24 Hours in the Life of HIV-1 in a T Cell Line.

HIV-1 infects CD4+ T cells and completes its replication cycle in approximately 24 hours. We employed repeated measurements in a standardized cell system and rigorous mathematical modeling to characterize the emergence of the viral replication intermediates and their impact on the cellular transcriptional response with high temporal resolution. We observed 7,991 (73%) of the 10,958 expressed genes to be modulated in concordance with key steps of viral replication. Fifty-two percent of the overall variability in the host transcriptome was explained by linear regression on the viral life cycle. This profound perturbation of cellular physiology was investigated in the light of several regulatory mechanisms, including transcription factors, miRNAs, host-pathogen interaction, and proviral integration. Key features were validated in primary CD4+ T cells, and with viral constructs using alternative entry strategies. We propose a model of early massive cellular shutdown and progressive upregulation of the cellular machinery to complete the viral life cycle.

Bacterial flagellin elicits innate immune defenses and cardiac dysfunction in vivo

Résumé Les agents pathogènes responsables d'infection entraînent chez l'hôte deux types de réponses immunes, la première, non spécifique, dite immunité innée, la seconde, spécifique à l'agent concerné, dite immunité adaptative. L'immunité innée, qui représente la première ligne de défense contre les pathogènes, est liée à la reconnaissance par les cellules de l'hôte de structures moléculaires propres aux micro-organismes (« Pathogen-Associated Molecular Patterns », PAMPs), grâce à des récepteurs membranaires et cytoplasmiques (« Pattern Recognition Receptors », PRRs) identifiant de manière spécifique ces motifs moléculaires. Les récepteurs membranaires impliqués dans ce processus sont dénommés toll-like récepteurs, ou TLRS. Lorsqu'ils sont activés par leur ligand spécifique, ces récepteurs activent des voies de signalisation intracellulaires initiant la réponse inflammatoire non spécifique et visant à éradiquer l'agent pathogène. Les deux voies de signalisation impliquées dans ce processus sont la voie des « Mitogen-Activated Protein Kinases » (MAPKs) et celle du « Nuclear Factor kappaB » (NF-κB), dont l'activation entraîne in fine l'expression de protéines de l'inflammation dénommées cytokines, ainsi que certaines enzymes produisant divers autres médiateurs inflammatoires. Dans certaines situations, cette réponse immune peut être amplifiée de manière inadéquate, entraînant chez l'hôte une réaction inflammatoire systémique exagérée, appelée sepsis. Le sepsis peut se compliquer de dysfonctions d'organes multiples (sepsis sévère), et dans sa forme la plus grave, d'un collapsus cardiovasculaire, définissant le choc septique. La défaillance circulatoire du choc septique touche les vaisseaux sanguins d'une part, le coeur d'autre part, réalisant un tableau de «dysfonction cardiaque septique », dont on connaît mal les mécanismes pathogéniques. Les bactéries à Gram négatif peuvent déclencher de tels phénomènes, notamment en libérant de l'endotoxine, qui active les voies de l'immunité innée par son interaction avec un toll récepteur, le TLR4. Outre l'endotoxine, la plupart des bactéries à Gram négatif relâchent également dans leur environnement une protéine, la flagelline, qui est le constituant majeur du flagelle bactérien, organelle assurant la mobilité de ces micro-organismes. Des données récentes ont indiqué que la flagelline active, dans certaines cellules, les voies de l'immunité innée en se liant au récepteur TLRS. On ne connaît toutefois pas les conséquences de l'interaction flagelline-TLRS sur le développement de l'inflammation et des dysfonctions d'organes au cours du sepsis. Nous avons par conséquent élaboré le présent travail en formulant l'hypothèse que la flagelline pourrait déclencher une telle inflammation et représenter ainsi un médiateur potentiel de la dysfonction d'organes au cours du sepsis à Gram négatif, en nous intéressant plus particulièrement àl'inflammation et à la dysfonction cardiaque. Dans la première partie de ce travail, nous avons étudié les effets de la flagelline sur l'activation du NF-κB et des MAPKs, et sur l'expression de cytokines inflammatoires au niveau du myocarde in vitro (cardiomyocytes en culture) et in vivo (injection de flagelline recombinante à des souris). Nous avons observé tout d'abord que le récepteur TLRS est fortement exprimé au niveau du myocarde. Nous avons ensuite démontré que la flagelline active la voie du NF-κB et des MAP kinases (p38 et JNK), stimule la production de cytokines et de chemokines inflammatoires in vitro et in vivo, et entraîne l'activation de polynucléaires neutrophiles dans le tissu cardiaque in vivo. Finalement, au plan fonctionnel, nous avons pu montrer que la flagelline entraîne une dilatation et une réduction aiguë de la contractilité du ventricule gauche chez la souris, reproduisant les caractéristiques de la dysfonction cardiaque septique. Dans la deuxième partie, nous avons déterminé la distribution du récepteur TLRS dans les autres organes majeurs de la souris (poumon, foie, intestin et rein}, et avons caractérisé dans ces organes l'effet de la flagelline sur l'activation du NF-κB et des MAPKs, l'expression de cytokines, et l'induction de l'apoptose. Nous avons démontré que le TLRS est exprimé de façon constitutive dans ces organes, et