Enteropathogenic Escherichia Coli Adherence Research Articles

Gut feelings: enteropathogenic E. coli (EPEC) interactions with the host.

Enteropathogenic Escherichia coli (EPEC) is a gram-negative bacterial pathogen that adheres to human intestinal epithelial cells, resulting in watery, persistent diarrhea. It subverts the host cell cytoskeleton, causing a rearrangement of cytoskeletal components into a characteristic pedestal structure underneath adherent bacteria. In contrast to other intracellular pathogens that affect the actin cytoskeleton from inside the host cytoplasm, EPEC remains extracellular and transmits signals through the host cell plasma membrane via direct injection of virulence factors by a "molecular syringe," the bacterial type III secretion system. One injected factor is Tir, which functions as the plasma membrane receptor for EPEC adherence. Tir directly links extracellular EPEC through the epithelial membrane and firmly anchors it to the host cell actin cytoskeleton, thereby initiating pedestal formation. In addition to stimulating actin nucleation and polymerization in the host cell, EPEC activates several other signaling pathways that lead to tight junction disruption, inhibition of phagocytosis, altered ion secretion, and immune responses. This review summarizes recent developments in our understanding of EPEC pathogenesis and discusses similarities and differences between EPEC pedestals, focal contacts, and Listeria monocytogenes actin tails.

Saccharomyces boulardii preserves the barrier function and modulates the signal transduction pathway induced in enteropathogenic Escherichia coli-infected T84 cells.

Use of the nonpathogenic yeast Saccharomyces boulardii in the treatment of infectious diarrhea has attracted growing interest. The present study designed to investigate the effect of this yeast on enteropathogenic Escherichia coli (EPEC)-associated disease demonstrates that S. boulardii abrogated the alterations induced by an EPEC strain on transepithelial resistance, [(3)H]inulin flux, and ZO-1 distribution in T84 cells. Moreover, EPEC-mediated apoptosis of epithelial cells was delayed in the presence of S. boulardii. The yeast did not modify the number of adherent bacteria but lowered by 50% the number of intracellular bacteria. Infection by EPEC induced tyrosine phosphorylation of several proteins in T84 cells, including p46 and p52 SHC isoforms, that was attenuated in the presence of S. boulardii. Similarly, EPEC-induced activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway was diminished in the presence of the yeast. Interestingly, inhibition of the ERK1/2 pathway with the specific inhibitor PD 98059 decreased EPEC internalization, suggesting that modulation of the ERK1/2 MAP pathway might account for the lowering of the number of intracellular bacteria observed in the presence of S. boulardii. Altogether, this study demonstrated that S. boulardii exerts a protective effect on epithelial cells after EPEC adhesion by modulating the signaling pathway induced by bacterial infection.

Molecular identification of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea in Londrina, Parana, Brazil

In this work, the prevalence of enteropathogenic Escherichia coli (EPEC) in children in Londrina-PR, Brazil, was evaluated by means of digoxigenin-labelled DNA probes which identify the plasmid responsible for EPEC adherence factor (EAF), and virulence genes for EPEC as bundle-forming pilus (bfp) and E. coli attaching-effacing factor (eae). In addition, the isolated strains were serotyped and tested for adherence to HEp-2 cells. From 102 children with diarrhoea, 19 strains hybridized with at least one probe, and eleven of them were identified as typical EPEC because they hybridized with the three probes used, showed a localized adherence (LA) pattern, and presented no genes for enterotoxins (ST and LT) or invasion as detected by PCR. Six of the typical EPEC strains belonged to the classical serotype O119:H6 (43%); in four strains O antigens could not be determined using antisera against O1 to O173, they were all ONT:H7 (29%); one strain belonged to O111:H6. Three strains were classified as atypical EPEC: O26H-, O111:H9 and O119:HNT. Strains O26H- and O111:H9 hybridized with the eae probe only and showed localized adherence like (LAL) pattern; strain O119:HNT hybridized with the bfp and eae probes, and showed a localized adherence/diffuse adherence (LA/DA) pattern after 6 h. A DA pattern was observed in two strains isolated from children with diarrhoea (ONT:H11 and O142:H34), which hybridized with the eae probe. From 46 controls, five strains hybridized with one or two probes, but none hybridized with all probes or presented the LA pattern. Three strains with the DA pattern hybridized with the eae probe. No EPEC strain belonging to classical EPEC serotypes was isolated from faeces of control children.

