Diarrhoeagenic Escherichia coli and Escherichia albertii in Brazil: pathotypes and serotypes over a 6-year period of surveillance.

Diarrhoeagenic Escherichia coli (DEC) is a leading cause of infectious diarrhoea worldwide. In recent years, Escherichia albertii has also been implicated as a cause of human enteric diseases. This study describes the occurrence of E. coli pathotypes and serotypes associated with enteric illness and haemolytic uremic syndrome (HUS) isolated in Brazil from 2011 to 2016. Pathotypes isolated included enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC). PCR of stool enrichments for DEC pathotypes was employed, and E. albertii was also sought. O:H serotyping was performed on all DEC isolates. A total of 683 DEC and 10 E. albertii strains were isolated from 5047 clinical samples. The frequencies of DEC pathotypes were 52.6% (359/683) for EPEC, 32.5% for EAEC, 6.3% for ETEC, 4.4% for EIEC and 4.2% for STEC. DEC strains occurred in patients from 3 months to 96 years old, but EPEC, EAEC and STEC were most prevalent among children. Both typical and atypical isolates of EPEC and EAEC were recovered and presented great serotype heterogeneity. HUS cases were only associated with STEC serotype O157:H7. Two E. albertii isolates belonged to serogroup O113 and one had the stx2f gene. The higher prevalence of atypical EPEC in relation to EAEC in community-acquired diarrhoea in Brazil suggests a shift in the trend of DEC pathotypes circulation as previously EAEC predominated. This is the first report of E. albertii isolation from active surveillance. These results highlight the need of continuing DEC and E. albertii surveillance, as a mean to detect changes in the pattern of pathotypes and serotypes circulation and provide useful information for intervention and control strategies.

Biofilm formation, invasiveness and colicinogeny in locus of enterocyte and effacement negative O113:H21 Shigatoxigenic Escherichia coli.

AIMS: Although Shiga toxins (Stx) are well-established virulence traits of O113:H21 Shigatoxigenic Escherichia coli (STEC) strains, a shortage in the knowledge of other virulence properties that may contribute to pathogenesis may exist in this serotype. This study investigated biofilm, invasiveness and colicinogeny capabilities in O113:H21 STEC isolated in Brazil, mostly from animal reservoirs. A search for genes that were reported to participate in the process of biofilm formation was also performed. METHODS AND RESULTS: The 34 O113:H21 STEC isolates analysed were assayed for biofilm production in polystyrene microplates. Genes for biofilm were investigated by PCR. Invasion of cell lineages was assessed in gentamicin protection assays and colicinogeny was investigated by phenotypic tests. Fifty per cent of the strains were biofilm formers, and 35% exhibited an invasive behaviour. The pattern of distribution of biofilm-related genes did not correlate with biofilm phenotypes observed, and a high percentage of the investigated strains were able to secrete colicins. CONCLUSION: Ability to form biofilm, invasiveness and colicinogeny is demonstrated for the first time in a collection of O113:H21 STEC. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to express three additional phenotypes besides Stx production may be a factor influencing the pathogenicity and persistence potential of O113:H21 STEC.

Serotypes, virulence markers and cell invasion ability of Shiga toxin-producing Escherichia coli strains isolated from healthy dairy cattle.

