inostroza J, Villanueva S, Mason K, Leiva LE, Sorensen RU
Effects of absorption with pneumococcal type 22F polysaccharide on maternal, cord blood, and infant immunoglobulin G antipneumococcal polysaccharide antibodies.
Clin Diagn Lab Immunol. 2005 Jun;12(6):722-6.
The aim of this study was to evaluate the effect of absorption with pneumococcal type 22F polysaccharide on antipneumococcal antibody titers in unimmunized Chilean pregnant women and on antibodies in their offspring at birth and 3, 6, and 12 months of age. Sera from 10 healthy pregnant women and from their offspring at birth and at 3, 6, and 12 months of age were studied. Immunoglobulin G antibodies against serotypes 1, 3, 4, 5, 6B, 9V, 14, 18, 19F, and 23F were measured by a standardized enzyme-linked immunosorbent assay method. All sera were absorbed with polysaccharide C, and aliquots of each serum were absorbed with polysaccharide 22F. Individual results were expressed in mug/ml based on the standard serum pool 89-SF. Absorption with polysaccharide 22F reduced antibody concentrations in all samples and to all 10 serotypes studied. Reduction was highest in maternal sera and in cord blood, but it was also present at 3, 6, and 12 months of age. The percent reduction ranged from 24% for serotype 14 to 50% for serotype 1 in maternal samples and from 20% for serotype 18C to 49% for serotype 4 in cord blood samples. The percentages of transplacental transmission were similar for nonabsorbed and absorbed maternal fetal pairs. Absorption with serotype 22F had a significant impact on antipneumococcal antibody concentrations in unimmunized pregnant women and in their offspring. Our results suggest that absorption with 22F polysaccharide needs to be performed in studies of transplacental transmission of antipneumococcal antibodies. [Abstract/Link to Full Text]

Pittman PR, Leitman SF, Oro JG, Norris SL, Marano NM, Ranadive MV, Sink BS, McKee KT
Protective antigen and toxin neutralization antibody patterns in anthrax vaccinees undergoing serial plasmapheresis.
Clin Diagn Lab Immunol. 2005 Jun;12(6):713-21.
Recipients of licensed anthrax vaccine (AVA; Biothrax) could serve as a source of hyperimmune plasma and immunoglobulin for therapy and prophylaxis. We measured serum antibodies during serial weekly to biweekly plasmapheresis in 38 individuals previously vaccinated with 4 to 27 doses of AVA. Immunoglobulin G (IgG) to protective antigen (PA) and toxin neutralization assay (TNA) antibody levels were highly correlated (r = 0.86930 and P < 0.0001 for anti-PA concentration versus TNA concentration). Significant decreases in antibody titer and concentration were observed over time when compared for the number of days from the last AVA injection (P < 0.0001 for both anti-PA and TNA concentration) and for the number of days from the first plasmapheresis (P = 0.0007 for anti-PA concentration and P = 0.0025 for TNA concentration). The rate of the decrease in total IgG concentration (half-life [t(1/2)] = 198.90 days after first plasmapheresis) was significantly less than the decrease in anti-PA IgG (t(1/2) = 63.53 days) (P < 0.0001), indicating that the reduction in anti-PA IgG was more likely due to natural decay than plasmapheresis. The time since the last injection and the time after initial plasmapheresis are important elements in considering an optimal schedule for collecting anthrax hyperimmune plasma. Good correlation between IgG to PA and TNA antibodies suggests that the anti-PA enzyme-linked immunosorbent assay can be used as a high-throughput screen for functional immune reactivity in donor plasma units. [Abstract/Link to Full Text]

Merlo A, Tenca C, Fais F, Battini L, Ciccone E, Grossi CE, Saverino D
Inhibitory receptors CD85j, LAIR-1, and CD152 down-regulate immunoglobulin and cytokine production by human B lymphocytes.
Clin Diagn Lab Immunol. 2005 Jun;12(6):705-12.
Class switching consists in the substitution of the heavy-chain constant region of immunoglobulin M (IgM) with that of IgG, IgA, or IgE. This enables antibodies to acquire new effector functions that are crucial to combat invading pathogens. Class switching usually requires engagement of CD40 on B cells by CD40 ligand (CD40L) on antigen-activated CD4(+) T cells and the production of cytokines. The process must be regulated tightly because abnormal IgG and IgA production favors the onset of autoimmunity, whereas increased switching to IgE leads to atopy. These inflammatory disorders can be triggered or exacerbated by costimulatory signals. Although thoroughly investigated on T cells, the roles of the inhibitory receptors CD85j, LAIR-1, and CD152 on B-cell functions have not been fully elucidated. In this study we show that cross-linking of the B-cell inhibitory receptors by specific monoclonal antibodies inhibits IgG and IgE production, reduces the percentage of IgG- and IgE-expressing B cells, and down-regulates interleukin 8 (IL-8), IL-10, and tumor necrosis factor alpha production. These effects were demonstrated using different B-cell stimulatory pathways (recall antigens, CD40L-transfected cells plus IL-4, and lipopolysaccharide plus IL-4). It thus appears that CD85j, LAIR-1, and CD152 play a central role for the control of IL-4-driven isotype switching. [Abstract/Link to Full Text]

Ampel NM, Nelson DK, Chavez S, Naus KA, Herman AB, Li L, Simmons KA, Pappagianis D
Preliminary evaluation of whole-blood gamma interferon release for clinical assessment of cellular immunity in patients with active coccidioidomycosis.
Clin Diagn Lab Immunol. 2005 Jun;12(6):700-4.
Assessment of the cellular immune response in coccidioidomycosis has epidemiologic and prognostic importance. Measurement of delayed-type hypersensitivity to skin testing has been used in the past to determine cellular immunity in coccidioidomycosis. However, no skin tests are currently available in the United States. Assay of gamma interferon (IFN-gamma) release in whole blood in response to incubation with antigen has been used to assess cellular immunity in tuberculosis. We used a similar assay using the coccidioidal antigen preparation T27K to measure the in vitro cellular immune responses among a cohort of 69 subjects with active coccidioidomycosis. IFN-gamma release was bimodal, with concentrations above and below 5 IU/ml. Using multivariate logistic regression, underlying disease and disseminated or chronic pulmonary coccidioidomycosis was significantly associated with the release of IFN-gamma at a concentration of <5 IU/ml (P = 0.02 or 0.05, respectively). In addition, the release IFN-gamma concentration was <5 IU/ml in all subjects with a clinical severity score of > or =6 (P = 0.02). The release IFN-gamma concentration correlated with expression of CD69 on T lymphocytes in an in vitro assay using T27K as the antigen (Spearman's rho = 0.59; P < 0.01). These results suggest that the IFN-gamma release assay with T27K as the antigen may be a useful clinical test for assessing cellular immunity in patients with active coccidioidomycosis. [Abstract/Link to Full Text]

Kroll JJ, Eichmeyer MA, Schaeffer ML, McOrist S, Harris DL, Roof MB
Lipopolysaccharide-based enzyme-linked immunosorbent assay for experimental use in detection of antibodies to Lawsonia intracellularis in pigs.
Clin Diagn Lab Immunol. 2005 Jun;12(6):693-9.
An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis. [Abstract/Link to Full Text]

Collins MT, Wells SJ, Petrini KR, Collins JE, Schultz RD, Whitlock RH
Evaluation of five antibody detection tests for diagnosis of bovine paratuberculosis.
Clin Diagn Lab Immunol. 2005 Jun;12(6):685-92.
Five diagnostic tests based on enzyme-linked immunosorbent assay (ELISA) technology for bovine paratuberculosis were evaluated by using individual serum or milk samples from 359 dairy cattle in seven paratuberculosis-free herds and 2,094 dairy cattle in seven Mycobacterium paratuberculosis-infected dairy herds. Three independent laboratories using three different culture procedures completed fecal cultures for M. paratuberculosis on these cattle and found 417 cows to be shedding M. paratuberculosis in their feces. An animal that was fecal culture positive for M. paratuberculosis by any of the three laboratories was considered a confirmed case of infection. The specificity of three ELISAs (two on serum and one on milk) was > or =99.8%. The specificity of the remaining two ELISAs, both done on serum, was 94.9 and 84.7%. Four of the five ELISAs evaluated produced similar sensitivity in detecting fecal culture-positive cattle (27.8 to 28.9%). Serum ELISA "D" had the lowest specificity (84.7%) and the highest sensitivity (44.5%), but if the cutoff value defining a positive test was changed from 125 to 250% (of the positive control) the sensitivity and specificity, 31.8 and 97.5%, respectively, were comparable to those of the other four assays. If the case definition for M. paratuberculosis infection was based on the culture results of a single laboratory instead of the combined results of three laboratories, ELISA sensitivity estimates were 45.7 to 50.0%. With the exception of ELISA D, assay agreement was high (kappa 0.66 to 0.85) for categorical assay interpretations (positive or negative), but linear regression of quantitative results showed low correlation coefficients (r(2) = 0.40 to 0.68) due to the fact that ELISA results for some cows were high in one assay but low in another assay. Likelihood ratio analysis showed a direct relationship between the magnitude of ELISA result and the odds of a cow shedding M. paratuberculosis in its feces. If used judiciously and interpreted quantitatively, these ELISAs are useful tools in support of paratuberculosis control programs in dairy herds. [Abstract/Link to Full Text]