Unidentified serogroups of enteropathogenic Escherichia coli (EPEC) associated with diarrhoea in infants in Londrina, Parana, Brazil

Digoxigenin-labelled DNA probes were used to characterise enteropathogenic Escherichia coli (EPEC) isolated in Londrina (Brazil) from faeces samples of 102 children with diarrhoea, and the results were compared with those obtained by serogrouping and adherence to HEp-2 cells. The probes employed detect the gene coding EPEC adherence factor (EAF) and the virulence genes for bundle-forming pilus (bfp) and entero-attaching-effacing (eae) factor. Twenty-one isolates hybridised with at least one probe, and 11 of them were classified as typical EPEC because they hybridised with all three probes, showed a pattern of localised adherence (LA) and carried no genes for enterotoxins (ST and LT) or invasion as detected by PCR. Six of the typical EPEC strains belonged to the classical serotype 0119:H6 and one to O111:H6; O antigens could not be determined in four strains with antisera against 01-0173. All typical EPEC strains carried a 70-MDa plasmid plus two other large plasmids. These data showed that typical EPEC virulence traits may be found in strains not belonging to classical serogroups/serotypes and that molecular identification is required for studying the epidemiology of diarrhoea in children.

Intimin from enteropathogenic Escherichia coli mediates remodelling of the eukaryotic cell surface.

Adhesion to cultured epithelial cells by enteropathogenic Escherichia coli (EPEC) is associated with extensive rearrangement of the host cell cytoskeleton. Evidence has been presented that EPEC adhesion is associated with activation of signal transduction pathways leading to production of a characteristic histopathological feature known as the attaching and effacing (A/E) lesion. A/E lesion formation requires intimin, an EPEC adhesion molecule and several EPEC secreted proteins (EspA, B, D and Tir) involved in cell signalling and protein translocation. In this study it is shown that HEp-2 cells respond during the early stages of infection with two wild-type EPEC strains (B171 and E2348/69) by producing microvillus-like processes (MLP) at the site of initial bacterial adherence. Intimin appears to play a key role in MLP elongation. At later stages of infection with these wild-type EPEC strains, when A/E lesions have formed, the MLP were reduced in number and length to appear as at time zero, and the cell surface in the vicinity of bacterial clusters appeared unaffected. In contrast, infection with EspA- or EspB-negative, but intimin-positive, EPEC strains (UMD872 and UMD864, respectively) resulted in enhanced MLP proliferation and formation of 'cage-like' structures engulfing the bacteria. Inoculating HEp-2 cells with intimin-coated latex spheres induced similar 'cage-like' structures. Caco-2 cells did not show intimin-induced microvillus elongation in response to EPEC infection, although microvillus effacement and reduction in number occurred. Similar phenomena appeared on B171 and E2348/69 infection of paediatric intestine using in vitro organ culture, i.e. elongated microvilli were seen in association with small colonies and at the periphery of large localized colonies, along with evidence of microvillus breakdown and debris in the colony centre. These results show that intimin activates signal transduction pathways involved in the remodelling of the eukaryotic cell surface, probably via binding to a receptor encoded by the host cell.

