Flagellin induces β-defensin 2 in human colonic ex vivo infection with enterohemorrhagic Escherichia coli

Enterohemorrhagic E. coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human β-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37 and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-κB, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8

Enterohemorrhagic Escherichia coli infection inhibits colonic thiamin pyrophosphate uptake via transcriptional mechanism.

Colonocytes possess a specific carrier-mediated uptake process for the microbiota-generated thiamin (vitamin B1) pyrophosphate (TPP) that involves the TPP transporter (TPPT; product of the SLC44A4 gene). Little is known about the effect of exogenous factors (including enteric pathogens) on the colonic TPP uptake process. Our aim in this study was to investigate the effect of Enterohemorrhagic Escherichia coli (EHEC) infection on colonic uptake of TPP. We used human-derived colonic epithelial NCM460 cells and mice in our investigation. The results showed that infecting NCM460 cells with live EHEC (but not with heat-killed EHEC, EHEC culture supernatant, or with non-pathogenic E. Coli) to lead to a significant inhibition in carrier-mediated TPP uptake, as well as in level of expression of the TPPT protein and mRNA. Similarly, infecting mice with EHEC led to a significant inhibition in colonic TPP uptake and in level of expression of TPPT protein and mRNA. The inhibitory effect of EHEC on TPP uptake by NCM460 was found to be associated with reduction in the rate of transcription of the SLC44A4 gene as indicated by the significant reduction in the activity of the SLC44A4 promoter transfected into EHEC infected cells. The latter was also associated with a marked reduction in the level of expression of the transcription factors CREB-1 and ELF3, which are known to drive the activity of the SLC44A4 promoter. Finally, blocking the ERK1/2 and NF-kB signaling pathways in NCM460 cells significantly reversed the level of EHEC inhibition in TPP uptake and TPPT expression. Collectively, these findings show, for the first time, that EHEC infection significantly inhibit colonic uptake of TPP, and that this effect appears to be exerted at the level of SLC44A4 transcription and involves the ERK1/2 and NF-kB signaling pathways

Enterohemorrhagic Escherichia coli colonization of human colonic epithelium in vitro and ex vivo

Enterohemorrhagic E. coli (EHEC) are important foodborne pathogens causing gastroenteritis and more severe complications such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by using in vitro organ culture (IVOC) of colonic biopsies and polarized T84 colon carcinoma cells. We showed for the first time that EHEC colonized human colonic biopsies by forming typical attaching/effacing (A/E) lesions which were dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the Translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Neither colonization of biopsies nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonize and form stable A/E lesions on the human colon which is likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models as they might not reflect the in vivo situation

Revisiting the STEC Testing Approach: Using espK and espV to Make Enterohemorrhagic Escherichia coli (EHEC) Detection More Reliable in Beef

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of “false positive” results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013

Revisiting the STEC Testing Approach: Using espK and espV to Make Enterohemorrhagic Escherichia coli (EHEC) Detection More Reliable in Beef

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of “false positive” results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013

Enterohemorrhagic Escherichia coli with particular attention to the German outbreak strain O104:H4

This review deals with the epidemiology and ecology of enterohemorrhagic Escherichia coli (EHEC), a subset of the verocytotoxigenic Escherichia coli (VTEC), and subsequently discusses its public health concern. Attention is also given to the outbreak strain O104:H4, which has been isolated as causative agent of the second largest outbreak of the hemolytic uremic syndrome worldwide, which started in Germany in May 2011. This outbreak strain is not an EHEC as such but possesses an unusual combination of EHEC and enteroaggregative E. coli (EAggEC) virulence properties

Prevalence and occurrence of zoonotic bacterial pathogens in surface waters determined by quantitative PCR