que l'injection de flagelline y déclenche les cascades de l'immunité innée et de processus apoptotiques. Finalement, nous avons également déterminé que la flagelline entraîne une augmentation significative de multiples cytokines dans le plasma une à six heures après son injection. En résumé, nos données démontrent que la flagelline bactérienne (a) entraîne une inflammation et une dysfonction importantes du myocarde et (b) active de manière très significative les mécanismes d'immunité innée dans les principaux organes et entraîne une réponse inflammatoire systémique. Par conséquent, la flagelline peut représenter un médiateur puissant de l'inflammation et de la dysfonction d'organes, notamment du coeur, au cours du choc septique déclenché par les bactéries à Gram négatif. Summary Pathogenic microorganisms trigger two kinds of immune responses in the host. The first one is immediate and non-specific and is termed innate immunity, whereas the second one, specifically targeted at the invading agent, is termed adaptative immunity. Innate immunity, which represents the first line of defense against invading pathogens, confers the host the ability to recognize molecular structures common to many microbial pathogens, ("Pathogen-Associated Molecular Patterns", PAMPs), through cytosolic or membrane-associated receptors ("Pattern Recognition Receptors", PRRs), the latter being represented by a family of receptors termed "toll-like receptors or TLRs". Once activated by the binding of their specific ligand, these receptors activate intracellular signaling pathways, which initiate the non-specific inflammatory response aimed at eradicating the pathogens. The two pathways implicated in this process are the mitogen-activated protein kinases (MAPK) and the nuclear factor kappa B (NF-κB) signaling pathways, whose activation elicit in fine the expression of inflammatory proteins termed cytokines, as well as various enzymes producing a wealth of additional inflammatory mediators. In some circumstances, the innate immune response can become amplified and dysregulated, triggering an overwhelming systemic inflammatory response in the host, identified as sepsis. Sepsis can be associated with multiple organ dysfunction (severe sepsis), and in its most severe form, with cardiovascular collapse, defming septic shock. The cardiovascular failure associated with septic shock affects blood vessels as well as the heart, resulting in a particular form of acute heart failure termed "septic cardiac dysfunction ", whose pathogenic mechanisms remain partly undefined. Gram-negative bacteria can initiate such phenomena, notably by releasing lipopolysaccharide (LPS), which activates innate immune signaling by interacting with its specific toll receptor, the TLR4. Besides LPS, most Gram-negative bacteria also release flagellin into their environment, which is the main structural protein of the bacterial flagellum, an appendage extending from the outer bacterial membrane, responsible for the motility of the microorganism. Recent data indicated that flagellin activate immune responses upon binding to its receptor, TLRS, in various cell types. However, the role of flagellin/TLRS interaction in the development of inflammation and organ dysfunction during sepsis is not known. Therefore, we designed the present work to address the hypothesis that flagellin might trigger such inflammatory responses and thus represent a potential mediator of organ dysfunction during Gram-negative sepsis, with a particular emphasis on cardiac inflammation and contractile dysfunction. In the first part of this work, we investigated the effects of flagellin on NF-κB and MAPK activation and the generation of pro-inflammatory mediators within the heart in vitro (cultured cardiomyocytes) and in vivo (injection of recombinant flagellin into mice). We first observed that TLRS protein is strongly expressed by the myocardium. We then demonstrated that flagellin activates NF-κB and MAP kinases (p38 and JNK), upregulates the transcription of pro-inflammatory cytokines and chemokines in vitro and in vivo, and stimulates the activation of polymorphonuclear neutrophils within the heart in vivo. Finally, we demonstrated that flagellin triggers acute cardiac dilation, and a significant reduction of left ventricular contractility, mimicking characteristics of clinical septic cardiac dysfunction. In the second part, we determined the TLRS distribution in other mice major organs (lung, liver, gut and kidney) and we characterized in these organs the effects of flagellin on NF-κB and MAPK activation, on the expression of pro-inflammatory çytokines, and on the induction of apoptosis. We demonstrated that TLRS protein is constitutively expressed and that flagellin activates prototypical innate immune responses and pro-apoptotic pathways in all these organs. Finally, we also observed that flagellin induces a significant increase of multiple cytokines in the plasma from 1 to 6 hours after its intravenous administration. Altogether, these data provide evidence that bacterial flagellin (a) triggers an important inflammatory response and an acute dysfunction of the myocardium, and (b) significantly activates the mechanisms of innate immunity in most major organs and elicits a systemic inflammatory response. In consequence, flagellin may represent a potent mediator of inflammation and multiple organ failure, notably cardiac dysfunction, during Gram-negative septic shock.