Taking the host cell hostage

Enteropathogenic Escherichia coli (EPEC) is a major cause of infant diarrhea in developing countries. This bacterium causes a characteristic lesion on epithelial cells known as ‘attaching and effacing’ (AE). In these lesions, the epithelial cell rearranges its cytoskeleton in response to bacterial signals, the microvilli are destroyed and pedestal-like structures are formed, above which the bacteria adheres tightly to the epithelial cell.The pedestal structures are formed in response to bacterial stimuli that induce localized actin rearrangements. In the absence of the adhesin intimin and/or Tir, its receptor, these pedestals are not completely formed. Now, Kalman and collaborators1xEnteropathogenic E. coli acts through WASP and Arp2/3 complex to form actin pedestals. Kalman, D. et al. Nat. Cell Biol. 1999; 1: 389–391Crossref | PubMedSee all References1 have shown that pedestal formation depends on localized recruitment and activation of two host-cell factors involved in actin polymerization: the heptameric Arp2/3 complex (Arp2/3c), and members of the Wiskott–Aldrich syndrome (WAS) family of proteins (WASP and N-WASP). WASP binds to and activates Arp2/3c and activated Arp2/3c nucleates actin polymerization. Recruitment of Arp2/3c depends on WASP and recruitment of WASP depends on its GTPase-binding domain (GBD), suggesting the involvement of a Rho family GTPase.Kalman et al. propose a model for pedestal formation in response to EPEC adherence, in which EPEC adheres and translocates Tir to the host cell by a type III secretion system. Tir is inserted into the host cell membrane, binds to intimin and directly or indirectly recruits and activates a host GTPase of the Chp subfamily. The Chp-like GTPase recruits WASP by binding the GBD domain of this protein, and WASP then recruits and activates Arp2/3c, stimulating actin nucleation and polymerization, driving membrane protusion and pedestal formation. Pedestal formation by EPEC therefore provides an excellent example of a pathogen recruiting host cell proteins for its own advantage.

Human Colostrum and Serum Contain Antibodies Reactive to the Intimin-Binding Region of the Enteropathogenic Escherichia coli Translocated Intimin Receptor

In Brazil, enteropathogenic Escherichia coli (EPEC) diarrhoea is endemic in young infants. A characteristic feature of EPEC adhesion to host cells is intimate attachment leading to the formation of distinctive "attaching and effacing" (A/E) lesions on mammalian cells. Two genes directly involved in intimate adhesion, eae and tir, encode the adhesion molecule intimin and its translocated receptor Tir, respectively. The intimin-binding domain of Tir was recently mapped to the middle part of the polypeptide (Tir-M), and the amino (Tir-N) and carboxy (Tir-C) termini were found to be located within infected host cells. Recently, it was shown that colostrum samples from mothers living in Sao Paulo contain IgA-class antibodies reactive with a number of proteins associated with EPEC virulence. It has also been shown that patients infected with verocytotoxin-producing E. coli O157 can produce antibodies to Tir. In the current study antibody responses to the different Tir domains were analyzed in sera and colostrum samples collected in an EPEC-endemic area of Brazil. Recombinant Tir, Tir-N, Tir-M, and Tir-C were expressed as His-tagged protein in E. coli BL21a and purified on nickel columns. Western blot analysis was used to investigate colostrum IgA- and serum IgG-class antibodies reactive with the Tir fragments. Anti-Tir IgG antibodies were detected in the serum of children, with (63%) or without (50%) diarrhoea. Anti-Tir IgA-class antibodies were detected in all the colostrum pools tested. With the use of both serum IgG- and colostrum IgA-class antibodies, an immunodominant domain of the Tir-polypeptide, Tir M, was identified. The intimin-binding region of Tir (Tir-M) is the immunodominant region of the polypeptide in humans. Both serum IgG-class and colostrum IgA-class antibodies reacted predominantly with the Tir-M domain.

The type IV bundle-forming pilus of enteropathogenic Escherichia coli undergoes dramatic alterations in structure associated with bacterial adherence, aggregation and dispersal.

BFP, a plasmid-encoded type IV bundle-forming pilus produced by enteropathogenic Escherichia coli (EPEC), has recently been shown to be associated with the aggregation of bacteria and dispersal of bacteria from bacterial microcolonies. In standard 3 h HEp-2 cell assays, EPEC adhere in localized microcolonies; after 6 h, bacterial microcolonies are no longer present, indicating that bacterial aggregation and dispersal occurs in vitro during EPEC adhesion to cultured epithelial cells. To examine the role of BFP in EPEC aggregation and dispersal, we examined HEp-2 cell adhesion of strain E2348/69 and defined E2348/69 mutants by immunofluorescence and immunoelectron microscopy. BFP was expressed initially as approximately 40 nm diameter pilus bundles that promoted bacteria-bacteria interaction and microcolony formation. BFP subsequently underwent a striking alteration in structural organization with the formation of much longer and thicker ( approximately 100 nm diameter) pilus bundles, which frequently aggregated laterally to form even thicker bundles often arranged in a loose three-dimensional network; EPEC dispersal from bacterial microcolonies was associated with this transformation of BFP from thin to thick bundles. Bacterial dispersal and transformation of BFP from thin to thick bundles did not occur with a bfpF mutant of strain E2348/69. It is concluded that BFP promotes both the formation and the dispersal of EPEC microcolonies, that the dispersal phase requires BfpF and that dispersal is associated with dramatic alterations in the structure of BFP bundles.

