Comparative genomic analysis and molecular examination of the diversity of enterotoxigenic Escherichia coli isolates from Chile

Enterotoxigenic Escherichia coli (ETEC) is one of the most common diarrheal pathogens in the low- and middle-income regions of the world, however a systematic examination of the genomic content of isolates from Chile has not yet been undertaken. Whole genome sequencing and comparative analysis of a collection of 125 ETEC isolates from three geographic locations in Chile, allowed the interrogation of phylogenomic groups, sequence types and genes specific to isolates from the different geographic locations. A total of 80.8% (101/125) of the ETEC isolates were identified in E. coli phylogroup A, 15.2% (19/125) in phylogroup B, and 4.0% (5/125) in phylogroup E. The over-representation of genomes in phylogroup A was significantly different from other global ETEC genomic studies. The Chilean ETEC isolates could be further subdivided into sub-clades similar to previously defined global ETEC reference lineages that had conserved multi-locus sequence types and toxin profiles. Comparison of the gene content of the Chilean ETEC identified genes that were unique based on geographic location within Chile, phylogenomic classifications or sequence type. Completion of a limited number of genomes provided insight into the ETEC plasmid content, which is conserved in some phylogenomic groups and not conserved in others. These findings suggest that the Chilean ETEC isolates contain unique virulence factor combinations and genomic content compared to global reference ETEC isolates

Highly conserved type 1 pili promote enterotoxigenic E. coli pathogen-host interactions

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens

Conservation and global distribution of non-canonical antigens in enterotoxigenic Escherichia coli

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) cause significant diarrheal morbidity and mortality in children of resource-limited regions, warranting development of effective vaccine strategies. Genetic diversity of the ETEC pathovar has impeded development of broadly protective vaccines centered on the classical canonical antigens, the colonization factors and heat-labile toxin. Two non-canonical ETEC antigens, the EtpA adhesin, and the EatA mucinase are immunogenic in humans and protective in animal models. To foster rational vaccine design that complements existing strategies, we examined the distribution and molecular conservation of these antigens in a diverse population of ETEC isolates. METHODS: Geographically diverse ETEC isolates (n = 1159) were interrogated by PCR, immunoblotting, and/or whole genome sequencing (n = 46) to examine antigen conservation. The most divergent proteins were purified and their core functions assessed in vitro. RESULTS: EatA and EtpA or their coding sequences were present in 57.0% and 51.5% of the ETEC isolates overall, respectively; and were globally dispersed without significant regional differences in antigen distribution. These antigens also exhibited \u3e93% amino acid sequence identity with even the most divergent proteins retaining the core adhesin and mucinase activity assigned to the prototype molecules. CONCLUSIONS: EtpA and EatA are well-conserved molecules in the ETEC pathovar, suggesting that they serve important roles in virulence and that they could be exploited for rational vaccine design

Modulation of porcine intestinal epitheliocytes immunetranscriptome response by Lactobacillus jensenii TL2937