AIM: The occurrence of virulence markers, serotypes and invasive ability were investigated in Shiga toxin-producing Escherichia coli (STEC) isolated from faecal samples of healthy dairy cattle at Rio de Janeiro State, Brazil. METHODS AND RESULTS: From 1562 stx-positive faecal samples, 105 STEC strains were isolated by immuno-magnetic separation (IMS) or plating onto MacConkey agar (MC) followed by colony hybridisation. Fifty (47·6%) strains belonged to nine serotypes (O8:H19, O22:H8, O22:H16, O74:H42, O113:H21, O141:H21, O157:H7, O171:H2 and ONT:H21). The prevalent serotypes were O157:H7 (12·4%), O113:H21 (6·7%) and O8:H19 (5·7%). Virulence genes were identified by polymerase chain reaction (PCR). E-hlyA (77·1%) was the more prevalent virulence marker, followed by espP (64·8%), saa (39%), eae (24·8%) and astA (21·9%). All O157:H7 strains carried the γ (gamma) variant of the locus of enterocyte effacement (LEE) genes and the stx2c gene, while the stx1/stx2 genotype prevailed among the eae-negative strains. None of the eae-positive STEC produced the localized adherence (LA) phenotype in HEp-2 or Caco-2 cells. However, intimate attachment (judged by the fluorescent actin staining test) was detected in some eae-positive strains, both in HEp-2 (23·1%) and in Caco-2 cells (11·5%). Most strains (87·5%) showed 'peripheral association' (PA) adherence phenotype to undifferentiated Caco-2 cells. Twenty-five (92·6%) of 27 strains invaded Caco-2 cells. The highest average value of invasion (9·6%) was observed among the eae-negative bovine strains from serotypes described in human disease. CONCLUSION: Healthy dairy cattle is a reservoir of STEC carrying virulence genes and properties associated with human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: Although reports of human disease associated with STEC are scarce in Brazil, the colonization of the animal reservoir by potentially pathogenic strains offers a significant risk to our population.

Shiga toxin-producing Escherichia coli in drinking water supplies of north Paraná State, Brazil.

AIM: To determine the occurrence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in drinking water supplies treated and untreated. METHODS AND RESULTS: Drinking water samples (n = 1850) were collected from 41 municipalities in the north of Paraná State between February 2005 and January 2006. Escherichia coli isolates (n = 300) were recovered from water and investigated for the presence of virulence markers related to STEC by PCR. STEC isolates recovered were then characterized for both phenotypic and genotypic traits. A total of 12 isolates (11 from untreated water and one from treated water) were positive for stx, including five positive for both stx1 and stx2, two positive for stx1 and five positive for stx2. None of the STEC isolates contained eae, but other virulence genes were observed such as ehxA (100%), saa (100%), lpfAO113 (75%), iha (42%), subAB (25%) and cdtV (8%). Multidrug resistance was identified in 25% of the STEC isolates. The 12 STEC isolates belonged to seven distinct serotypes and pulsed-field gel electrophoresis typing revealed the presence of two clusters and two clones in this region. CONCLUSION: Drinking water, especially from untreated water supplies, can be source of STEC strains potentially pathogenic for humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation of the drinking water supplies for pathogenic E. coli, as STEC, may be useful to prevent waterborne outbreaks.

Comparative analysis of rapid agglutination latex test using single-chain antibody fragments (scFv) versus the gold standard Vero cell assay for Shiga toxin (Stx) detection.

The latex agglutination test using single-chain antibody fragments (scFvStx1 and scFvStx2) coupled to latex particles, was compared with the gold standard Vero cell assay for Shiga toxin (Stx) detection, aiming to estimate the diagnosis potential of these scFv fragments in a rapid and straightforward test. The latex complexes identified the presence of the toxins up to a 1:8 dilution in the majority of the evaluated strains. Moreover, the Stx concentration was indirectly determined in Stx-producing Escherichia coli (STEC) strains, allowing detection limit inference. A Stx dilution curve was constructed, and the data was analyzed in a non-linear model by second-order polynomial regression for prediction (p-value of 0.001 and a R2 above 0.98 were considered for correlations). The detection limit was 30 ng/mL for Stx1 and 10 ng/mL for Stx2. The scFvStx1 and scFvStx2 coupled to latex nanoparticles provide a toxin assay with a competitive Stx detection limit, which has a low cost and short execution time. The diagnostic method proposed here, using, for the first time, recombinant antibody fragments, raises the possibility of developing a more affordable test to be used in the routine detection and surveillance of STEC infections.