Increased levels of intracellular calcium are not required for the formation of attaching and effacing lesions by enteropathogenic and enterohemorrhagic Escherichia coli.

Elevated concentrations of intracellular calcium ([Ca]i) have been implicated as an important signalling event during attaching and effacing (A/E) lesion formation by enteropathogenic Escherichia coli (EPEC). The highly localized nature of the cytoskeletal and cell surface alterations occurring during A/E lesion formation suggests that there should be equally localized EPEC-induced signalling events. To analyze further the calcium responses to infection of HEp-2 cells by EPEC, we employed calcium-imaging fluorescence microscopy, which allows both temporal and spatial measurements of [Ca]i in live cells. Using this imaging technique, not only were we unable to detect any significant elevation in [Ca]i at sites of A/E EPEC adhesion, but, with several different classical EPEC and enterohemorrhagic E. coli (EHEC) strains and three different infection procedures, each of which resulted in extensive A/E bacterial adhesion, we were unable to detect any significant alterations in [Ca]i in infected cells compared to uninfected cells. In addition, chelation of intracellular free calcium with bis-(aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) did not, as previously reported, prevent A/E lesion formation. We conclude that increased [Ca]i are not required for A/E lesion formation by EPEC and EHEC.

Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro.

Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 (eae deficient), CVD206(pCVD438) (eae-complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a TnphoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206 (pCVD438) (both Int+ BFP+) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int+ BFP-) adhered and caused A/E lesions although bacteria adhered in "flat," two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int- BFP+) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.

Activation of host cell protein kinase C by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) consists of a group of diarrhea-producing E. coli strains, common in developing countries, which do not produce classical toxins and are not truly invasive. EPEC strains adhere to mammalian cells in an intimate fashion, trigger a localized increase in intracellular calcium levels, and elevate inositol phosphate production. We hypothesized that these mediators could activate host cell protein kinase C (PKC) and tested this idea in vitro with two cultured human cell lines, HeLa cells and T84 cells. Using a recently described subculturing protocol to "induce" or accelerate EPEC adherence, we infected the cells with EPEC at a multiplicity of infection of approximately 100:1 for 30 to 60 min. Under these conditions, EPEC E2348 increased membrane-bound PKC activity 1.5- to 2.3-fold in HeLa cells and T84 cells, respectively. The increase in membrane-bound PKC activity was accompanied by a decrease in cytosolic PKC activity in EPEC-infected HeLa cells. Nonadherent laboratory E. coli strains such as HB101 and H.S. failed to trigger any consistent change in PKC production, similar to the nonadherent mutant strains derived from E2348, JPN15 (plasmid cured) and CVD206 (eaeA). In addition, immunoblots performed on extracts of T84 cells with a monoclonal antibody against PKC-alpha showed an increased PKC content in membranes of EPEC-infected cells. Finally, EPEC-infected T84 cells showed a 60% increase in responsiveness to the E. coli heat-stable toxin. We conclude that mediators produced in response to EPEC adherence activate PKC in intestinal and nonintestinal cells.

Genetic and phenotypic analysis of Escherichia coli with enteropathogenic characteristics isolated from Seattle children.

Coliform colonies from children whose stools were submitted for microbiologic analysis were studied prospectively to determine the frequency of shedding of enteropathogenic Escherichia coli (EPEC). In total, 2225 isolates from 445 patients were probed with eaeA (encoding intimin) and the EAF (EPEC adherence factor) probe, and adherence and actin-aggregating phenotypes were determined. Twenty-five patients (5.6%) shed non-O157:H7 eaeA+ EAF- E. coli. Of these 25 patients, isolates from 5 produced Shiga toxins and from 3 possessed bfpA (encoding the bundle-forming pilus) sequences. Non-O157:H7 eaeA+ E. coli from 21 (84%) of 25 patients adhered locally to and aggregated actin in HeLa cells. Four patients shed nonadherent EAF+ eaeA- E. coli. Non-O157:H7 eaeA+ and EAF- isolates belonged to diverse electrophoretic types and classical and nonclassical enteropathogenic serotypes. EPEC are relatively common in stools submitted for analysis in this North American pediatric hospital. Their etiologic role in childhood diarrhea warrants elucidation.