In order to evaluate probiotic strains applicable for the beneficial immunomodulation of the porcine gut (immunobiotics), we previously developed a porcine intestinal epitheliocyte cell line (PIE cells). Here, transcriptomic studies using PIE cells were performed considering that this information would be valuable for understanding the mechanisms involved in the protective activity of the immunobiotic strain Lactobacillus jensenii TL2937 against intestinal inflammatory damage in pigs. In addition, those studies would provide criteria for selecting biomarkers for the screening of new immunobiotic strains. We performed microarray analysis to investigate the transcriptomic response of PIE cells to the challenge with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs) and, the changes induced by L. jensenii TL2937 in that response. The approach allowed us to obtain a global overview of the immune genes involved in the response of PIE cells to heat-stable ETEC PAMPs. We observed that L. jensenii TL2937 differently modulated gene expression in ETEC PAMPs-challenged PIE cells. Microarray and RT-PCR analysis indicated that the most remarkable changes in PIE cells transcriptomic profile after heat-stable ETEC PAMPs challenge were observed in chemokines, adhesion molecules, complement and coagulation cascades factors. In addition, an anti-inflammatory effect triggered by TL2937 strain in PIE cells was clearly demonstrated. The decrease in the expression of chemokines (CCL8, CXCL5, CXCL9, CXCL10, and CXCL11), complement (C1R, C1S, C3, and CFB), and coagulation factors (F3) by L. jensenii TL2937 supports our previous reports on the immunoregulatory effect of this strain. These results provided clues for the better understanding of the mechanism underlying host-immunobiotic interaction in the porcine host. The comprehensive transcriptomic profiles of PIE cells provided by our analyses successfully identified a group of genes, which could be used as prospective biomarkers for the screening and evaluation of new anti-inflammatory immunobiotics for the prevention of inflammatory intestinal disorders in pigs.Fil: Kobayashi, Hisakazu. Tohoku University; JapónFil: Albarracín, Leonardo Miguel. Tohoku University; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Sato, Nana. Tohoku University; JapónFil: Kanmani, Paulraj. Tohoku University; JapónFil: Kober, Humayun A.K.M.. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University. Department of Dairy and Poultry Science; BangladeshFil: Ikeda-Ohtsubo, Wakako. Tohoku University; JapónFil: Suda, Yoshihito. Miyagi University; JapónFil: Nochi, Tomonori. Tohoku University; JapónFil: Aso, Hisashi. Tohoku University; JapónFil: Makino, Seiya. Meiji Co., Ltd. Food Science Research Labs.; JapónFil: Kano, Hiroshi. Meiji Co., Ltd. Food Science Research Labs.; JapónFil: Ohkawara, Sou. Meiji Seika Pharma Co., Ltd. Agricultural and Veterinary Division; JapónFil: Saito, Tadao. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó

Dynamic interactions of a conserved enterotoxigenic Escherichia coli adhesin with intestinal mucins govern epithelium engagement and toxin delivery

At present, there is no vaccine for enterotoxigenic Escherichia coli (ETEC), an important cause of diarrheal illness. Nevertheless, recent microbial pathogenesis studies have identified a number of molecules produced by ETEC that contribute to its virulence and are novel antigenic targets to complement canonical vaccine approaches. EtpA is a secreted two-partner adhesin that is conserved within the ETEC pathovar. EtpA interacts with the tips of ETEC flagella to promote bacterial adhesion, toxin delivery, and intestinal colonization by forming molecular bridges between the bacteria and the epithelial surface. However, the nature of EtpA interactions with the intestinal epithelium remains poorly defined. Here, we demonstrate that EtpA interacts with glycans presented by transmembrane and secreted intestinal mucins at epithelial surfaces to facilitate pathogen-host interactions that culminate in toxin delivery. Moreover, we found that a major effector molecule of ETEC, the heat-labile enterotoxin (LT), may enhance these interactions by stimulating the production of the gel-forming mucin MUC2. Our studies suggest, however, that EtpA participates in complex and dynamic interactions between ETEC and the gastrointestinal mucosae in which host glycoproteins promote bacterial attachment while simultaneously limiting the epithelial engagement required for effective toxin delivery. Collectively, these data provide additional insight into the intricate nature of ETEC interactions with the intestinal epithelium that have potential implications for rational approaches to vaccine design

Evidence for antibiotic induced Clostridium perfringens diarrhoea

Clostridium difficile is a well documented cause of antibiotic associated diarrhoea in hospitalised patients, but may account for only approximately 20% of all cases. This leader reviews the current knowledge and understanding of the pathogenesis, epidemiology, and diagnosis of non-food borne Clostridium perfringens diarrhoea. Although enterotoxigenic C perfringens has been implicated in some C difficile negative cases of antibiotic associated diarrhoea, C perfringens enterotoxin detection methods are not part of the routine laboratory investigation of such cases. Testing for C perfringens enterotoxin in faecal samples from patients with antibiotic associated diarrhoea and sporadic diarrhoea on a routine basis would have considerable resource implications. Therefore, criteria for initiating investigations and optimum laboratory tests need to be established. In addition, establishing the true burden of C perfringens antibiotic associated diarrhoea is important before optimum control and treatment measures can be defined