New role for the <em>ibeA</em> gene in H2O2 stress resistance of <em>Escherichia coli</em>

International audienceibeA is a virulence factor found in some Extra-intestinal Pathogenic Escherichia coli (ExPEC) strains from the B2 phylogenetic group, and more particularly in newborn meningitic and avian pathogenic strains. It was shown to be involved in the invasion process of the newborn meningitic strain RS218. In a previous work, we showed that in the APEC strain BEN2908 isolated from a colibacillosis case, ibeA was rather involved in adhesion to eukaryotic cells by modulating type 1 fimbriae synthesis. In this study, we demonstrate a new role for ibeA in oxidative stress resistance. We showed that an ibeA mutant of E. coli BEN2908 was more sensitive than its wild type counterpart to H(2)O(2) killing. This phenotype was also observed in a mutant deleted for the whole GimA genomic region carrying ibeA and might be linked to alterations in the expression of a subset of genes involved in the oxidative stress response. We also showed that RpoS expression was not altered by the ibeA deletion. Moreover, the transfer of an ibeA-expressing plasmid into an E. coli K-12 strain, expressing or not type 1 fimbriae, rendered it more resistant to an H(2)O(2) challenge. Altogether, these results show that ibeA by itself is able to confer an increased H(2)O(2) resistance to E. coli. This feature could partly explain the role played by ibeA in the virulence of pathogenic strains

Cellular and humoral immune response to recombinant Escherichia coli OmpA in cows.

The outer membrane protein (Omp) A is a major constituent of the outer membrane of Escherichia coli. This protein has been used in several vaccine development studies, but seldom with a view to vaccinating against mastitis. The objective of this study was to investigate the immunogenicity of E. coli OmpA and its vaccine potential for cows. Both the humoral and cellular immune responses were investigated. The gene for OmpA of the mastitis-causing strain P4 was cloned and expressed, and the recombinant protein (rEcOmpA) purified. Cows were immunized twice with rEcOmpA with adjuvant one month apart by the systemic route. Before immunization, few antibodies to rEcOmpA were detected, and there was little production of IL-17A in a whole blood stimulation assay (WBA) with rEcOmpA. Antibodies to rEcOmpA were induced by immunization. These antibodies were not able to react with E. coli P4, but reacted with a rough P4 mutant prepared by inactivating the rfb locus. This suggests that the complete LPS O-chain precluded the accessibility of antibodies to their target at the outer membrane. The cellular immune response appeared to be biased towards a Th17-type, as more IL-17A than IFN-γ was produced in the OmpA-specific WBA. There was a good correlation between antibody titers and the production of IL-17A in the WBA. The intramammary instillation of rEcOmpA elicited a slight local inflammatory response which was not related to the WBA. Overall, the interest of OmpA as vaccine immunogen was not established, although other experimental conditions (dose, adjuvant, route) need to be investigated to conclude definitively. The study pointed to several important issues such as the accessibility of OmpA to antibodies and the weakness of Th1-type response induced by OmpA

Extraintestinal Pathogenic Escherichia coli Strains of Avian and Human Origin: Link between Phylogenetic Relationships and Common Virulence Patterns▿

Extraintestinal pathogenic Escherichia coli (ExPEC) strains of human and avian origin show similarities that suggest that the avian strains potentially have zoonotic properties. However, the phylogenetic relationships between avian and human ExPEC strains are poorly documented, so this possibility is difficult to assess. We used PCR-based phylotyping and multilocus sequence typing (MLST) to determine the phylogenetic relationships between 39 avian pathogenic E. coli (APEC) strains of serogroups O1, O2, O18, and O78 and 51 human ExPEC strains. We also compared the virulence genotype and pathogenicity for chickens of APEC strains and human ExPEC strains. Twenty-eight of the 30 APEC strains of serogroups O1, O2, and O18 were classified by MLST into the same subcluster (B2-1) of phylogenetic group B2, whereas the 9 APEC strains of serogroup O78 were in phylogenetic groups D (3 strains) and B1 (6 strains). Human ExPEC strains were closely related to APEC strains in each of these three subclusters. The 28 avian and 25 human strains belonging to phylogenetic subcluster B2-1 all expressed the K1 antigen and presented no significant differences concerning the presence of other virulence factors. Moreover, human strains of this phylogenetic subcluster were highly virulent for chicks, so no host specificity was identified. Thus, APEC strains of serotypes O1:K1, O2:K1, and O18:K1 belong to the same highly pathogenic clonal group as human E. coli strains of the same serotypes isolated from cases of neonatal meningitis, urinary tract infections, and septicemia. These APEC strains constitute a potential zoonotic risk

Escherichia coli mastitis strains: In vitro phenotypes and severity of infection in vivo.