Surface properties of diarrhoeagenic Escherichia coli isolates.

The surface properties of various Escherichia coli isolates associated with diarrhoeal illness were compared by aqueous partitioning between polyethylene glycol (PEG) and Dextran phases. Two well characterised strains of enteropathogenic E. coli (EPEC) were found to be very hydrophobic, based on the critical polymer concentration. EPEC strain E2348 cured of the EPEC adherence factor (EAF) plasmid had much reduced surface hydrophobicity. Partitioning of a series of diarrhoeagenic E. coli strains demonstrated that the majority of EAF+ EPEC strains were significantly more hydrophobic than EAF- EPEC strains. E. coli strains defined as enteroaggregative on the basis of hybridisation with a specific DNA probe showed much greater heterogeneity in their partitioning behaviour, possibly indicating that the AAF/I pili were not expressed in all strains. The E. coli K-12 strain used as a transformation host for adhesion studies had very low surface hydrophobicity but had a detectable negative charge. No alteration in these properties was observed when transformed with EPEC and recombinant plasmids known to specify adherence to tissue culture cells.

Clonal structure and virulence factors in strains of Escherichia coli of the classic serogroup O55.

Virulence properties and genetic variation as determined by multilocus enzyme electrophoresis were studied in 70 strains of Escherichia coli 055, a common serogroup of enteropathogenic E. coli (EPEC), a major cause of infantile diarrhea in developing countries. Nearly 40% of the strains were originally isolated in Brazil and represented serotypes 055:H6, 055:H7, and 055:H51 and nonmotile (055:H-) strains. The analysis of electrophoretic variants of 20 enzymes defined seven distinct electrophoretic types (ETs). ET 1 was represented by 41% of the strains, including strains which usually hybridized with DNA probes for the intimin gene (eaeA), the EPEC adherence plasmid (EAF), and the gene for the pilin subunit of the bundle-forming pilus (bfpA). The ET 1 strains were also typically serotype 055:H6, displayed localized adherence (LA) in tissue culture assays, and were positive in the fluorescent-actin staining test for intimate cell adherence. These same characteristics were observed in the closely related ETs 2 to 4, which clustered in the same branch as ET 1. No known virulence marker could be identified in ET 6. ET 5 included 23 strains, all of which carried the eaeA gene but otherwise displayed a striking array of distinct virulence traits. This ET was represented by 055:H7 strains with phenotypes as diverse as the simultaneous expression of LA and diffuse adherence and the ability to form a newly described adherence pattern, called LA-like adherence. The results suggest that ET 5 marks a special pathogenic clone with a propensity to acquire virulence factors which may facilitate the emergence of new pathogenic strains.

Use of gene probes and adhesion tests to characterise Escherichia coli belonging to enteropathogenic serogroups isolated in the United Kingdom

Nine hundred and twenty-five Escherichia coli isolates from cases of diarrhoea in the United Kingdom and belonging to enteropathogenic E. coli (EPEC) O serogroups were examined for virulence properties. The tests included adhesion to HEp-2 cells, the fluorescence actin staining (FAS) test (which correlates with the ability to cause attaching and effacing lesions) and DNA hybridisations with probes to detect sequences for eaeA (E. coli attaching and effacing factor), EAF (EPEC adherence factor), verocytotoxins VT1 and VT2, enteroaggregative E. coli and diffusely adherent E. coli. The O serogroups examined were 18, 26, 44, 55, 86, 111, 114, 119, 125, 126, 127, 128 and 142. Six hundred and sixty strains (71.4%) hybridised with at least one of the DNA probes. Over 80% of strains in O serogroups 26, 55, 119, 125, 127 and 142 and 41% of strains of serogroups 86, 111, 114, 126 and 128 hybridised with the eae probe and most showed localised attachment and were FAS-positive. However, <10% of these eae probe-positive strains hybridised with the EAF probe. Eighty-four of 232 strains in O serogroups 44, 86, 111, and 126 were enteroaggregative. VT genes were detected in 57 of 402 strains in O serogroups 26, 55, 111 and 128. Identification of EPEC by serogrouping was shown to be an effective method of identifying strains with pathogenic potential, although the organisms were diverse in their properties.