Vel�zquez B, Massaldi H, Battistoni J, Chabalgoity JA
Construction and expression of recombinant streptolysin-o and preevaluation of its use in immunoassays.
Clin Diagn Lab Immunol. 2005 May;12(5):683-4.
Commercially available immunoassays for assessment of anti-streptolysin-O antibodies use native streptolysin-O obtained by a complex process. We prepared a biologically active recombinant streptolysin-O with higher yield and a simpler purification process. An enzyme-linked immunosorbent assay developed with this recombinant showed good correlation with a commercial test, suggesting that it could be suitable for immunoassays. [Abstract/Link to Full Text]

Seet RC, Lau LG, Tambyah PA
Strongyloides hyperinfection and hypogammaglobulinemia.
Clin Diagn Lab Immunol. 2005 May;12(5):680-2.
We report strongyloides hyperinfection in two patients with generalized hypogammaglobulinemia from multiple myeloma and nephrotic syndrome, despite a significant strongyloides-specific immunoglobulin G (IgG) response. In contrast to reports on animals, where human IgG was shown to be a protective antibody, our observation suggests that in humans, immunity to the infective-stage larvae is not protective against the autoinfective larvae, which are the causative agents of strongyloides hyperinfection. [Abstract/Link to Full Text]

Wildner G, Diedrichs-Moehring M
Multiple autoantigen mimotopes of infectious agents induce autoimmune arthritis and uveitis in lewis rats.
Clin Diagn Lab Immunol. 2005 May;12(5):677-9.
We found multimolecular antigenic mimicry of arthritogenic autoantigens and peptides from several other "self" or foreign antigens sharing amino acid sequence homologies. Many of these new mimotopes induced arthritis and/or uveitis upon immunization in Lewis rats, indicating a role for multiple antigens in the initiation of a certain autoimmune disease. [Abstract/Link to Full Text]

Matsunaga H, Tanaka S, Sasao F, Nishino Y, Takeda M, Tomonaga K, Ikuta K, Amino N
Detection by radioligand assay of antibodies against Borna disease virus in patients with various psychiatric disorders.
Clin Diagn Lab Immunol. 2005 May;12(5):671-6.
Using a radioligand assay, which preserves the natural form of the antigen, antibodies against Borna disease virus nucleoprotein and phosphoprotein were detected in 11 and 19 sera of 171 psychiatric patients, respectively. Compared with results by Western blotting, three and nine sera were concordantly positive, respectively. The four sera showing the highest levels of antibodies by radioligand assay were all negative by Western blotting; however, dilution and inhibition tests supported the positive results. Our results suggest the importance of conformational structure to detect human anti-Borna disease virus antibodies. [Abstract/Link to Full Text]

Kremer JR, Muller CP
Evaluation of commercial assay detecting specific immunoglobulin g in oral fluid for determining measles immunity in vaccinees.
Clin Diagn Lab Immunol. 2005 May;12(5):668-70.
A commercial assay for detection of measles immunoglobulin G (IgG) in oral fluid was evaluated in a highly vaccinated cohort using serum IgG as gold standard. In contrast to previous studies from cohorts protected by natural immunity, antibody prevalence was significantly underestimated (-7.4%; confidence interval: -1.5 to -13.2%; P = 0.01) due to a reduced sensitivity when antibody levels were low. [Abstract/Link to Full Text]

Gibbs SE, Hoffman DM, Stark LM, Marlenee NL, Blitvich BJ, Beaty BJ, Stallknecht DE
Persistence of antibodies to West Nile virus in naturally infected rock pigeons (Columba livia).
Clin Diagn Lab Immunol. 2005 May;12(5):665-7.
Wild caught rock pigeons (Columba livia) with antibodies to West Nile virus were monitored for 15 months to determine antibody persistence and compare results of three serologic techniques. Antibodies persisted for the entire study as detected by epitope-blocking enzyme-linked immunosorbent assay and plaque reduction neutralization test. Maternal antibodies in squabs derived from seropositive birds persisted for an average of 27 days. [Abstract/Link to Full Text]

Formento JL, Berra E, Ferrua B, Magn� N, Simos G, Brahimi-Horn C, Pouyss�gur J, Milano G
Enzyme-linked immunosorbent assay for pharmacological studies targeting hypoxia-inducible factor 1alpha.
Clin Diagn Lab Immunol. 2005 May;12(5):660-4.
Hypoxia-inducible factor 1 (HIF-1) activates the transcription of a wide range of genes related to oxygen delivery and metabolic adaptation under hypoxic (low-oxygen) conditions. HIF-1 is, in fact, a heterodimer of two subunits, HIF-1alpha and HIF-1beta. The only analytical methods available for measuring HIF-1alpha levels in tumors are immunohistochemistry and Western blotting. Immunohistochemistry has the advantage of allowing the identification and direct examination of HIF-1alpha-expressing cells, but has the intrinsic limitation, as for Western blotting, of being nonquantitative. We developed and validated an enzyme-linked immunosorbent assay (ELISA) approach to measure HIF-1alpha levels in cultured tumor cell lines in vitro. HIF-1alpha was expressed in thirteen tumor cell lines grown under hypoxic conditions; however, the levels differed strongly between cell lines. These data point to intrinsic differences between cell lines for the induction of HIF-1alpha under hypoxic conditions. The ELISA developed in the present study is thus an interesting alternative to other analytical methods used to measure HIF-1alpha protein levels and should be useful in preclinical pharmacological studies targeting HIF-1alpha. [Abstract/Link to Full Text]

Paldanius M, Bloigu A, Alho M, Leinonen M, Saikku P
Prevalence and persistence of Chlamydia pneumoniae antibodies in healthy laboratory personnel in Finland.
Clin Diagn Lab Immunol. 2005 May;12(5):654-9.
The rates of Chlamydia pneumoniae seroconversions suggesting acute primary infections or reinfections and the prevalences of antibodies were followed up among healthy laboratory workers. Annual serum samples were collected from 47 persons in Helsinki from 1958 to 1990 and from 40 persons in Oulu from 1994 to 1999. C. pneumoniae species-specific immunoglobulin G (IgG), IgA, and IgM antibodies were measured by microimmunofluorescence (MIF) in 407 sera from Helsinki. The 185 sera collected in Oulu were tested both by MIF and by commercial enzyme immunoassay (EIA). During the follow-up periods of 31 years in Helsinki and 6 years in Oulu, seroconversions were demonstrated by MIF in 45% and 15% of the study groups, respectively. In Helsinki 9% of the persons seroconverted twice during the follow-up period. By MIF, the total incidence rate per 100 person-years at risk was 6.9 in Helsinki and 4.9 in Oulu, and annual incidence rates varied from 0 to 15.4. By EIA, annual incidence rates in Oulu varied from 0 to 10.8. The seroconversions by MIF were usually not confirmed by EIA and vice versa. Prevalence and persistence rates, respectively, of IgA antibodies were higher in EIA (62% and 26%) than in MIF (26% and 17%), whereas the figures for IgG were quite similar. The prevalence of IgG and IgA antibodies was higher in older persons than in younger ones. The presence of antibodies did not offer protection from reinfection. [Abstract/Link to Full Text]

Boarino A, Scalone A, Gradoni L, Ferroglio E, Vitale F, Zanatta R, Giuffrida MG, Rosati S
Development of recombinant chimeric antigen expressing immunodominant B epitopes of Leishmania infantum for serodiagnosis of visceral leishmaniasis.
Clin Diagn Lab Immunol. 2005 May;12(5):647-53.
Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts. [Abstract/Link to Full Text]

Spencer JA, Deinnocentes P, Moyana EM, Guarino AJ, Ellison SE, Bird RC, Blagburn BL
Cytokine gene expression in response to SnSAG1 in horses with equine protozoal myeloencephalitis.
Clin Diagn Lab Immunol. 2005 May;12(5):644-6.
Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome seen in horses from the Americas and is mainly caused by Sarcocystis neurona. Recently, a 29-kDa surface antigen from S. neurona merozoites was identified as being highly immunodominant on a Western blot. This antigen has been sequenced and cloned, and the expressed protein has been named SnSAG1. In a previous study, cell-mediated immune responses to SnSAG1 were shown to be statistically significantly reduced in horses with EPM in comparison to EPM-negative control horses. It therefore appears as though the parasite is able to induce immunosuppression towards parasite-derived antigens as parasite-specific responses are decreased. Isolated peripheral blood lymphocytes from 21 EPM (cerebrospinal fluid [CSF] Western blot)-negative horses with no clinical signs and 21 horses with clinical signs of EPM (CSF Western blot positive) were cocultured with SnSAG1 for 48 and 72 h, and the effect on cytokine production was investigated by means of reverse transcriptase PCR. Cytokines assayed include gamma interferon (IFN-gamma), tumor necrosis factor alpha, interleukin (IL)-2, IL-4, and IL-6. beta-Actin was used as the housekeeping gene. A Wilcoxon signed-rank test of the findings indicated that there was a statistically significant decrease in IFN-gamma production after 48 h in culture for samples from horses with clinical disease. There was also a statistically significant increase in IL-4 production after 72 h in culture for samples from horses with EPM. These results further support the notion that this parasite is able to subvert the immune system in horses with clinical disease. [Abstract/Link to Full Text]