Application of molecular techniques in the study of Staphylococcus aureus clonal evolution - A Review

Staphylococcus aureus is an important agent of healthcare-associated and community-acquired infections. A major characteristic of this microorganism is the ability to develop resistance to antimicrobial agents. Several molecular techniques have been applied for the characterization of S. aureus in epidemiological studies. In the present review, we discuss the application of molecular techniques for typing S. aureus strains and describe the nomenclature and evolution of epidemic clones of this important pathogen.

Serologic evidence of human metapneumovirus circulation in Uruguay

First identified in 2001, the human metapneumovirus (hMPV), is a respiratory tract pathogen that affects young children, elderly, and immunocompromised patients. The present work represents the first serologic study carried out in Uruguay. It was performed with the purpose of obtaining serological evidence of hMPV circulation in Uruguay and to contribute to the few serologic reports described until now. Sixty nine serum samples collected between 1998 and 2001 by vein puncture from patients without respiratory symptoms or underlying pathology aged 6 days to 60 years were examined using an indirect immunofluorescence assay (IFA). The global seropositivity rate of the samples was 80% (55/69). Rates of 60% (15/25) and 91% (40/44) were observed for the pediatric and adult cohorts, respectively. Results obtained from a longitudinal analysis of 6 children aged 6 days to 18 months are discussed. These results are a clear evidence of hMPV circulation in Uruguay, at least since 1998, and reinforce the previous data on worldwide circulation of this virus.

The glycoinositol-phospholipids of Phytomonas.

The Phytomonas spp. are trypanosomatid parasites of plants. A polar glycolipid fraction of a Phytomonas sp., isolated from the plant Euphorbia characias and grown in culture, was fractionated into four major glycolipid species (Phy 1-4). The glycolipids were analysed by chemical and enzymic modifications, composition and methylation analyses, electrospray mass spectrometry and microsequencing after HNO2 deamination and NaB3H4 reduction. The water-soluble headgroup of the Phy2 glycolipid was also analysed by 1H NMR. All four glycolipids were shown to be glycoinositol-phospholipids (GIPLs) with phosphatidylinositol (PI) moieties containing the fully saturated alkylacylglycerol lipids 1-O-hexadecyl-2-O-palmitoylglycerol and 1-O-hexadecyl-2-O-stearoylglycerol. The structures of the Phy 1-4 GIPLs are: Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6PI, Glc alpha 1-2(NH2-CH2CH2-HPO4-)Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6PI, [formula: see text] Glc alpha 1-2(NH2CH2CH2-HPO4-)Man alpha 1-2Man alpha 1-6Man alpha 1-4(NH2-CH2CH2-HPO4-)GlcN alpha 1-6PI [formula: see text] and Glc alpha 1-2Glc alpha 1-2(NH2CH2-CH2-HPO4-)Man alpha 1-2Man alpha 1-6Man alpha 1-4(NH2CH2CH2-HPO4-)-GlcN alpha 1-6PI. [formula: see text] The Phytomonas GIPLs represent a novel series of structures. This is the first description of the chemical structure of cell-surface molecules of this plant pathogen. The Phytomonas GIPLs are compared with those of other trypanosomatid parasites and are discussed with respect to trypanosomatid phylogenetic relationships.

