Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups.
The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers. (+info)
Enteropathogenic Escherichia coli (EPEC) infection of the human small intestine induces severe watery diarrhoea linked to a rather weak inflammatory response despite EPEC's in vivo capacity to disrupt epithelial barrier function. Here, we demonstrate that EPEC flagellin triggers the secretion of the pro-inflammatory cytokine, interleukin (IL)-8, from small (Caco-2) and large (T84) intestinal epithelia model systems. Interestingly, IL-8 secretion required basolateral infection of T84 cells implying that flagellin must penetrate the epithelial barrier. In contrast, apical infection of Caco-2 cells induced IL-8 secretion but less potently than basolateral infections. Importantly, infection of Caco-2, but not T84 cells rapidly inhibited IL-8 secretion by a mechanism dependent on the delivery of effectors through a translocation system encoded on the locus of enterocyte effacement (LEE). Moreover, EPEC prevents the phosphorylation-associated activation of multiple kinase pathways regulating IL-8 gene transcription by a mechanism apparently independent of LEE-encoded effectors and four non-LEE-encoded effectors. Crucially, our studies reveal that EPEC inhibits the capacity of the cells to secrete IL-8 in response to bacterial antigens and inflammatory cytokines prior to disrupting barrier function by a distinct mechanism. Thus, these findings also lend themselves to a plausible mechanism to explain the absence of a strong inflammatory response in EPEC-infected humans. (+info)
The possible influence of LuxS in the in vivo virulence of rabbit enteropathogenic Escherichia coli.
Attaching and effacing (A/E) organisms, such as rabbit enteropathogenic Escherichia coli (EPEC), human EPEC or enterohemorrhagic E. coli (EHEC) share attaching and effacing phenotype and LEE pathogenicity island responsible for A/E. The present study was undertaken to investigate the impact of the LuxS quorum sensing (QS) signaling system in vitro and in vivo pathogenicity of A/E organisms using rabbit EPEC (rEPEC) strain E22 (O103:H2). Analysis of the bioluminescence indicated abolished production of the QS signal AI-2 by luxS mutant (E22DeltaluxS). Strain E22Deltalux also exhibited impaired expression of several normally secreted proteins and reduced adherence to cultured HeLa cells. Complementation of the intact luxS gene to E22DeltaluxS restored secreted protein expression comparable to the WT type but not adherence to HeLa cells. In experimentally infected rabbits, the isogenic luxS mutant induced clinical illness and intimate adherence to the intestinal mucosa, albeit to a less extent, comparable to that seen with the parent virulent strain. It is worth noting that reduced fecal bacterial shedding, mucosal adherence and improved cumulative weight gain were seen for the mutant strain complemented with luxS when compared to the WT. It appears that the luxS gene is not essential for in vivo pathogenicity by rEPEC where exogenous QS signals are present in the gut. The impact of AI-2 provided by multicopy plasmid on bacterial virulence is discussed. (+info)
The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin.
Synthetic N-acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc-specific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants (K(assoc)), to synthetic LacNAc-Benzene and LacNAc-O(CH(2))(8)CONH(2) glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express alpha bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the alpha bundlin monomer. Collectively, these results suggest that alpha bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of alpha bundlin-expressing EPEC strains to host intestinal epithelial cells. (+info)
Comparison between O serotyping method and multiplex real-time PCR to identify diarrheagenic Escherichia coli in Taiwan.
To compare the diarrheagenic Escherichia coli (DEC) identifications obtained between traditional O serotyping and modern virulence gene detection assays, we developed a multiplex real-time PCR assay by detecting six specific virulence genes for enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). Among 261 clinical diarrheal stool samples, a total of 137 suspected DEC (sDEC) isolates were identified by the use of commercially available antisera. The most prevalent serogroups were O1 (12/137; 8.7%), O25 (9/137; 6.5%), and O44 (9/137; 6.5%). The specific virulence genes for the 137 sDEC isolates were analyzed by the multiplex real-time PCR assay. Fifteen (10.9%) of 137 isolates were confirmed to be true DEC strains, indicating that the serotypic markers did not correlate with the specific virulence genes. ETEC (66.7%) was the most prevalent, followed by EIEC (20%) and EPEC (13.3%). No EHEC strains were identified in the specimens. Four novel serotypes were found in the study: two in EPEC strains (O111:H9 and O63:H6) and two in EIEC strains (O63:H9 and O169:H9). In conclusion, the real-time PCR assay considerably reduces the high false-positive rate from the use of serotyping alone, and thus, it is suggested that serogrouping-based methods are inadequate for the identification of DEC isolates, although they are useful for the identification of a limited number of serogroups. In addition, ETEC, EPEC, and EIEC strains were present in 5.7% (15/261) of the diarrheal patients in northern Taiwan in 2006. (+info)
Distribution, functional expression, and genetic organization of Cif, a phage-encoded type III-secreted effector from enteropathogenic and enterohemorrhagic Escherichia coli.