Carey VJ, Pahwa S, Weinberg A
Reliability of CD4 quantitation in human immunodeficiency virus-positive children: implications for definition of immunologic response to highly active antiretroviral therapy.
Clin Diagn Lab Immunol. 2005 May;12(5):640-3.
Our objective was to develop data-based algorithms for definition of immunologic response to AIDS therapies in pediatric patients, taking account of T-cell subset measurement errors. The study design involved cross-protocol analysis of 2,148 enrollees in six completed Pediatric AIDS Clinical Trials Group trials. We used standard quantitation of T-cell subsets; linear modeling with mean-dependent measurement error variance was used to develop 95% tolerance limits for change in CD4%. For individuals with a CD4% of approximately 25%, the measurement error-based 95% tolerance interval ranges from 15% to 35%, whereas for individuals with a CD4% of approximately 5%, the tolerance interval ranges from 3% to 7%. When pairs of CD4% measures taken within a time interval of less than 30 days are averaged to estimate steady-state CD4%, tolerance interval width decreases by approximately 30%. A simple graphical tool that provides a data-based criterion for immunologic response over and above variation ascribable to T-cell measurement error is provided. Variability in CD4% due to measurement error is substantial, increases with level of CD4%, and complicates assessment of immunologic response to therapy. Replicates of CD4% measures could be used to improve precision of interpretation of CD4% measures. [Abstract/Link to Full Text]

Higgins DP, Hemsley S, Canfield PJ
Association of uterine and salpingeal fibrosis with chlamydial hsp60 and hsp10 antigen-specific antibodies in Chlamydia-infected koalas.
Clin Diagn Lab Immunol. 2005 May;12(5):632-9.
Infection by Chlamydia pneumoniae or Chlamydia pecorum commonly causes chronic, fibrotic disease of the urogenital tracts of female koalas. Studies of humans have associated titers of serum immunoglobulin G (IgG) against chlamydial hsp60 and hsp10 antigens with chronic infection, salpingeal fibrosis, and tubal infertility. To determine whether a similar relationship exists in Chlamydia-infected koalas, samples were collected opportunistically from 34 wild female koalas and examined by gross pathology and histopathology, PCR, and immunohistochemistry for Chlamydia spp. and enzyme-linked immunosorbent assay for serological responses to chlamydial hsp10 and hsp60 antigens. Greater anti-hsp titers occurred in Chlamydia-infected koalas with fibrous occlusion of the uterus or uterine tube than in other Chlamydia-infected koalas (for hsp10 IgG, P = 0.005; for hsp60 IgG, P = 0.001; for hsp10 IgA, P = 0.04; for hsp60 IgA, P = 0.09). However, as in humans, some koalas with tubal occlusion had low titers. Among Chlamydia-infected koalas with tubal occlusion, those with low titers were more likely to have an active component to their ongoing uterine or salpingeal inflammation (P = 0.007), such that the assay predicted, with 79% sensitivity and 92% specificity, tubal occlusion where an active component of inflammation was absent. Findings of this study permit advancement of clinical and epidemiological studies of host-pathogen-environment interactions and pose intriguing questions regarding the significance of the Th1/Th2 paradigm and antigen-presenting and inflammation-regulating capabilities of uterine epithelial cells and the roles of latency and reactivation of chlamydial infections in pathogenesis of upper reproductive tract disease of koalas. [Abstract/Link to Full Text]

Lambert JS, Moye J, Plaeger SF, Stiehm ER, Bethel J, Mofenson LM, Mathieson B, Kagan J, Rosenblatt H, Paxton H, Suter H, Landay A
Association of selected phenotypic markers of lymphocyte activation and differentiation with perinatal human immunodeficiency virus transmission and infant infection.
Clin Diagn Lab Immunol. 2005 May;12(5):622-31.
This study of a subset of women and infants participating in National Institutes of Health Pediatric AIDS Clinical Trials Group protocol 185 evaluated lymphocyte phenotypic markers of immune activation and differentiation to determine their association with the likelihood of human immunodeficiency virus (HIV) transmission from the women to their infants and the potential for early identification and/or prognosis of infection in the infants. Lymphocytes from 215 human immunodeficiency virus type 1 (HIV)-infected women and 192 of their infants were analyzed by flow cytometry with an extended three-color panel of monoclonal antibodies. Women who did not transmit to their infants tended to have higher CD4+ T cells. Most notably, levels of total CD8+ T cells and CD8+ CD38+ cells made significant independent contributions to predicting the risk of mother-to-child transmission. Adjusting for HIV-1 RNA level at entry, a one percentage-point increase in these marker combinations was associated with a nine percent increase in the likelihood of maternal transmission. Total as well as naive CD4+ T cells were significantly higher in uninfected than infected infants. Total CD8+ cells, as well as CD8+ cells positive for HLA-DR+, CD45 RA+ HLA-DR+, and CD28+ HLA-DR+ were elevated in infected infants. Detailed immunophenotyping may be helpful in predicting which pregnant HIV-infected women are at increased risk of transmitting HIV to their infants. Increasing differences in lymphocyte subsets between infected and uninfected infants became apparent as early as six weeks of age. Detailed immunophenotyping may be useful in supporting the diagnosis of HIV infection in infants with perinatal HIV exposure. [Abstract/Link to Full Text]

Abel K, Wang Y, Fritts L, Sanchez E, Chung E, Fitzgerald-Bocarsly P, Krieg AM, Miller CJ
Deoxycytidyl-deoxyguanosine oligonucleotide classes A, B, and C induce distinct cytokine gene expression patterns in rhesus monkey peripheral blood mononuclear cells and distinct alpha interferon responses in TLR9-expressing rhesus monkey plasmacytoid dendritic cells.
Clin Diagn Lab Immunol. 2005 May;12(5):606-21.
To determine if deoxycytidyl-deoxyguanosine oligonucleotides (CpG ODN) can be used effectively as nonspecific inducers of innate immune defenses for preventative or therapeutic interventions in infectious disease models for nonhuman primates, the present study evaluated the response of rhesus monkey peripheral blood mononuclear cells to three different synthetic CpG ODN classes by defining the cytokine gene expression patterns and by characterizing IFN-alpha/beta responses. Depending on the type and dose of CpG ODN used for stimulation, distinct gene expression patterns were induced. CpG ODN class A (CpG-A ODN) and CpG-C ODN, but not CpG-B ODN, were potent inducers of alpha interferon (IFN-alpha), and this response was due to IFN-alpha production by TLR9-positive plasmacytoid dendritic cells. Importantly, there was a dose-dependent increase in IFN-alpha responses to CpG-A ODN but a dose-dependent decrease in IFN-alpha responses by CpG-B ODN. The most sustained IFN-alpha response was induced by CpG-A ODN and was associated with a stronger induction of interferon regulatory factor 7 and the induction of several interferon-stimulated genes. In contrast, and independent of the dose, CpG-B ODN were the weakest inducers of IFN-alpha but the most potent inducers of proinflammatory cytokines. CpG-C ODN induced cytokine gene expression patterns that were intermediate between those of CpG-A and CpG-B ODN. Thus, the different types of CpG ODN induce different post-TLR9 signaling pathways that result in distinct cytokine gene expression patterns. Based on these findings, A and C class CpG ODN, but not B class CpG ODN, may be particularly suited for use as therapeutic or prophylactic antiviral interventions. [Abstract/Link to Full Text]

Aaberge IS, Oster P, Helland OS, Kristoffersen AC, Ypma E, H�iby EA, Feiring B, N�kleby H
Combined administration of meningococcal serogroup B outer membrane vesicle vaccine and conjugated serogroup C vaccine indicated for prevention of meningococcal disease is safe and immunogenic.
Clin Diagn Lab Immunol. 2005 May;12(5):599-605.
MenBvac and Menjugate are safe and efficacious vaccines. The purpose of this study was to evaluate safety and immunogenicity of the combination (MenB/C) of the lyophilized active components of the conjugated group C vaccine Menjugate when reconstituted with the full liquid group B outer membrane vesicle vaccine MenBvac compared to MenBvac and Menjugate given separately. At 6-week intervals, healthy adults were given one dose of MenB/C followed by two doses of MenBvac (MenB/C group), three doses of MenBvac (MenB group), or one dose of Menjugate and two doses of placebo (MenC group). Injection site reactions were frequent in all groups. However, most reactions were short lasting and mild or moderate in intensity, and the vaccines were found to be well tolerated, with no vaccine-related serious adverse events. MenB/C was immunogenic with regard to both serogroup B and C meningococci. Both the serum bactericidal assay and the enzyme-linked immunosorbent assay analyses showed that the immune responses of the combination vaccine were similar to the immune responses of its separate components MenBvac and Menjugate for both serogroup B and C. In conclusion, the combined MenB/C vaccine is safe and immunogenic. The two vaccines do not interact negatively with each other and can easily be administered in the same syringe. The induced immune responses suggest that the combined vaccine is likely to confer protection against systemic group B disease caused by the vaccine strain as well as against group C meningococcal disease. [Abstract/Link to Full Text]

