Yolande Bertin

Enterohaemorrhagic Escherichia coli gains a competitive advantage by using ethanolamine as a nitrogen source in the bovine intestinal content

The bovine gastrointestinal tract is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Characterization of nutrients that promote the carriage of these pathogens by the ruminant would help to develop ecological strategies to reduce their survival in the bovine gastrointestinal tract. In this study, we show for the first time that free ethanolamine (EA) constitutes a nitrogen source for the O157:H7 EHEC strain EDL933 in the bovine intestinal content because of induction of the eut (ethanolamine utilization) gene cluster. In contrast, the eut gene cluster is absent in the genome of most species constituting the mammalian gut microbiota. Furthermore, the eutB gene (encoding a subunit of the enzyme that catalyses the release of ammonia from EA) is poorly expressed in non-pathogenic E. coli. Accordingly, EA is consumed by EHEC but is poorly metabolized by endogenous microbiota of the bovine small intestine, including commensal E. coli. Interestingly, the capacity to utilize EA as a nitrogen source confers a growth advantage to E. coli O157:H7 when the bacteria enter the stationary growth phase. These data demonstrate that EHEC strains take advantage of a nitrogen source that is not consumed by the resident microbiota, and suggest that EA represents an ecological niche favouring EHEC persistence in the bovine intestine.

Stx2 Subtyping of Shiga Toxin-Producing Escherichia coli Isolated from Cattle in France: Detection of a New Stx2 Subtype and Correlation with Additional Virulence Factors

ABSTRACT At least 11 Stx2 variants produced by Shiga toxin-producingEscherichia coli (STEC) isolated from patients and animals have been described. The Stx2 subtyping of STEC isolated from healthy cows positive for stx2 (n = 104) or stx2 and stx1(n = 63) was investigated. Stx2vh-b, Stx2 (renamed Stx2-EDL933), and Stx2vh-a were the subtypes mostly detected among the bovine isolates (39.5, 39, and 25.5%, respectively). Stx2e was not present, and subtypes included in the Stx2d group (Stx2d-OX3a, Stx2d-O111, and Stx2d-Ount) were found infrequently among the isolates examined (8.5%). A combination of two distinct Stx2 subtypes was observed among 23.5% of the strains. For the first time, a combination of three subtypes (Stx2-EDL933/Stx2vh-b/Stx2d and Stx2vh-a/Stx2vh-b/Stx2d) was detected (3.5% of the isolates). In addition, bovine STEC harboring stx1 and one or two stx2 genes appeared highly cytotoxic toward Vero cells. A new Stx2 subtype (Stx2-NV206), present among 14.5% of the isolates, showed high cytotoxicity for Vero cells. Two amino acid residues (Ser-291 and Glu-297) important for the activation of Stx2 by human intestinal mucus were conserved on the Stx2-NV206 A subunit. The gene encoding Ehx enterohemolysin was prominent among STEC harboringstx2-EDL933 alone (78%) or a combination ofstx2-EDL933 andstx2vh-b (85%). In addition, Stx2-EDL933 and/or Stx2vh-b subtypes were highly associated with other putative virulence factors such as Stx1 and EspP extracellular serine protease, but not with EAST1 enterotoxin.

Molecular Analysis of Shiga Toxin-Producing Escherichia coli Strains Isolated from Hemolytic-Uremic Syndrome Patients and Dairy Samples in France

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) has been associated with food-borne diseases ranging from uncomplicated diarrhea to hemolytic-uremic syndrome (HUS). While most outbreaks are associated with E. coli O157:H7, about half of the sporadic cases may be due to non-O157:H7 serotypes. To assess the pathogenicity of STEC isolated from dairy foods in France, 40 strains isolated from 1,130 raw-milk and cheese samples were compared with 15 STEC strains isolated from patients suffering from severe disease. The presence of genes encoding Shiga toxins (stx1, stx2, and variants), intimin (eae and variants), adhesins (bfp, efa1), enterohemolysin (ehxA), serine protease (espP), and catalase-peroxidase (katP) was determined by PCR and/or hybridization. Plasmid profiling, ribotyping, and pulsed-field gel electrophoresis (PFGE) were used to further compare the strains at the molecular level. A new stx2 variant, stx2-CH013, associated with an O91:H10 clinical isolate was identified. The presence of the stx2, eae, and katP genes, together with a combination of several stx2 variants, was clearly associated with human-pathogenic strains. In contrast, dairy food STEC strains were characterized by a predominance of stx1, with a minority of isolates harboring eae, espP, and/or katP. These associations may help to differentiate less virulent STEC strains from those more likely to cause disease in humans. Only one dairy O5 isolate had a virulence gene panel identical to that of an HUS-associated strain. However, the ribotype and PFGE profiles were not identical. In conclusion, most STEC strains isolated from dairy products in France showed characteristics different from those of strains isolated from patients.