Neutrophil transepithelial migration: role of toll-like receptors in mucosal inflammation

The symptomatic phases of many inflammatory diseases are characterized by migration of large numbers of neutrophils (PMN) across a polarized epithelium and accumulation within a lumen. For example, acute PMN influx is common in diseases of the gastrointestinal system (ulcerative colitis, Crohn's disease, bacterial enterocolitis, gastritis), hepatobiliary system (cholangitis, acute cholecystitis), respiratory tract (bronchial pneumonia, bronchitis, cystic fibrosis, bronchiectasis), and urinary tract (pyelonephritis, cystitis). Despite these observations, the molecular basis of leukocyte interactions with epithelial cells is incompletely understood. In vitro models of PMN transepithelial migration typically use N-formylated bacterial peptides such as fMLP in isolation to drive human PMNs across epithelial monolayers. However, other microbial products such as lipopolysaccharide (LPS) are major constituents of the intestinal lumen and have potent effects on the immune system. In the absence of LPS, we have shown that transepithelial migration requires sequential adhesive interactions between the PMN beta2 integrin CD11b/CD18 and JAM protein family members. Other epithelial ligands appear to be abundantly represented as fucosylated proteoglycans. Further studies indicate that the rate of PMN migration across mucosal surfaces can be regulated by the ubiquitously expressed transmembrane protein CD47 and microbial-derived factors, although many of the details remain unclear. Current data suggests that Toll-like receptors (TLR), which recognize specific pathogen-associated molecular patterns (PAMPs), are differentially expressed on both leukocytes and mucosal epithelial cells while serving to modulate leukocyte-epithelial interactions. Exposure of epithelial TLRs to microbial ligands has been shown to result in transcriptional upregulation of inflammatory mediators whereas ligation of leukocyte TLRs modulate specific antimicrobial responses. A better understanding of these events will hopefully provide new insights into the mechanisms of epithelial responses to microorganisms and ideas for therapies aimed at inhibiting the deleterious consequences of mucosal inflammation.