Chen HY, Lu Y, Howard T, Anderson D, Fong PY, Hu WP, Chia CP, Guan M
Comparison of a new immunochromatographic test to enzyme-linked immunosorbent assay for rapid detection of immunoglobulin m antibodies to hepatitis e virus in human sera.
Clin Diagn Lab Immunol. 2005 May;12(5):593-8.
An immunochromatographic test for rapid detection of IgM antibodies in patients with acute hepatitis E infection was developed utilizing the well-characterized recombinant protein EP2.1 and monoclonal antibody 4B2. The new rapid test based on a novel reverse-flow technology was able to generate a positive result within 2 to 3 min. Our study showed that this test was able to detect anti-HEV IgM antibodies in 96.7% of the patient samples tested (n = 151) while maintaining an excellent specificity of 98.6% with samples from various patient or healthy control groups (total n = 208). Furthermore, this rapid test gave a good specificity of 90.9% when tested with rheumatoid factor (RF)-positive sera (RF value of < or =850 IU/ml; n = 11) although a higher concentration of RF in samples might cause cross-reactivity. The new test has a good agreement of 97.2% with a kappa value of 0.943 when compared with a reference enzyme-linked immunosorbent assay. The positive predictive value and the negative predictive value for the rapid test thus reached 98.0 and 97.6%, respectively. This is the first rapid, point-of-care test for hepatitis E and will be especially useful for the diagnosis of acute hepatitis E virus infection in field and emergency settings and in resource-poor countries. [Abstract/Link to Full Text]

Giardina PC, Longworth E, Evans-Johnson RE, Bessette ML, Zhang H, Borrow R, Madore D, Fernsten P
Analysis of human serum immunoglobulin G against O-acetyl-positive and O-acetyl-negative serogroup W135 meningococcal capsular polysaccharide.
Clin Diagn Lab Immunol. 2005 May;12(5):586-92.
The capsular polysaccharide of Neisseria meningitidis serogroup W135 is expressed in both O-acetyl-positive (OA+) and O-acetyl-negative (OA-) forms. This study investigates the impact of OA status (OA+ versus OA-) on serological measurements of anti-W135 immunoglobulin G (IgG) antibodies in immunized adults. W135-specific serum antibody assignments were made for 28 postimmunization sera from adults by enzyme-linked immunosorbent assay using the meningococcal standard reference serum CDC1992. The established IgG concentration in micrograms per milliliter ([IgG]microg/ml) for CDC1992 against OA+ antigen (16.2 microg/ml) was used as a reference to assign a concentration of 10.13 microg/ml IgG against OA- antigen by cross-standardization. Overall, the IgG assignments for these sera were higher against OA+ antigen (geometric mean concentration [GMC] = 7.16 microg/ml) than against OA- antigen (GMC = 2.84 microg/ml). However, seven sera showed higher specific [IgG]microg/ml values against the OA+ antigen than against the OA- antigen. These sera were also distinguished by the inability of fluid-phase OA- antigen to compete for antibody binding to OA+ solid-phase antigen. Although there was no overall difference in functional activity measured by complement-mediated serum bactericidal assay (SBA) against OA+ and OA- target bacteria (geometric mean titers of 9,642 and 9,045, respectively), three serum specimens showed a large difference in SBA antibody titers against OA+ versus OA- W135 target bacteria, which may reflect different epitope specificities for these sera. Our data indicate that, for some sera, the agreement in anti-OA+ versus anti-OA- W135 IgG assignments is serum specific and does not reflect the functional (killing) activity in vitro. [Abstract/Link to Full Text]

Jeong KY, Kim WK, Lee JS, Lee J, Lee IY, Kim KE, Park JW, Hong CS, Ree HI, Yong TS
Immunoglobulin E reactivity of recombinant allergen Tyr p 13 from Tyrophagus putrescentiae homologous to fatty acid binding protein.
Clin Diagn Lab Immunol. 2005 May;12(5):581-5.
The storage mite, Tyrophagus putrescentiae, is one of the important causes of allergic disorders. Fifteen allergenic components were demonstrated in storage mite by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, but only the group 2 allergen Tyr p 2 has been cloned and characterized. In this study, we attempted to identify and characterize new allergens from T. putrescentiae, which is a dominant species of storage mite in Korea. Expressed sequence tags were analyzed to identify possible storage mite allergens, and the cDNA sequence encoding a protein homologous to fatty acid binding protein, a mite group 13 allergen, was identified and named Tyr p 13. Its deduced amino acid sequence showed 61.1 to 85.3% identity with other mite group 13 allergens. The recombinant protein was expressed in Escherichia coli using a pET 28b vector system, and its allergenicity was investigated by enzyme-linked immunosorbent assay (ELISA). The recombinant allergen was detected in 5 of 78 (6.4%) T. putrescentiae-positive sera tested, and it inhibited 61.9% of immunoglobulin E binding to crude extract at an inhibitor concentration of 10 mug/ml by inhibition ELISA using serum from the patient who showed the strongest reaction by ELISA. In this study, a novel allergen was identified in T. putrescentiae. This allergen could be helpful for more-detailed characterizations of storage mite allergy. [Abstract/Link to Full Text]

Chevrier MR, Ryan AE, Lee DY, Zhongze M, Wu-Yan Z, Via CS
Boswellia carterii extract inhibits TH1 cytokines and promotes TH2 cytokines in vitro.
Clin Diagn Lab Immunol. 2005 May;12(5):575-80.
Traditional herbal formulas used to treat inflammatory arthritis in China and India include Boswellia carterii or Boswellia serrata. They both contain boswellic acids (BAs) which have been shown to exhibit anti-inflammatory and antiarthritic properties. This study tests the hypothesis that mixtures of BAs derived from B. carterii have immunomodulatory properties. B. carterii plant resin obtained from China was prepared as an ethanol extract, and the presence of seven BAs was confirmed by column chromatography, high-performance liquid chromatography, and UV laser desorption/ionization tandem mass spectroscopy. The extract was then tested for its ability to alter in vitro production of TH1 cytokines (interleukin-2 [IL-2] and gamma interferon) and TH2 cytokines (IL-4 and IL-10) by murine splenocytes. Delivery of the resin extract using ethanol as a solvent resulted in significant cellular toxicity not seen with the addition of ethanol alone. By contrast, delivery of the resin extract using a sesame oil solvent resulted in a dose-dependent inhibition of TH1 cytokines coupled with a dose-dependent potentiation of TH2 cytokines. These results indicate that a purified mixture of BAs from B. carterii plant resin exhibits carrier-dependent immunomodulatory properties in vitro. [Abstract/Link to Full Text]

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Recent Articles in Clinical Microbiology Reviews

Tanyuksel M, Petri WA
Laboratory diagnosis of amebiasis.
Clin Microbiol Rev. 2003 Oct;16(4):713-29.
The detection of Entamoeba histolytica, the causative agent of amebiasis, is an important goal of the clinical microbiology laboratory. To assess the scope of E. histolytica infection, it is necessary to utilize accurate diagnostic tools. As more is discovered about the molecular and cell biology of E. histolytica, there is great potential for further understanding the pathogenesis of amebiasis. Molecular biology-based diagnosis may become the technique of choice in the future because establishment of these protozoa in culture is still not a routine clinical laboratory process. In all cases, combination of serologic tests with detection of the parasite (by antigen detection or PCR) offers the best approach to diagnosis, while PCR techniques remain impractical in many developing country settings. The detection of amebic markers in serum in patients with amebic colitis and liver abscess appears promising but is still only a research tool. On the other hand, stool antigen detection tests offer a practical, sensitive, and specific way for the clinical laboratory to detect intestinal E. histolytica. All the current tests suffer from the fact that the antigens detected are denatured by fixation of the stool specimen, limiting testing to fresh or frozen samples. [Abstract/Link to Full Text]