Heterogeneity of Shiga Toxin-Producing Escherichia coli Strains Isolated from Hemolytic-Uremic Syndrome Patients, Cattle, and Food Samples in Central France

ABSTRACT A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like thestx genes or the plasmids (stx2-restriction fragment length polymorphism [RFLP], stx2 gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx2-RFLP andstx2 variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx2-RFLP,stx2 variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.

Association of Virulence Genotype with Phylogenetic Background in Comparison to Different Seropathotypes of Shiga Toxin-Producing Escherichia coli Isolates

ABSTRACT The distribution of virulent factors (VFs) in 287 Shiga toxin-producing Escherichia coli (STEC) strains that were classified according to Karmali et al. into five seropathotypes (M. A. Karmali, M. Mascarenhas, S. Shen, K. Ziebell, S. Johnson, R. Reid-Smith, J. Isaac-Renton, C. Clark, K. Rahn, and J. B. Kaper, J. Clin. Microbiol. 41:4930-4940, 2003) was investigated. The associations of VFs with phylogenetic background were assessed among the strains in comparison with the different seropathotypes. The phylogenetic analysis showed that STEC strains segregated mainly in phylogenetic group B1 (70%) and revealed the substantial prevalence (19%) of STEC belonging to phylogenetic group A (designated STEC-A). The presence of virulent clonal groups in seropathotypes that are associated with disease and their absence from seropathotypes that are not associated with disease support the concept of seropathotype classification. Although certain VFs (eae, stx2-EDL933, stx2-vha, and stx2-vhb) were concentrated in seropathotypes associated with disease, others (astA, HPI, stx1c, and stx2-NV206) were concentrated in seropathotypes that are not associated with disease. Taken together with the observation that the STEC-A group was exclusively composed of strains lacking eae recovered from seropathotypes that are not associated with disease, the“ atypical” virulence pattern suggests that STEC-A strains comprise a distinct category of STEC strains. A practical benefit of our phylogenetic analysis of STEC strains is that phylogenetic group A status appears to be highly predictive of“ nonvirulent” seropathotypes.

Differential expression of stx2 variants in Shiga toxin-producing Escherichia coli belonging to seropathotypes A and C.

Only a subset of Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens, but the characteristics that account for differences in pathogenicity are not well understood. In this study, we investigated the distribution of the stx variants coding for Stx2 and its variants in highly virulent STEC of seropathotype A and low-pathogenic STEC of seropathotype C. We analysed and compared transcription of the corresponding genes, production of Shiga toxins, and stx-phage release in basal as well as in induced conditions. We found that the stx(2) variant was mainly associated with strains of seropathotype A, whereas most of the strains of seropathotype C possessed the stx(2-vhb) variant, which was frequently associated with stx(2), stx(2-vha) or stx(2c). Levels of stx(2) and stx(2)-related mRNA were higher in strains belonging to seropathotype A and in those strains of seropathotype C that express the stx(2) variant than in the remaining strains of seropathotype C. The stx(2-vhb) genes were the least expressed, in basal as well as in induced conditions, and in many cases did not seem to be carried by an inducible prophage. A clear correlation was observed between stx mRNA levels and stx-phage DNA in the culture supernatants, suggesting that most stx(2)-related genes are expressed only when they are carried by a phage. In conclusion, some relationship between stx(2)-related gene expression in vitro and the seropathotype of the STEC strains was observed. A higher expression of the stx(2) gene and a higher release of its product, in basal as well as in induced conditions, was observed in pathogenic strains of seropathotype A. A subset of strains of seropathotype C shows the same characteristics and could be a high risk to human health.

Localization of the insertion site and pathotype determination of the locus of enterocyte effacement of shiga toxin-producing Escherichia coli strains.