CHARACTERISATION OF ARABIDOPSIS THALIANA PERMEABLE CUTICLE MUTANTS SHOWING A STRONG RESISTANCE TO BOTRYTIS CINEREA

La cuticule des plantes, composée de cutine, un polyester lipidique complexe et de cires cuticulaires, couvre l'épiderme de la plupart des parties aériennes des plantes. Elle est constituée d'une barrière hydrophobique primaire qui minimise les pertes en eau et en soluté et protège l'organisme de différents stress environnementaux tels que les rayons UV, la dessiccation et l'infection par des pathogènes. Elle est aussi impliquée dans la délimitation des organes durant le développement. La cutine est un polyester qui, dans la plupart des espèces végétales, est principalement composé d'acides gras ω-hydroxylés composé de 16 à 18 carbones. Cependant, la cutine des feuilles d'Arabidopsis a une composition différente et est principalement constituée d'acides dicarboxyliques à 16-18 carbones. Les cires sont présentes dans le polyester de la cutine ou le recouvrent. Chez Arabidopsis, un nombre de mutants, tel que 1er, bdg, hth, att1, wbc11, et des plantes transgéniques avec différents changement dans la structure de la cuticule dans les feuilles et la tige, ont récemment été décrits et servent d'outils pour étudier la relation entre la structure et la fonction de la cuticule.7 mutants d'Arabidopsis ont été isolés par une méthode de coloration qui permet de détecter une augmentation dans la perméabilité cuticulaire. Ces mutants ont été appelés pec pour permeable cuticle.Pour la première partie de mon projet, j'ai principalement travaillé avec pec9/bre1 (permeable cuticle 9/botrytis resistance 1). PEC9/BRE1 a été identifié comme étant LACS2 (LONG CHAIN ACYL-CoA SYNTHETASE 2). Dans ce mutant, la cuticule n'est pas visible sous microscopie électronique et la quantité en acides gras omega- hydroxylés et en leurs dérivés est fortement réduite. Ces altérations conduisent à une plus grande perméabilité de la cuticule qui est mise en évidence par une plus grande sensibilité à la sécheresse et aux xénobiotiques et une coloration plus rapide par bleu de toluidine. Le mutant Iacs2 démontre aussi une grande capacité de résistance à l'infection du champignon nécrotrophique B. cinerea. Cette résistance est due à l'extrusion sur les feuilles d'un composé antifongique durant l'infection. Ce travail a été publié dans EMBO journal (Bessire et al., 2007, EMBO Journal).Mon second projet était principalement concentré sur pec1, un autre mutant isolé par le premier crible. La caractérisation de pec1 a révélé des phénotypes similaires à ceux de Iacs2, mais à chaque fois dans des proportions moindres : sensibilité accrue à la sécheresse et aux herbicides, plus grande perméabilité au bleu de toluidine et au calcofluor white, altération de la structure cuticulaire et résistance à B. cinerea à travers la même activité antifongique. PEC1 a été identifié comme étant AtPDR4. Ce gène code pour un transporteur ABC de la famille PDR ("Pleiotropic Drugs Resistance") qui sont des transporteurs ayants un large spectre de substrats. Le mutant se différencie de Iacs2, en cela que la composition en acides gras de la cuticule n'est pas autant altérée. C'est principalement le dihydroxypalmitate des fleurs dont la quantité est réduite. L'expression du gène marqué avec une GFP sous le contrôle du promoteur endogène a permis de localiser le transporteur au niveau de la membrane plasmique des cellules de l'épiderme, de manière polaire. En effet, la protéine est principalement dirigée vers l'extérieure de la plante, là où se trouve la cuticule, suggérant une implication d'AtPDR4 dans le transport de composants de la cuticule. Ce travail est actuellement soumis à Plant Cell.Une étude phylogénétique a aussi montré qu'AtPDR4 était très proche d'OsPDR6 du riz. Le mutant du riz a d'ailleurs montré des phénotypes de nanisme et de perméabilité similaire au mutant chez Arabidopsis.AbstractThe cuticle, consisting principally of cutin and cuticular waxes, is a hydrophobic layer of lipidic nature, which covers all aerial parts of plants and protects them from different abiotic and biotic stresses. Recently, the research in this area has given us a better understanding of the structure and the formation of the cuticle. The Arabidopsis mutants permeable cuticle 1 (peel) and botrytis resistance 1 (brel) were identified in two screens to identify permeable cuticles. The screens used the fluorescent dye calcofluor to measure permeability and also resistance to the fungal pathogen Botrytis. These mutants have highly permeable cuticle characteristics such as higher water loss, intake of chemicals through the cuticle, higher resistance to Botrytis cinerea infection, and organ fusion.BRE1 was cloned and found to be LACS2, a gene previously identified which is important in the formation and biosynthetic pathway of the cuticle. In brel, the amount of the major component of cutin in Arabidopsis leaves and stems, dicarboxylic acids, is five times lower than in the wild type. Moreover, the permeability of the cuticle allows the release of antifungal compounds at the leaf surface that inhibits the growth of two necrotrophic fungi: Botrytis cinerea and Sclerotinia sclerotiorum.PEC1 was identified as AtPDR4, a gene that codes for a plasma membrane transporter of the Pleiotropic Drug Resistance family, a sub-family of the ABC- transporters. AtPDR4 is strongly expressed in the epidermis of expanding tissues. In the epidermis it is located in a polar manner on the external plasma membrane, facing the cuticle. Analysis of the monomer composition of the cutin reveals that in this mutant the amount of hydroxy-acids and dihydroxy-palmitate is 2-3 times lower in flowers, in which organ these cutin monomers are the major components. Thus AtPDR4 is thought to function as a putative cutin monomer transporter.