Kocan KM, de la Fuente J, Guglielmone AA, Mel�ndez RD
Antigens and alternatives for control of Anaplasma marginale infection in cattle.
Clin Microbiol Rev. 2003 Oct;16(4):698-712.
Anaplasmosis, a tick-borne cattle disease caused by the rickettsia Anaplasma marginale, is endemic in tropical and subtropical areas of the world. The disease causes considerable economic loss to both the dairy and beef industries worldwide. Analyses of 16S rRNA, groESL, and surface proteins have resulted in the recent reclassification of the order Rickettsiales. The genus Anaplasma, of which A. marginale is the type species, now also includes A. bovis, A. platys, and A. phagocytophilum, which were previously known as Ehrlichia bovis, E. platys, and the E. phagocytophila group (which causes human granulocytic ehrlichiosis), respectively. Live and killed vaccines have been used for control of anaplasmosis, and both types of vaccines have advantages and disadvantages. These vaccines have been effective in preventing clinical anaplasmosis in cattle but have not blocked A. marginale infection. Thus, persistently infected cattle serve as a reservoir of infective blood for both mechanical transmission and infection of ticks. Advances in biochemical, immunologic, and molecular technologies during the last decade have been applied to research of A. marginale and related organisms. The recent development of a cell culture system for A. marginale provides a potential source of antigen for the development of improved killed and live vaccines, and the availability of cell culture-derived antigen would eliminate the use of cattle in vaccine production. Increased knowledge of A. marginale antigen repertoires and an improved understanding of bovine cellular and humoral immune responses to A. marginale, combined with the new technologies, should contribute to the development of more effective vaccines for control and prevention of anaplasmosis. [Abstract/Link to Full Text]

Ewing SA, Panciera RJ
American canine hepatozoonosis.
Clin Microbiol Rev. 2003 Oct;16(4):688-97.
American canine hepatozoonosis (ACH) is a tick-borne disease that is spreading in the southeastern and south-central United States. Characterized by marked leukocytosis and periosteal bone proliferation, ACH is very debilitating and often fatal. Dogs acquire infection by ingesting nymphal or adult Gulf Coast ticks (Amblyomma maculatum) that, in a previous life stage, ingested the parasite in a blood meal taken from some vertebrate intermediate host. ACH is caused by the apicomplexan Hepatozoon americanum and has been differentiated from Old World canine hepatozoonosis caused by H. canis. Unlike H. canis, which is transmitted by the ubiquitous brown dog tick (Rhipicephalus sanguineus), H. americanum is essentially an accidental parasite of dogs, for which Gulf Coast ticks are not favored hosts. The geographic portrait of the disease parallels the known distribution of the Gulf Coast tick, which has expanded in recent years. Thus, the endemic cycle of H. americanum involves A. maculatum as definitive host and some vertebrate intermediate host(s) yet to be identified. Although coyotes (Canis latrans) are known to be infected, it is not known how important this host is in maintaining the endemic cycle. This review covers the biology of the parasite and of the tick that transmits it and contrasts ACH with classical canine hepatozoonosis. Clinical aspects of the disease are discussed, including diagnosis and treatment, and puzzling epidemiologic issues are examined. Brief consideration is given to the potential for ACH to be used as a model for study of angiogenesis and of hypertrophic osteoarthropathy. [Abstract/Link to Full Text]

Koch AL
Bacterial wall as target for attack: past, present, and future research.
Clin Microbiol Rev. 2003 Oct;16(4):673-87.
When Bacteria, Archaea, and Eucarya separated from each other, a great deal of evolution had taken place. Only then did extensive diversity arise. The bacteria split off with the new property that they had a sacculus that protected them from their own turgor pressure. The saccular wall of murein (or peptidoglycan) was an effective solution to the osmotic pressure problem, but it then was a target for other life-forms, which created lysoymes and beta-lactams. The beta-lactams, with their four-member strained rings, are effective agents in nature and became the first antibiotic in human medicine. But that is by no means the end of the story. Over evolutionary time, bacteria challenged by beta-lactams evolved countermeasures such as beta-lactamases, and the producing organisms evolved variant beta-lactams. The biology of both classes became evident as the pharmaceutical industry isolated, modified, and produced new chemotherapeutic agents and as the properties of beta-lactams and beta-lactamases were examined by molecular techniques. This review attempts to fit the wall biology of current microbes and their clinical context into the way organisms developed on this planet as well as the changes arising since the work done by Fleming. It also outlines the scientific advances in our understanding of this broad area of biology. [Abstract/Link to Full Text]

Reid G, Jass J, Sebulsky MT, McCormick JK
Potential uses of probiotics in clinical practice.
Clin Microbiol Rev. 2003 Oct;16(4):658-72.
Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. There is now mounting evidence that selected probiotic strains can provide health benefits to their human hosts. Numerous clinical trials show that certain strains can improve the outcome of intestinal infections by reducing the duration of diarrhea. Further investigations have shown benefits in reducing the recurrence of urogenital infections in women, while promising studies in cancer and allergies require research into the mechanisms of activity for particular strains and better-designed trials. At present, only a small percentage of physicians either know of probiotics or understand their potential applicability to patient care. Thus, probiotics are not yet part of the clinical arsenal for prevention and treatment of disease or maintenance of health. The establishment of accepted standards and guidelines, proposed by the Food and Agriculture Organization of the United Nations and the World Health Organization, represents a key step in ensuring that reliable products with suitable, informative health claims become available. Based upon the evidence to date, future advances with single- and multiple-strain therapies are on the horizon for the management of a number of debilitating and even fatal conditions. [Abstract/Link to Full Text]

Meijer E, Boland GJ, Verdonck LF
Prevention of cytomegalovirus disease in recipients of allogeneic stem cell transplants.
Clin Microbiol Rev. 2003 Oct;16(4):647-57.
The main risk factors for cytomegalovirus (CMV) disease in recipients of allogeneic stem cell transplants (SCT) are recipient CMV seropositivity and acute graft-versus-host disease. Currently, two antiviral strategies, prophylactic or preemptive antiviral treatment, are used for prevention of CMV disease. Preemptive treatment is most favorable when short-term (14-day) treatment is applied. Several methods are available for monitoring of CMV reactivation. PCR-based CMV DNA detection assays are the most sensitive methods; however, the clinical benefit of this high sensitivity is unclear. Even more, there is lack of clarity whether PCR tests can better be performed with plasma, whole blood, or peripheral blood leukocyte samples. Recovery of a CMV-specific CD8(+) cytotoxic-T-lymphocyte (CTL) response is necessary for preventing CMV reactivation and disease. Reconstitution of absolute CMV-specific CTL counts to values above 10 x 10(6) to 20 x 10(6) CTLs/liter is associated with protection from CMV disease. In the near future, preemptive therapy might be withheld in patients with CMV reactivation who are shown to have adequate CMV-specific cytotoxic T-cell levels. Antiviral therapy with (val)acyclovir has been studied only as prophylactic treatment for prevention of CMV infection. High-dose oral valacyclovir is more effective than acyclovir when used in addition to preemptive treatment of CMV reactivation with ganciclovir or foscarnet. Three antiviral drugs have been tested for preemptive therapy of CMV reactivation and/or treatment of CMV disease. Although intravenous ganciclovir is considered the drug of choice, foscarnet has similar efficacy and less toxicity, especially hematologic toxicity. Cidofovir has not been tested extensively, but so far the results are disappointing. Oral valganciclovir for preemptive treatment of SCT recipients is currently being studied. In addition to antiviral therapy, adoptive immunotherapy with CMV-specific cytotoxic T cells as prophylactic or preemptive therapy is a very elegant strategy; however, generation of these cells is expensive and time-consuming, and therefore the therapy is not available at every transplantation center. Magnetic selection of CMV-specific CD8(+) T cells from peripheral blood by using HLA class I-peptide tetramers may be very promising, making this strategy more accessible. [Abstract/Link to Full Text]

Janssens S, Beyaert R
Role of Toll-like receptors in pathogen recognition.
Clin Microbiol Rev. 2003 Oct;16(4):637-46.
The innate immune system relies on a vast array of non-clonally expressed pattern recognition receptors for the detection of pathogens. Pattern recognition receptors bind conserved molecular structures shared by large groups of pathogens, termed pathogen-associated molecular patterns. The Toll-like receptors (TLRs) are a recently discovered family of pattern recognition receptors which show homology with the Drosophila Toll protein and the human interleukin-1 receptor family. Engagement of different TLRs can induce overlapping yet distinct patterns of gene expression that contribute to an inflammatory response. The TLR family is characterized by the presence of leucine-rich repeats and a Toll/interleukin-1 receptor-like domain, which mediate ligand binding and interaction with intracellular signaling proteins, respectively. Most TLR ligands identified so far are conserved microbial products which signal the presence of an infection, but evidence for some endogenous ligands that might signal other danger conditions has also been obtained. Molecular mechanisms for pathogen-associated molecular pattern recognition still remain elusive but seem to be more complicated than initially anticipated. In most cases, direct binding of microbial ligands to TLRs still has to be demonstrated. Moreover, Drosophila TLRs bind endogenous ligands, generated through a proteolytic cascade in response to an infection. In the case of endotoxin, recognition involves a complex of TLR4 and a number of other proteins. Moreover, TLR heterodimerization further extends the spectrum of ligands and modulates the response towards specific ligands. The fact that TLR expression is regulated in both a cell type- and stimulus-dependent fashion further contributes to the complexity. [Abstract/Link to Full Text]