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049 E. coli strains of human, animal, and environmental origins. A total of 115 E. coli isolates from diverse origins and geographic locations carried cif. The presence of cif was tightly associated with the LEE, since all the cif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population of E. coli but positively selected within the population of LEE-positive strains. Nonetheless, 66% of cif-positive E. coli strains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in the cif gene. The passenger region of the prophages carrying cif was highly variable and showed various combinations of IS elements and genes coding for other effectors such as nleB, nleC, nleH, nleG, espJ, and nleA/espI (some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism. (+info)
Effect of zinc in enteropathogenic Escherichia coli infection.
Enteropathogenic Escherichia coli (EPEC) infection triggers the release of ATP from host intestinal cells, and the ATP is broken down to ADP, AMP, and adenosine in the lumen of the intestine. Ecto-5'-nucleotidase (CD73) is the main enzyme responsible for the conversion of 5'-AMP to adenosine, which triggers fluid secretion from host intestinal cells and also has growth-promoting effects on EPEC bacteria. In a recent study, we examined the role of the host enzyme CD73 in EPEC infection by testing the effect of ecto-5'-nucleotidase inhibitors. Zinc was a less potent inhibitor of ecto-5'-nucleotidase in vitro than the nucleotide analog alpha,beta-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than alpha,beta-methylene-ADP. This discrepancy between the in vitro and in vivo potencies of the two inhibitors prompted us to search for potential targets of zinc other than ecto-5'-nucleotidase. Zinc, at concentrations that produced little or no inhibition of EPEC growth, caused a decrease in the expression of EPEC protein virulence factors, such as bundle-forming pilus (BFP), EPEC secreted protein A, and other EPEC secreted proteins, and reduced EPEC adherence to cells in tissue culture. The effects of zinc were not mimicked by other transition metals, such as manganese, iron, copper, or nickel, and the effects were not reversed by an excess of iron. Quantitative real-time PCR showed that zinc reduced the abundance of the RNAs encoded by the bfp gene, by the plasmid-encoded regulator (per) gene, by the locus for the enterocyte effacement (LEE)-encoded regulator (ler) gene, and by several of the esp genes. In vivo, zinc reduced EPEC-induced fluid secretion into ligated rabbit ileal loops, decreased the adherence of EPEC to rabbit ileum, and reduced histopathological damage such as villus blunting. Some of the beneficial effects of zinc on EPEC infection appear to be due to the action of the metal on EPEC bacteria as well as on the host. (+info)
Influence of geographical origin, host animal and stx gene on the virulence characteristics of Escherichia coli O26 strains.
The influence of geographical origin, host animal and presence of the stx gene on the virulence of Escherichia coli O26 strains from ruminants was determined in this study. A clear association was found between the virulence profile and geographical origin of Shiga-toxigenic E. coli (STEC) O26 strains, with UK STEC O26 strains harbouring virtually identical profiles, whilst central European strains showed considerable heterogeneity in plasmid-encoded genes. The former group were also more likely to be non-motile and katP gene positive. Comparison of UK STEC and atypical enteropathogenic E. coli (aEPEC) O26 strains showed that the presence of the stx1 gene was positively correlated with the presence of espP and katP genes and negatively associated with the presence of the yagP-yagT region and with rhamnose fermentation. In contrast to the uniform profiles of STEC O26 strains from ruminants in the UK, aEPEC O26 strains of bovine and ovine origin showed diverse profiles both within and between groups, and could not be separated into discrete groups. These results indicate that the characteristics of UK O26 strains from ruminants are distinct from those of O26 strains from ruminants and humans in other regions in central Europe. Such differences are expected to influence the zoonotic potential of this pathogen and the subsequent incidence of O26-associated human disease. (+info)