Detection and molecular characterization of group A rotavirus from hospitalized children in Rio de Janeiro, Brazil, 2004

Rotavirus is a major cause of infantile acute diarrhea, causing about 440,000 deaths per year, mainly in developing countries. The World Health Organization has been recommending the assessment of rotavirus burden and strain characterization as part of the strategies of immunization programs against this pathogen. In this context, a prospective study was made on a sample of 134 children with acute diarrhea and severe dehydration admitted to venous fluid therapy in two state hospitals in Rio de Janeiro, Brazil, from February to September 2004. Rotavirus where detected by polyacrylamide gel electrophoresis (PAGE) and by an enzyme-linked immunoassay to rotavirus and adenovirus (EIARA) in 48% of the children. Positive samples for group A rotavirus (n = 65) were analyzed by reverse transcription/heminested multiplex polymerase chain reaction to determine the frequency of G and [P] genotypes and, from these, 64 samples could be typed. The most frequent G genotype was G1 (58%) followed by G9 (40%). One mixed infection (G1/G9) was detected. The only [P] genotype identified was [8]. In order to estimate the rotavirus infection frequency in children who acquired diarrhea as hospital infection in those hospitals, we studied 24 patients, detecting the pathogen in 41% of them. This data suggest that genotype G9 is an important genotype in Rio de Janeiro, with implications to the future strategies of vaccination against rotavirus, reinforcing the need of continuous monitoring of circulating strains of the pathogen, in a surveillance context.

Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization.

Glomalean fungi induce and colonize symbiotic tissue called arbuscular mycorrhiza on the roots of most land plants. Other fungi also colonize plants but cause disease not symbiosis. Whole-transcriptome analysis using a custom-designed Affymetrix Gene-Chip and confirmation with real-time RT-PCR revealed 224 genes affected during arbuscular mycorrhizal symbiosis. We compared these transcription profiles with those from rice roots that were colonized by pathogens (Magnaporthe grisea and Fusarium moniliforme). Over 40% of genes showed differential regulation caused by both the symbiotic and at least one of the pathogenic interactions. A set of genes was similarly expressed in all three associations, revealing a conserved response to fungal colonization. The responses that were shared between pathogen and symbiont infection may play a role in compatibility. Likewise, the responses that are different may cause disease. Some of the genes that respond to mycorrhizal colonization may be involved in the uptake of phosphate. Indeed, phosphate addition mimicked the effect of mycorrhiza on 8% of the tested genes. We found that 34% of the mycorrhiza-associated rice genes were also associated with mycorrhiza in dicots, revealing a conserved pattern of response between the two angiosperm classes.

Biomarkers of human gastrointestinal tract regions.

Dysregulation of intestinal epithelial cell performance is associated with an array of pathologies whose onset mechanisms are incompletely understood. While whole-genomics approaches have been valuable for studying the molecular basis of several intestinal diseases, a thorough analysis of gene expression along the healthy gastrointestinal tract is still lacking. The aim of this study was to map gene expression in gastrointestinal regions of healthy human adults and to implement a procedure for microarray data analysis that would allow its use as a reference when screening for pathological deviations. We analyzed the gene expression signature of antrum, duodenum, jejunum, ileum, and transverse colon biopsies using a biostatistical method based on a multivariate and univariate approach to identify region-selective genes. One hundred sixty-six genes were found responsible for distinguishing the five regions considered. Nineteen had never been described in the GI tract, including a semaphorin probably implicated in pathogen invasion and six novel genes. Moreover, by crossing these genes with those retrieved from an existing data set of gene expression in the intestine of ulcerative colitis and Crohn's disease patients, we identified genes that might be biomarkers of Crohn's and/or ulcerative colitis in ileum and/or colon. These include CLCA4 and SLC26A2, both implicated in ion transport. This study furnishes the first map of gene expression along the healthy human gastrointestinal tract. Furthermore, the approach implemented here, and validated by retrieving known gene profiles, allowed the identification of promising new leads in both healthy and disease states.

Increased resistance to first-line agents among bacterial pathogens isolated from urinary tract infections in Latin America: time for local guidelines?