Zintl A, Mulcahy G, Skerrett HE, Taylor SM, Gray JS
Babesia divergens, a bovine blood parasite of veterinary and zoonotic importance.
Clin Microbiol Rev. 2003 Oct;16(4):622-36.
Babesia divergens is an intraerythrocytic protozoan parasite, transmitted by the tick Ixodes ricinus, and is the main agent of bovine babesiosis in Europe. It is not only a cause of significant loss to the cattle industry; it can also infect immunocompromised humans, causing medical emergencies characterized by rapid fulmination and parasitemias that may exceed 70%. The current emphasis in Europe on sustainable agriculture and extensification is likely to lead to an increase in vector tick populations with increased risk of infection. Despite the veterinary and zoonotic importance of this parasite, relatively little research has been carried out on B. divergens, and many questions regarding the parasite's epidemiology and the host's response remain unanswered. A better understanding of the species' biology and host-parasite interactions may lead to improved control mechanisms and new trends in vaccine and antibabesial drug development. This review provides the first comprehensive summary of B. divergens biology, including its morphology, life cycle, and host specificity, and the current state of knowledge of both human and bovine infections. [Abstract/Link to Full Text]

Andrews T, Sullivan KE
Infections in patients with inherited defects in phagocytic function.
Clin Microbiol Rev. 2003 Oct;16(4):597-621.
Patients with defects in phagocytic function are predisposed to intracellular microorganisms and typically have early dissemination of the infection. Recognition of the underlying disorder and aggressive antimicrobial therapy has been beneficial for the patients. Improved understanding of the pathophysiology has also affected patient management by allowing specific, targeted immunomodulatory intervention. The disorders described in this review are not common but have had a significant impact on our understanding of the role of phagocytic cells in host defense. Conversely, understanding the role of the neutrophil and macrophage in infection has benefited not just the patients described in this review but also other patients with similar disease processes. [Abstract/Link to Full Text]

De Clercq E
Clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of DNA virus and retrovirus infections.
Clin Microbiol Rev. 2003 Oct;16(4):569-96.
The acyclic nucleoside phosphonates HPMPC (cidofovir), PMEA (adefovir), and PMPA (tenofovir) have proved to be effective in vitro (cell culture systems) and in vivo (animal models and clinical studies) against a wide variety of DNA virus and retrovirus infections: cidofovir against herpesvirus (herpes simplex virus types 1 and 2 varicella-zoster virus, cytomegalovirus [CMV], Epstein-Barr virus, and human herpesviruses 6, 7, and 8), polyomavirus, papillomavirus, adenovirus, and poxvirus (variola virus, cowpox virus, vaccinia virus, molluscum contagiosum virus, and orf virus) infections; adefovir against herpesvirus, hepadnavirus (human hepatitis B virus), and retrovirus (human immunodeficiency virus types 1 [HIV-1] and 2 [HIV-2], simian immunodeficiency virus, and feline immunodeficiency virus) infections; and tenofovir against both hepadnavirus and retrovirus infections. Cidofovir (Vistide) has been officially approved for the treatment of CMV retinitis in AIDS patients, tenofovir disoproxil fumarate (Viread) has been approved for the treatment of HIV infections (i.e., AIDS), and adefovir dipivoxil (Hepsera) has been approved for the treatment of chronic hepatitis B. Nephrotoxicity is the dose-limiting side effect for cidofovir (Vistide) when used intravenously (5 mg/kg); no toxic side effects have been described for adefovir dipivoxil and tenofovir disoproxil fumarate, at the approved doses (Hepsera at 10 mg orally daily and Viread at 300 mg orally daily). [Abstract/Link to Full Text]

Henderson DK
Managing occupational risks for hepatitis C transmission in the health care setting.
Clin Microbiol Rev. 2003 Jul;16(3):546-68.
Hepatitis C virus (HCV) infection is a significant contemporary health problem in the United States and elsewhere. Because it is primarily transmitted via blood, hepatitis C infection presents risks for both nosocomial transmission to patients and occupational spread to health care workers. Recent insights into the pathogenesis, immunopathogenesis, natural history, and treatment of infection caused by this unique flavivirus provide a rationale for the use of new strategies for managing occupational hepatitis C infections when they occur. This article reviews this developing information. Recently published data demonstrate success rates in the treatment of "acute hepatitis C syndrome" that approach 100\%, and although these studies are not directly applicable to all occupational infections, they may provide important clues to optimal management strategies. In addition, the article delineates approaches to the prevention of occupational exposures and also addresses the difficult issue of managing HCV-infected health care providers. The article summarizes currently available data about the nosocomial epidemiology of HCV infection and the magnitude of risk and discusses several alternatives for managing exposure and infection. No evidence supports the use of immediate postexposure prophylaxis with immunoglobulin, immunomodulators, or antiviral agents. Based on the very limited data available, the watchful waiting and preemptive therapy strategies described in detail in this article represent reasonable interim approaches to the complex problem of managing occupational HCV infections, at least until more definitive data are obtained. [Abstract/Link to Full Text]

Bode L, Ludwig H
Borna disease virus infection, a human mental-health risk.
Clin Microbiol Rev. 2003 Jul;16(3):534-45.
This article focuses on human Borna disease virus (BDV) infections, most notably on the development of valid diagnostic systems, which have arisen as a major research issue in the past decade. The significance of a novel modular triple enzyme-linked immunosorbent assay that is capable of specifically measuring anti-BDV antibodies as well as major structural proteins N (p40) and P (p24) in the blood, either as free antigens in the plasma or as antibody-bound circulating immune complexes (CICs), is explained. The impact of CICs and plasma antigen, which indicate periods of antigenemia in the course of BDV infection, along with other infection markers that are still in use is discussed. The review further provides new insight into possible links of BDV to human diseases, summarizing cross-sectional and longitudinal data which correlate acute depression with the presence and amount of antigen and CICs. Moreover, BDV prevalence in healthy people is reevaluated, suggesting that this was previously underestimated. Antiviral efficacy of amantadine, in vivo and in vitro, is outlined as well, with emphasis on wild-type (human and equine) versus laboratory strains. Finally, the pros and cons of the association of BDV with human disease, as detailed in the literature, are critically discussed and related to our data and concepts. This article supports existing correlative evidence for a pathogenic role of BDV infection in particular human mental disorders, in analogy to what has been proven for a variety of animal species. [Abstract/Link to Full Text]

Noverr MC, Erb-Downward JR, Huffnagle GB
Production of eicosanoids and other oxylipins by pathogenic eukaryotic microbes.
Clin Microbiol Rev. 2003 Jul;16(3):517-33.
Oxylipins are oxygenated metabolites of fatty acids. Eicosanoids are a subset of oxylipins and include the prostaglandins and leukotrienes, which are potent regulators of host immune responses. Host cells are one source of eicosanoids and oxylipins during infection; however, another potential source of eicosanoids is the pathogen itself. A broad range of pathogenic fungi, protozoa, and helminths produce eicosanoids and other oxylipins by novel synthesis pathways. Why do these organisms produce oxylipins? Accumulating data suggest that phase change and differentiation in these organisms are controlled by oxylipins, including prostaglandins and lipoxygenase products. The precise role of pathogen-derived eicosanoids in pathogenesis remains to be determined, but the potential link between pathogen eicosanoids and the development of TH2 responses in the host is intriguing. Mammalian prostaglandins and leukotrienes have been studied extensively, and these molecules can modulate Th1 versus Th2 immune responses, chemokine production, phagocytosis, lymphocyte proliferation, and leukocyte chemotaxis. Thus, eicosanoids and oxylipins (host or microbe) may be mediators of a direct host-pathogen "cross-talk" that promotes chronic infection and hypersensitivity disease, common features of infection by eukaryotic pathogens. [Abstract/Link to Full Text]

Bennett JW, Klich M
Mycotoxins.
Clin Microbiol Rev. 2003 Jul;16(3):497-516.
Mycotoxins are secondary metabolites produced by microfungi that are capable of causing disease and death in humans and other animals. Because of their pharmacological activity, some mycotoxins or mycotoxin derivatives have found use as antibiotics, growth promotants, and other kinds of drugs; still others have been implicated as chemical warfare agents. This review focuses on the most important ones associated with human and veterinary diseases, including aflatoxin, citrinin, ergot akaloids, fumonisins, ochratoxin A, patulin, trichothecenes, and zearalenone. [Abstract/Link to Full Text]

Smith I
Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence.
Clin Microbiol Rev. 2003 Jul;16(3):463-96.
Tuberculosis (TB), one of the oldest known human diseases. is still is one of the major causes of mortality, since two million people die each year from this malady. TB has many manifestations, affecting bone, the central nervous system, and many other organ systems, but it is primarily a pulmonary disease that is initiated by the deposition of Mycobacterium tuberculosis, contained in aerosol droplets, onto lung alveolar surfaces. From this point, the progression of the disease can have several outcomes, determined largely by the response of the host immune system. The efficacy of this response is affected by intrinsic factors such as the genetics of the immune system as well as extrinsic factors, e.g., insults to the immune system and the nutritional and physiological state of the host. In addition, the pathogen may play a role in disease progression since some M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines and drugs and more specific and rapid diagnostics. Researchers are utilizing information obtained from the complete sequence of the M. tuberculosis genome and from new genetic and physiological methods to identify targets in M. tuberculosis that will aid in the development of these sorely needed antitubercular agents. [Abstract/Link to Full Text]