The prevalence and concentrations of Campylobacter jejuni, Salmonella spp. and enterohaemorrhagic E. coli (EHEC) were investigated in surface waters in Brisbane, Australia using quantitative PCR (qPCR) based methodologies. Water samples were collected from Brisbane City Botanic Gardens (CBG) Pond, and two urban tidal creeks (i.e., Oxley Creek and Blunder Creek). Of the 32 water samples collected, 8 (25%), 1 (3%), 9 (28%), 14 (44%), and 15 (47%) were positive for C. jejuni mapA, Salmonella invA, EHEC O157 LPS, EHEC VT1, and EHEC VT2 genes, respectively. The presence/absence of the potential pathogens did not correlate with either E. coli or enterococci concentrations as determined by binary logistic regression. In conclusion, the high prevalence, and concentrations of potential zoonotic pathogens along with the concentrations of one or more fecal indicators in surface water samples indicate a poor level of microbial quality of surface water, and could represent a significant health risk to users. The results from the current study would provide valuable information to the water quality managers in terms of minimizing the risk from pathogens in surface waters

Extensive genomic diversity and selective conservation of virulence determinants in enterohemorrhagic Escherichia coli strains of O157 and non O157 serotypes

Background: Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe food-borne illness in humans. The chromosome of O157 consists of 4.1 Mb backbone sequences shared by benign E. coli K-12, and 1.4 Mb O157-specific sequences encoding many virulence determinants, such as Shiga toxin genes (stx genes) and the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to distinct clonal lineages from O157 also cause similar illness in humans. According to the "parallel" evolution model, they have independently acquired the major virulence determinants, the stx genes and LEE. However, the genomic differences between O157 and non-O157 EHECs have not yet been systematically analyzed. Results: Using microarray and whole genome PCR scanning analyses, we performed a whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with O157. In non-O157 EHEC strains, although genome sizes were similar with or rather larger than O157 and the backbone regions were well conserved, O157-specific regions were very poorly conserved. Around only 20% of the O157- specific genes were fully conserved in each non-O157 serotype. However, the non-O157 EHECs contained a significant number of virulence genes that are found on prophages and plasmids in O157, and also multiple prophages similar to, but significantly divergent from, those in O157. Conclusion: Although O157 and non-O157 EHECs have independently acquired a huge amount of serotype- or strain-specific genes by lateral gene transfer, they share an unexpectedly large number of virulence genes. Independent infections of similar but distinct bacteriophages carrying these virulence determinants are deeply involved in the evolution of O157 and non-O157 EHECs

Escherichia coli EHEC Germany outbreak preliminary functional annotation using BG7 system

We have annotated the European outbreak E. coli EHEC genome sequenced by BGI (6-2-2011) and assembled with MIRA by Nick Loman (6-2-2011 ). Our system BG7, Bacterial Genome annotation of Era7 Bioinformatics, predicts ORFs and annotates them based on fragments of similarity with Uniprot proteins. We have predicted 6327 genes, 6156 encoding proteins y 171 corresponding to ribosomal and tRNA. Based on the preliminary results of our semi-automated method of annotation we have selected some predicted proteins with potential implications in pathogenicity and virulence.
There are 33 predicted genes annotated as toxins and we have found three putative hemolysins: Hemolysin E, a putative hemolysin expression modulating protein and a channel protein, hemolysin III family. We have found 31 predicted genes that could be related to specific antibiotic resistance: beta-lactamic, aminoglycoside, macrolide, polymyxin, tetracycline, fosfomycin and deoxycholate, novobiocin, chloramphenicol, bicyclomycin, norfloxacin and enoxacin and 6-mercaptopurine. This strain is rich in adhesion, secretion systems, pathogenicity and virulence related proteins. It seems to have a restriction-modification system, many proteins involved in Fe transport and utilization (siderophores as aerobactin and enterobactin), lysozyme, one inhibitor of pancreatic serine proteases, proteins involved in anaerobic respiration, antimicrobial peptides, and proteins involved in quorum sensing and biofilm formation that could confer competitive advantage to this strain