Emerging resistance phenotypes and antimicrobial resistance rates among pathogens recovered from community-acquired urinary tract infections (CA-UTI) is an increasing problem in specific regions, limiting therapeutic options. As part of the SENTRY Antimicrobial Surveillance Program, a total of 611 isolates were collected in 2003 from patients with CA-UTI presenting at Latin American medical centers. Each strain was tested in a central laboratory using Clinical Laboratory Standard Institute (CLSI) broth microdilution methods with appropriate controls. Escherichia coli was the leading pathogen (66%), followed by Klebsiella spp. (7%), Proteus mirabilis (6.4%), Enterococcus spp. (5.6%), and Pseudomonas aeruginosa (4.6%). Surprisingly high resistance rates were recorded for E. coli against first-line orally administered agents for CA-UTI, such as ampicillin (53.6%), TMP/SMX (40.4%), ciprofloxacin (21.6%), and gatifloxacin (17.1%). Decreased susceptibility rates to TMP/SMX and ciprofloxacin were also documented for Klebsiella spp. (79.1 and 81.4%, respectively), and P. mirabilis (71.8 and 84.6%, respectively). For Enterococcus spp., susceptibility rates to ampicillin, chloramphenicol, ciprofloxacin, and vancomycin were 88.2, 85.3, 55.9, and 97.1%, respectively. High-level resistance to gentamicin was detected in 24% of Enterococcus spp. Bacteria isolated from patients with CA-UTI in Latin America showed limited susceptibility to orally administered antimicrobials, especially for TMP/SMX and fluoroquinolones. Our results highlight the need for developing specific CA-UTI guidelines in geographic regions where elevated resistance to new and old compounds may influence prescribing decisions.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Host and invader impact of transfer of the clc genomic island into Pseudomonas aeruginosa PAO1

Genomic islands, large potentially mobile regions of bacterial chromosomes, are a major contributor to bacteria evolution. Here, we investigated the fitness cost and phenotypic differences between the bacterium Pseudomonas aeruginosa PAO1 and a derivative carrying one integrated copy of the clc element, a 103-kb genomic island [and integrative and conjugative element (ICE)] originating in Pseudomonas sp. strain B13 and a close relative of genomic islands found in clinical and environmental isolates of P. aeruginosa. By using a combination of whole genome transcriptome profiling, phenotypic arrays, competition experiments, and biofilm formation studies, only few differences became apparent, such as reduced biofilm growth and fourfold stationary phase repression of genes involved in acetoin metabolism in PAO1 containing the clc element. In contrast, PAO1 carrying the clc element acquired the capacity to grow on 3-chlorobenzoate and 2-aminophenol as sole carbon and energy substrates. No fitness loss >1% was detectable in competition experiments between PAO1 and PAO1 carrying the clc element. The genes from the clc element were not silent in PAO1, and excision was observed, although transfer of clc from PAO1 to other recipient bacteria was reduced by two orders of magnitude. Our results indicate that newly acquired mobile DNA not necessarily invoke an important fitness cost on their host. Absence of immediate detriment to the host may have contributed to the wide distribution of genomic islands like clc in bacterial genomes

In Vivo Systematic Analysis of Candida albicans Zn2-Cys6 Transcription Factors Mutants for Mice Organ Colonization.

The incidence of fungal infections in immuno-compromised patients increased considerably over the last 30 years. New treatments are therefore needed against pathogenic fungi. With Candida albicans as a model, study of host-fungal pathogen interactions might reveal new sources of therapies. Transcription factors (TF) are of interest since they integrate signals from the host environment and participate in an adapted microbial response. TFs of the Zn2-Cys6 class are specific to fungi and are important regulators of fungal metabolism. This work analyzed the importance of the C. albicans Zn2-Cys6 TF for mice kidney colonization. For this purpose, 77 Zn2-Cys6 TF mutants were screened in a systemic mice model of infection by pools of 10 mutants. We developed a simple barcoding strategy to specifically detect each mutant DNA from mice kidney by quantitative PCR. Among the 77 TF mutant strains tested, eight showed a decreased colonization including mutants for orf19.3405, orf19.255, orf19.5133, RGT1, UGA3, orf19.6182, SEF1 and orf19.2646, and four an increased colonization including mutants for orf19.4166, ZFU2, orf19.1685 and UPC2 as compared to the isogenic wild type strain. Our approach was validated by comparable results obtained with the same animal model using a single mutant and the revertant for an ORF (orf19.2646) with still unknown functions. In an attempt to identify putative involvement of such TFs in already known C. albicans virulence mechanisms, we determined their in vitro susceptibility to pH, heat and oxidative stresses, as well as ability to produce hyphae and invade agar. A poor correlation was found between in vitro and in vivo assays, thus suggesting that TFs needed for mice kidney colonization may involve still unknown mechanisms. This large-scale analysis of mice organ colonization by C. albicans can now be extended to other mutant libraries since our in vivo screening strategy can be adapted to any preexisting mutants.

Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.