Bryant AE
Biology and pathogenesis of thrombosis and procoagulant activity in invasive infections caused by group A streptococci and Clostridium perfringens.
Clin Microbiol Rev. 2003 Jul;16(3):451-62.
Group A streptococcal necrotizing fasciitis/myonecrosis and Clostridium perfringens gas gangrene are two of the most fulminant gram-positive infections in humans. Tissue destruction associated with these infections progresses rapidly to involve an entire extremity. Multiple-organ failure is common, and morbidity and mortality remain high. Systemic activation of coagulation and dysregulation of the anticoagulation pathways contribute to the pathogenesis of many diverse disease entities of infectious etiology, and it has been our hypothesis that microvascular thrombosis contributes to reduced tissue perfusion, hypoxia, and subsequent regional tissue necrosis and organ failure in these invasive gram-positive infections. This article reviews the coagulation, anticoagulation, and fibrinolytic systems from cellular players to cytokines to novel antithrombotic therapies and discusses the mechanisms contributing to occlusive microvascular thrombosis and tissue destruction in invasive group A streptococcal and C. perfringens infections. A thorough understanding of these mechanisms may suggest novel therapeutic targets for patients with these devastating infections. [Abstract/Link to Full Text]

Vakulenko SB, Mobashery S
Versatility of aminoglycosides and prospects for their future.
Clin Microbiol Rev. 2003 Jul;16(3):430-50.
Aminoglycoside antibiotics have had a major impact on our ability to treat bacterial infections for the past half century. Whereas the interest in these versatile antibiotics continues to be high, their clinical utility has been compromised by widespread instances of resistance. The multitude of mechanisms of resistance is disconcerting but also illuminates how nature can manifest resistance when bacteria are confronted by antibiotics. This article reviews the most recent knowledge about the mechanisms of aminoglycoside action and the mechanisms of resistance to these antibiotics. [Abstract/Link to Full Text]

van der Flier M, Geelen SP, Kimpen JL, Hoepelman IM, Tuomanen EI
Reprogramming the host response in bacterial meningitis: how best to improve outcome?
Clin Microbiol Rev. 2003 Jul;16(3):415-29.
Despite effective antibiotic therapy, bacterial meningitis is still associated with high morbidity and mortality in both children and adults. Animal studies have shown that the host inflammatory response induced by bacterial products in the subarachnoid space is associated with central nervous system injury. Thus, attenuation of inflammation early in the disease process might improve the outcome. The feasibility of such an approach is demonstrated by the reduction in neurologic sequelae achieved with adjuvant dexamethasone therapy. Increased understanding of the pathways of inflammation and neuronal damage has suggested rational new targets to modulate the host response in bacterial meningitis, but prediction of which agents would be optimal has been difficult. This review compares the future promise of benefit from the use of diverse adjuvant agents. It appears unlikely that inhibition of a single proinflammatory mediator will prove useful in clinical practice, but several avenues to reprogram a wider array of mediators simultaneously are encouraging. Particularly promising are efforts to adjust combinations of cytokines, to inhibit neuronal apoptosis and to enhance brain repair. [Abstract/Link to Full Text]

Van Amersfoort ES, Van Berkel TJ, Kuiper J
Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock.
Clin Microbiol Rev. 2003 Jul;16(3):379-414.
Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins. [Abstract/Link to Full Text]

Clarke SC, Haigh RD, Freestone PP, Williams PH
Virulence of enteropathogenic Escherichia coli, a global pathogen.
Clin Microbiol Rev. 2003 Jul;16(3):365-78.
Enteropathogenic Escherichia coli (EPEC) remains an important cause of diarrheal disease worldwide. Research into EPEC is intense and provides a good virulence model of other E. coli infections as well as other pathogenic bacteria. Although the virulence mechanisms are now better understood, they are extremely complex and much remains to be learnt. The pathogenesis of EPEC depends on the formation of an ultrastructural lesion in which the bacteria make intimate contact with the host apical enterocyte membrane. The formation of this lesion is a consequence of the ability of EPEC to adhere in a localized manner to the host cell, aided by bundle-forming pili. Tyrosine phosphorylation and signal transduction events occur within the host cell at the lesion site, leading to a disruption of the host cell mechanisms and, consequently, to diarrhea. These result from the action of highly regulated EPEC secreted proteins which are released via a type III secretion system, many genes of which are located within a pathogenicity island known as the locus of enterocyte effacement. Over the last few years, dramatic increases in our knowledge of EPEC virulence have taken place. This review therefore aims to provide a broad overview of and update to the virulence aspects of EPEC. [Abstract/Link to Full Text]

Duchini A, Goss JA, Karpen S, Pockros PJ
Vaccinations for adult solid-organ transplant recipients: current recommendations and protocols.
Clin Microbiol Rev. 2003 Jul;16(3):357-64.
Recipients of solid-organ transplantation are at risk of severe infections due to their life-long immunosuppression. Despite emerging evidence that vaccinations are safe and effective among immunosuppressed patients, most vaccines are still underutilized in these patients. The efficacy, safety, and protocols of several vaccines in this patient population are poorly understood. Timing of vaccination appears to be critical because response to vaccinations is decreased in patients with end-stage organ disease and in the first 6 months after transplantation. For these reasons, the primary immunizations should be given before transplantation, as early as possible during the course of disease. Vaccination strategy should include vaccination of household contacts and health care workers at transplant centers unless contraindicated. No conclusive data are available on the use of immunoadjuvants and screening for protective titers. Most vaccines appear to be safe in solid-organ transplantation recipients, but live vaccines should be avoided until further studies are available. The risk of rejection appears minimal. Recommended vaccines include pneumovax, hepatitis A and B, influenza, and tetanus-diphtheria. We outline specific protocols and recommendations in this particular patient population. Specific contraindications exist for other vaccines, such as yellow fever, oral polio vaccine, bacillus Calmette-Guerin, and vaccinia. We conclude that solid-organ recipients will benefit from consistent immunization practices. Further studies are recommended to improve established protocols in this patient population. [Abstract/Link to Full Text]

Tortoli E
Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s.
Clin Microbiol Rev. 2003 Apr;16(2):319-54.
The advancement of genetic techniques has greatly boosted taxonomic studies in recent years. Within the genus Mycobacterium, 42 new species have been detected since 1990, most of which were grown from clinical samples. Along with species for which relatively large numbers of strains have been reported, some of the new species of mycobacteria have been detected rarely or even only once. From the phenotypic point of view, among the new taxa, chromogens exceed nonchromogens while the numbers of slowly and rapidly growing species are equivalent. Whereas conventional identification tests were usually inconclusive, an important role was played by lipid analyses and in particular by high-performance liquid chromatography. Genotypic investigations based on sequencing of 16S rRNA gene have certainly made the most important contribution. The investigation of genetic relatedness led to the redistribution of the species previously included in the classically known categories of slow and rapid growers into new groupings. Within slow growers, the intermediate branch related to Mycobacterium simiae and the cluster of organisms related to Mycobacterium terrae have been differentiated; among rapid growers, the group of thermotolerant mycobacteria has emerged. The majority of species are resistant to isoniazid and, to a lesser extent, to rifampin. Many of the new species of mycobacteria are potentially pathogenic, and there are numerous reports of their involvement in diseases. Apart from disseminated and localized diseases in immunocompromised patients, the most frequent infections in immunocompetent people involve the lungs, skin, and, in children, cervical lymph nodes. The awareness of such new mycobacteria, far from being a merely speculative exercise, is therefore important for clinicians and microbiologists. [Abstract/Link to Full Text]

Artz AS, Ershler WB, Longo DL
Pneumococcal vaccination and revaccination of older adults.
Clin Microbiol Rev. 2003 Apr;16(2):308-18.
As individuals advance in age, the risk of infection, bacteremia, and mortality caused by Streptococcus pneumoniae rises. Retrospective data demonstrate that the licensed penumococcal polysaccharide vaccine (PPV) is effective in older persons in reducing serotype-specific invasive disease. PPV demonstrates good immunogenicity in older adults, generally comparable to that in younger subjects, although certain cohorts respond less well. The response to PPV is T cell independent, however, and does not elicit immunologic memory. The duration of the anti-capsular polysaccharide antibody response appears to wane as early as 3 years after vaccination. In older persons, revaccination induces an antibody response, although it may not be as strong as that from the initial vaccine. While revaccination of older adults has been recommended, clinical efficacy has not yet been proven. Measures of antibody function may be at least as important in determining protection as are quantitative antibody levels. Additional studies of immunogenicity, particularly regarding revaccination, will facilitate the design of an optimal pneumococcal vaccination policy. Research into conjugate- and protein-based pneumococcal vaccines, which elicit T-cell-dependent responses and induce immunologic memory, is needed in older persons. In the meantime, administering to PPV to recommended groups should be a public health priority. [Abstract/Link to Full Text]