Mastitis remains a major infection of dairy cows and an important issue for dairy farmers and the dairy industry, in particular infections due to Escherichia coli strains. So far, properties specific to E. coli causing mastitis remain ill defined. In an attempt to better understand the properties required for E. coli to trigger mastitis, we used a range of in vitro assays to phenotypically characterize four E. coli strains, including the prototypical E. coli mastitis strain P4, possessing different relative abilities to cause mastitis in a mouse model. Our results indicate that a certain level of serum resistance might be required for colonization of the mammary gland. Resistance to neutrophil killing is also likely to contribute to a slower clearance of bacteria and higher chances to colonize the udder. In addition, we show that the four different strains do induce a pro-inflammatory response by mammary epithelial cells but with different intensities. Interestingly, the prototypical mastitis strain P4 actually induces the less intense response while it is responsible for the most severe infections in vivo. Altogether, our results suggest that different strategies can be used by E. coli strains to colonize the mammary gland and cause mastitis

ICEEc2, a New Integrative and Conjugative Element Belonging to the pKLC102/PAGI-2 Family, Identified in Escherichia coli Strain BEN374▿ †

The diversity of the Escherichia coli species is in part due to the large number of mobile genetic elements that are exchanged between strains. We report here the identification of a new integrative and conjugative element (ICE) of the pKLC102/PAGI-2 family located downstream of the tRNA gene pheU in the E. coli strain BEN374. Indeed, this new region, which we called ICEEc2, can be transferred by conjugation from strain BEN374 to the E. coli strain C600. We were also able to transfer this region into a Salmonella enterica serovar Typhimurium strain and into a Yersinia pseudotuberculosis strain. This transfer was then followed by the integration of ICEEc2 into the host chromosome downstream of a phe tRNA gene. Our data indicated that this transfer involved a set of three genes encoding DNA mobility enzymes and a type IV pilus encoded by genes present on ICEEc2. Given the wide distribution of members of this family, these mobile genetic elements are likely to play an important role in the diversification of bacteria

A Metabolic Operon in Extraintestinal Pathogenic Escherichia coli Promotes Fitness under Stressful Conditions and Invasion of Eukaryotic Cells▿ †

We identified a carbohydrate metabolic operon (frz) that is highly associated with extraintestinal pathogenic Escherichia coli (ExPEC) strains. The frz operon codes for three subunits of a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) transporter of the fructose subfamily, for a transcriptional activator of PTSs of the MgA family, for two type II ketose-1,6-bisphosphate aldolases, for a sugar-specific kinase (repressor, open reading frame, kinase family [ROK]), and for a protein of the cupin superfamily. We proved that the frz operon promotes bacterial fitness under stressful conditions, such as oxygen restriction, late stationary phase of growth, or growth in serum or in the intestinal tract. Furthermore, we showed that frz is involved in adherence to and internalization in human type II pneumocytes, human enterocytes, and chicken liver cells by favoring the ON orientation of the fim operon promoter and thus acting on the expression of type 1 fimbriae, which are the major ExPEC adhesins. Both the PTS activator and the metabolic enzymes encoded by the frz operon are involved in these phenotypes

Mammary antigen-specific inflammatory response.

<p>A) Time-course of cell concentrations in milk (SCC) after infusion of 3 μg rEcOmpA through the teat canal of immunized and control cows. B) Time-course of cell concentrations in milk (SCC) after infusion of 9 μg rEcOmpA through the teat canal of immunized and control cows. Data are median values and interquartile (Q1; Q3).</p

Correlations between humoral and cellular responses to rEcOmpA.

<p>Correlations between humoral and cellular responses to rEcOmpA.</p

Humoral response to immunization with rEcOmpA.

<p>A) The response to immunization with rEcOmpA (on days 0 and 30) was assessed by ELISA using whole heat-killed smooth (P4) and rough (P4Δrfb) bacteria as coating antigen. Data are median values (12 immunized cows) and interquartile (Q1 and Q3). B) Reference curve of the pooled serum in ELISA with <i>E</i>. <i>coli</i> P4 and P4Δrfb as coating antigens and secondary antibody to bovine IgG(H+L) revealing IgG and IgM antibodies. C) Titration of natural antibodies detected before immunization, showing the correlation between titers of antibodies to <i>E</i>. <i>coli</i> P4 and P4Δrfb. D) Titration of antibodies to <i>E</i>. <i>coli</i> P4Δrfb in the IgM isotype and IgG1 and IgG2 subisotypes. As different reference sera, and different secondary antibodies were used for IgM and IgG antibodies, only titers variation can be compared, not their absolute values.</p