A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation.

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement.

A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus

Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA). LA is dependent upon the presence of 50-70 MDa plasmids, termed EPEC adherence factor (EAF) plasmids. Expression of an EAF plasmid-encoded type IV fimbria, the bundle-forming pilus (BFP), is associated with the LA phenotype. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10 kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes. The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described. The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred. This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein. Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG-inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. coli strain, but is insufficient for LA. This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E. coli strain with a defined set of genes. The BFP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.

Characteristics of Escherichia coli strains belonging to enteropathogenic E. coli serogroups isolated in Italy from children with diarrhea

Fifty-five Escherichia coli strains belonging to enteropathogenic E. coli (EPEC) serogroups were examined for phenotypic and genetic factors associated with virulence. The strains were isolated in Italy from children with diarrhea and identified as EPEC by clinical laboratories using commercially available antisera. O:H serotyping showed that 35 strains (27 of O26, O111, and O128 serogroups) belonged to 11 serotypes considered to be classical EPEC O:H serotypes. The other 20 isolates were classified as 15 nonclassical EPEC O:H serotypes. All the potential EPEC virulence factors associated with bacterial adhesion (localized adherence, fluorescentactin staining test positivity, presence of the attaching and effacing [eaeA] gene), the production of verotoxin, and the positivity with the enterohemorrhagic E. coli probe were significantly more frequent among isolates belonging to classical than nonclassical serotypes. Strains displaying an aggregative adhesion and hybridizing with the enteroaggregative DNA probe were found in serogroups O86, O111, and O126. Verotoxin-producing isolates belonged to serogroups O26, O111, and O128. Only one of the isolates hybridized with the EPEC adherence factor (EAF) probe, but 33 strains gave positive results with the eae probe, confirming that the former is more suitable in epidemiological studies in European countries. These results indicate that up to 75% of strains identified as EPEC by commercial antisera may possess potential virulence properties and/or belong to classical EPEC O:H serotypes and suggest that O grouping is still a useful diagnostic tool for presumptive identification of diarrheagenic E. coli in clinical laboratories.

Adherence patterns and DNA probe types of Escherichia coli isolated from diarrheal patients in China.

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P.R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).

Genotypic and phenotypic characterization of Escherichia coli isolates from dogs manifesting attaching and effacing lesions.

Thirteen Escherichia coli isolates from dogs manifesting attaching and effacing lesions were characterized genetically with respect to the presence of the following virulence determinants associated with human enteropathogenic E. coli (EPEC): eaeA, encoding the outer membrane protein intimin; eaeB, which is necessary for inducing signal transduction; bfpA, encoding the bundle-forming pilus; and the EAF (stands for EPEC adherence factor) plasmid. These isolates were also analyzed phenotypically with respect to adherence to mammalian cells in vivo and in vitro. Nine of these 13 isolates were found to be eaeA positive by PCR: four of these nine were eaeB positive. The 5' end, but not the 3' end, of the eaeA gene was amplified by PCR when primers derived from the eaeA gene of EPEC were used. Six and eight of these 13 isolates were found to be bfpA positive and EAF positive, respectively. The bfpA gene and EAF locus were found on high-molecular-weight plasmids, whereas the eaeA and eaeB genes were chromosomally located when present. Only one canine E. coli isolate, 4221, which was positive for eaeA, eaeB, bfpA, and EAF, adhered to HEp-2 cells in a localized manner and was positive in the fluorescence actin staining test. The nine eaeA-positive isolates adhered to the mucosal surface of piglet ileal explants and induced some microvillus effacement. However, when tested in experimentally inoculated gnotobiotic piglets, isolate 4221 did not induce attaching and effacing lesions at any level of the intestinal tract. Our results indicate that canine E. coli isolates associated with attaching and effacing lesions share some properties with human EPEC but form a heterogeneous group.