Marciano-Cabral F, Cabral G
Acanthamoeba spp. as agents of disease in humans.
Clin Microbiol Rev. 2003 Apr;16(2):273-307.
Acanthamoeba spp. are free-living amebae that inhabit a variety of air, soil, and water environments. However, these amebae can also act as opportunistic as well as nonopportunistic pathogens. They are the causative agents of granulomatous amebic encephalitis and amebic keratitis and have been associated with cutaneous lesions and sinusitis. Immuno compromised individuals, including AIDS patients, are particularly susceptible to infections with Acanthamoeba. The immune defense mechanisms that operate against Acanthamoeba have not been well characterized, but it has been proposed that both innate and acquired immunity play a role. The ameba's life cycle includes an active feeding trophozoite stage and a dormant cyst stage. Trophozoites feed on bacteria, yeast, and algae. However, both trophozoites and cysts can retain viable bacteria and may serve as reservoirs for bacteria with human pathogenic potential. Diagnosis of infection includes direct microscopy of wet mounts of cerebrospinal fluid or stained smears of cerebrospinal fluid sediment, light or electron microscopy of tissues, in vitro cultivation of Acanthamoeba, and histological assessment of frozen or paraffin-embedded sections of brain or cutaneous lesion biopsy material. Immunocytochemistry, chemifluorescent dye staining, PCR, and analysis of DNA sequence variation also have been employed for laboratory diagnosis. Treatment of Acanthamoeba infections has met with mixed results. However, chlorhexidine gluconate, alone or in combination with propamidene isethionate, is effective in some patients. Furthermore, effective treatment is complicated since patients may present with underlying disease and Acanthamoeba infection may not be recognized. Since an increase in the number of cases of Acanthamoeba infections has occurred worldwide, these protozoa have become increasingly important as agents of human disease. [Abstract/Link to Full Text]

Despommier D
Toxocariasis: clinical aspects, epidemiology, medical ecology, and molecular aspects.
Clin Microbiol Rev. 2003 Apr;16(2):265-72.
Toxocariasis is caused by a series of related nematode species (ascarids) that routinely infect dogs and cats throughout the world. The eggs from these ascarids are common environmental contaminants of human habitation, due largely to the fact that many kinds of dogs and cats serve as pets, while countless others run wild throughout the streets of most urban centers. The eggs, present in dog and cat feces, become infectious within weeks after they are deposited in the local environment (e.g., sandboxes, city parks, and public beaches, etc.). Humans, particularly children, frequently ingest these eggs by accident and become infected. Infection in humans, in contrast to their definitive hosts, remains occult, often resulting in disease caused by the migrating larval stages. Visceral larva migrans (VLM) and ocular larva migrans (OLM) are two clinical manifestations that result in definable syndromes and present as serious health problems wherever they occur. Diagnosis and treatment of VLM and OLM are difficult. These issues are summarized in this review, with emphasis on the ecology of transmission and control of spread to both humans and animals through public health initiatives employing treatment of pets and environmental intervention strategies that limit the areas that dogs and cats are allowed within the confines of urban centers. [Abstract/Link to Full Text]

Henrickson KJ
Parainfluenza viruses.
Clin Microbiol Rev. 2003 Apr;16(2):242-64.
Human parainfluenza viruses (HPIV) were first discovered in the late 1950s. Over the last decade, considerable knowledge about their molecular structure and function has been accumulated. This has led to significant changes in both the nomenclature and taxonomic relationships of these viruses. HPIV is genetically and antigenically divided into types 1 to 4. Further major subtypes of HPIV-4 (A and B) and subgroups/genotypes of HPIV-1 and HPIV-3 have been described. HPIV-1 to HPIV-3 are major causes of lower respiratory infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. Each subtype can cause somewhat unique clinical diseases in different hosts. HPIV are enveloped and of medium size (150 to 250 nm), and their RNA genome is in the negative sense. These viruses belong to the Paramyxoviridae family, one of the largest and most rapidly growing groups of viruses causing significant human and veterinary disease. HPIV are closely related to recently discovered megamyxoviruses (Hendra and Nipah viruses) and metapneumovirus. [Abstract/Link to Full Text]

Heikkinen T, Chonmaitree T
Importance of respiratory viruses in acute otitis media.
Clin Microbiol Rev. 2003 Apr;16(2):230-41.
Acute otitis media is usually considered a simple bacterial infection that is treated with antibiotics. However, ample evidence derived from studies ranging from animal experiments to extensive clinical trials supports a crucial role for respiratory viruses in the etiology and pathogenesis of acute otitis media. Viral infection of the upper respiratory mucosa initiates the whole cascade of events that finally leads to the development of acute otitis media as a complication. The pathogenesis of acute otitis media involves a complex interplay between viruses, bacteria, and the host's inflammatory response. In a substantial number of children, viruses can be found in the middle-ear fluid either alone or together with bacteria, and recent studies indicate that at least some viruses actively invade the middle ear. Viruses appear to enhance the inflammatory process in the middle ear, and they may significantly impair the resolution of otitis media. Prevention of the predisposing viral infection by vaccination against the major viruses would probably be the most effective way to prevent acute otitis media. Alternatively, early treatment of the viral infection with specific antiviral agents would also be effective in reducing the occurrence of acute otitis media. [Abstract/Link to Full Text]

Fredriksson-Ahomaa M, Korkeala H
Low occurrence of pathogenic Yersinia enterocolitica in clinical, food, and environmental samples: a methodological problem.
Clin Microbiol Rev. 2003 Apr;16(2):220-9.
While Yersinia enterocolitica is an important pathogen, which can cause yersiniosis in humans and animals, its epidemiology remains obscure. The pig is the major reservoir of pathogenic Y. enterocolitica of bioserotype 4/O:3, the most common type found in humans. Y. enterocolitica is thought to be a significant food-borne pathogen, although pathogenic isolates have seldom been recovered from foods. The low isolation rate of this pathogenic bacterium in natural samples, including clinical, food, and environmental samples, may be due to the limited sensitivity of culture methods. During the last decade, numerous DNA-based methods, such as PCR and colony hybridization assays, have been designed to detect pathogenic Y. enterocolitica in natural samples more rapidly and with better sensitivity than can be achieved by culture methods. In addition, the occurrence of pathogenic Y. enterocolitica in natural samples is clearly higher with PCR than with culture methods. The methods available for detection of pathogenic Y. enterocolitica in natural samples are reviewed in this article. [Abstract/Link to Full Text]

Friedman H, Newton C, Klein TW
Microbial infections, immunomodulation, and drugs of abuse.
Clin Microbiol Rev. 2003 Apr;16(2):209-19.
The use of recreational drugs of abuse has generated serious health concerns. There is a long-recognized relationship between addictive drugs and increased levels of infections. Studies of the mechanisms of actions of these drugs became more urgent with the advent of AIDS and its correlation with abused substances. The nature and mechanisms of immunomodulation by marijuana, opiates, cocaine, nicotine, and alcohol are described in this review. Recent studies of the effects of opiates or marijuana on the immune system have demonstrated that they are receptor mediated, occurring both directly via specific receptors on immune cells and indirectly through similar receptors on cells of the nervous system. Findings are also discussed that demonstrate that cocaine and nicotine have similar immunomodulatory effects, which are also apparently receptor mediated. Finally, the nature and mechanisms of immunomodulation by alcohol are described. Although no specific alcohol receptors have been identified, it is widely recognized that alcohol enhances susceptibility to opportunistic microbes. The review covers recent studies of the effects of these drugs on immunity and on increased susceptibility to infectious diseases, including AIDS. [Abstract/Link to Full Text]

Gilbert P, McBain AJ
Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance.
Clin Microbiol Rev. 2003 Apr;16(2):189-208.
There has recently been much controversy surrounding the increased use of antibacterial substances in a wide range of consumer products and the possibility that, as with antibiotics, indiscriminate use of biocides might contribute to the overall pattern of susceptibility in the general environment and in the clinic. Such speculation, based on the isolation of resistant mutants from in vitro monoculture experiments, is not reflected by an emergence of biocide-resistant strains in vivo. This review provides a broad coverage of the biocide and resistance literature and evaluates the potential risks, perceived from such laboratory monoculture experiments, against evidence gathered over 50 years of field studies. An explanation for the continued effectiveness of broad-spectrum biocidal agents against the decline in efficacy of therapeutic agents is provided based on the fitness costs of resistance and the ubiquity of naturally occurring substances that possess antibacterial effect. While we conclude from this review of the literature that the incorporation of antibacterial agents into a widening sphere of personal products has had little or no impact on the patterns of microbial susceptibility observed in the environment, the associated risks remain finite. The use of such products should therefore be associated with a clear demonstration of added value either to consumer health or to the product life. Hygienic products should therefore be targeted to applications for which the risks have been established. [Abstract/Link to Full Text]

Recent Articles in Infection and Immunity

Choi KS, Webb T, Oelke M, Scorpio DG, Dumler JS
Differential innate immune cell activation and proinflammatory response in Anaplasma phagocytophilum infection.
Infect Immun. 2007 Jun;75(6):3124-30.
Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The critical role of gamma interferon (IFN-gamma) for induction of severe inflammatory histopathology, even in the absence of a significant bacterial load, was previously demonstrated in a murine mode