ABSTRACT Of 220 Shiga toxin-producing Escherichia coli (STEC) strains collected in central France from healthy cattle, food samples, and asymptomatic children, 12 possessed the eae gene included in the locus of enterocyte effacement (LEE) pathogenicity island. Based on gene typing, we observed 7 different eae espA espB tir pathotypes among the 12 STEC strains and described the new espAβv variant. As previously observed, the O157 serogroup is associated with eaeγ, O26 is associated with eaeβ, and O103 is associated with eaeε. However, the unexpected eaeζ allele was detected in 5 of the 12 isolates. PCR amplification and pulsed-field gel electrophoresis using the I-CeuI endonuclease followed by Southern hybridization indicated that the LEE was inserted in the vicinity of the selC (three isolates), pheU (two isolates), or pheV (six isolates) tRNA gene. Six isolates harbored two or three of these tRNA loci altered by the insertion of integrase genes (CP4-int and/or int-phe), suggesting the insertion of additional foreign DNA fragments at these sites. In spite of great genetic diversity of LEE pathotypes and LEE insertion sites, bovine strains harbor alleles of LEE genes that are frequently found in clinical STEC strains isolated from outbreaks and sporadic cases around the world, underscoring the potential risk of the bovine strains on human health.

Carbohydrate utilization by enterohaemorrhagic Escherichia coli O157:H7 in bovine intestinal content

The bovine gastrointestinal (GI) tract is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Characterization of nutrients preferentially used by EHEC in the bovine intestine would help to develop ecological strategies to reduce EHEC carriage. However, the carbon sources that support the growth of EHEC in the bovine intestine are poorly documented. In this study, a very low concentration of glucose, the most abundant monomer included in the cattle dietary polysaccharides, was detected in bovine small intestine contents (BSIC) collected from healthy cows at the slaughterhouse. Six carbohydrates reported to be included in the mucus layer covering the enterocytes [galactose, N-acetyl-glucosamine (GlcNAc), N-acetyl- galactosamine (GalNAc), fucose, mannose and N-acetyl neuraminic acid (Neu5Ac)] have been quantified for the first time in BSIC and accounted for a total concentration of 4.2 mM carbohydrates. The genes required for enzymatic degradation of the six mucus-derived carbohydrates are highly expressed during the exponential growth of the EHEC strain O157:H7 EDL933 in BSIC and are more strongly induced in EHEC than in bovine commensal E. coli. In addition, EDL933 consumed the free monosaccharides present in the BSIC more rapidly than the resident microbiota and commensal E. coli, indicating a competitive ability of EHEC to catabolize mucus-derived carbohydrates in the bovine gut. Mutations of EDL933 genes required for the catabolism of each of these sugars have been constructed, and growth competitions of the mutants with the wild-type strain clearly demonstrated that mannose, GlcNAc, Neu5Ac and galactose catabolism confers a high competitive growth advantage to EHEC in BSIC and probably represents an ecological niche for EHEC strains in the bovine small intestine. The utilization of these mucus-derived monosaccharides by EDL933 is apparently required for rapid growth of EHEC in BSIC, and for maintaining a competitive growth rate as compared with that of commensal E. coli. The results suggest a strategy for O157:H7 E. coli survival in the bovine intestine, whereby EHEC rapidly consumes mucus-derived carbohydrates that are poorly consumed by bacteria belonging to the resident intestinal microbiota, including commensal E. coli.

Serotyping, stx2 subtyping, and characterization of the locus of enterocyte effacement island of shiga toxin-producing Escherichia coli and E. coli O157:H7 strains isolated from the environment in France.

ABSTRACT Twenty-seven Shiga toxin-producing Escherichia coli (STEC) strains were isolated from 207 stx-positive French environmental samples. Ten of these strains were positive for stx1, and 24 were positive for stx2 (10 were positive for stx2vh-a or stx2vh-b, 19 were positive for stx2d, and 15 were positive for stx2e). One strain belonged to serotype O157:H7, and the others belonged to serogroups O2, O8, O11, O26, O76, O103, O113, O121, O141, O166, and O174. The environment is a reservoir in which new clones of STEC that are pathogenic for humans can emerge.

Pilins of fimbrial adhesins of different member species of enterobacteriaceae are structurally similar to the C-terminal half of adhesin proteins

The structural relatedness of pilins and the C‐terminal half of adhesin proteins in different member species of Enterobacteriaceae was deduced from their two‐dimensional sequence analysis using the hydrophobic cluster analysis (HCA) and secondary structure predictions from the profile network Hei‐Delberg program (PHD). Despite a large evolutionary distance between the two protein families, we show that pilins and the C‐terminal domain of adhesins have a similar folding that can serve as modules for pilus assembly.