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil.

AIMS: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. METHODS AND RESULTS: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. CONCLUSIONS: This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans.

The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

Outbreaks of cholera-like diarrhoea caused by enterotoxigenic Escherichia coli in the Brazilian Amazon Rainforest.

The relationship between enteropathogens and severe diarrhoea in the Brazilian Amazon is poorly understood. In 1998, outbreaks of acute diarrhoea clinically diagnosed as cholera occurred in two small villages localized far from the main cholera route in the Brazilian rainforest. PCR was performed on some enteropathogens and heat-labile (LT) and/or heat-stable (STh) toxin genes, the virulence determinants of enterotoxigenic Escherichia coli (ETEC), were detected. Further characterization of ETEC isolates revealed the presence of two clones, one from each outbreak. One presenting serotype O167:H5 harboured LT-I and STh toxin genes and expressed the CS5CS6 colonization factor. The other, a non-typeable serotype, was positive for the LT-I gene and expressed the CS7 colonization factor. The current study demonstrates the importance of molecular diagnosis in regions such as the Amazon basin, where the enormous distances and local support conditions make standard laboratory diagnosis difficult. Here we also show that the mis-identified cholera cases were in fact associated with ETEC strains. This is the first report of ETEC, molecularly characterized as the aetiological agent of severe diarrhoea in children and adults in the Brazilian Amazon Rainforest.

Serotypes and virulence markers of Shiga toxin-producing Escherichia coli (STEC) isolated from dairy cattle in São Paulo State, Brazil.

In order to determine the occurrence, serotypes and virulence markers of Shiga toxin-producing Escherichia coli (STEC) strains, 153 fecal samples of cattle randomly selected from six dairy farms in Sao Paulo State, Brazil, were examined for Shiga toxin (Stx) production by the Vero cell assay. Feces were directly streaked onto MacConkey Sorbitol Agar and incubated at 37 degrees C overnight. Sorbitol-negative colonies (maximum 20) and up to 10 sorbitol-positive colonies from each plate were subcultured onto presumptive diagnostic medium IAL. Sorbitol-negative isolates were screened with O157 antiserum for identification of O157:H7 E. coli. Isolates presenting cytotoxic activity were submitted to colony hybridization assays with specific DNA probes for stx1, stx2, eae, Ehly and astA genes. The isolation rate of STEC ranged from 3.8 to 84.6% depending on the farm analysed. STEC was identified in 25.5% of the animals, and most of them (64.1%) carried a single STEC serotype. A total of 202 STEC isolates were recovered from the animals, and except for the 2 O157:H7 isolates all the others expressed cytotoxic activity. The great majority of the STEC isolates carried both stx1 and stx2 genes (114/202, 56.4%) or stx2 (82/202, 40.6%); and whereas the Ehly sequence occurred in most of them (88%) eae was only observed in O157:H7 and O111:HNM isolates. Serotypes O113:H21, O178:H19 and O79:H14 were the most frequent STEC serotypes identified and widely distributed among animals from different farms, while others such as O77:H18, O88:H25 and O98:H17 occurred only in particular farms. This is the first report on the occurrence of STEC in dairy cattle in Sao Paulo State, and the results point to substantial differences in rate of isolation, serotypes and genetic profile of STEC that has been previously described among beef cattle in our community. Moreover, to our knowledge O79:H14 and O98:H17 represent new STEC serotypes, while O178:H19 has only been recently reported in Spain.

Characterization of enterotoxigenic Escherichia coli by random amplification of polymorphic DNA.

Two enterotoxigenic Escherichia coli (ETEC) strains (H10407 and 4011-1) were characterized by random amplification of polymorphic DNA (RAPD) profiles using 10-mer oligonucleotides with diverse GC content. All tested primers yielded arrays of amplified DNA products ranging in size from 200 to 3000 bp. The effects of annealing temperature, template concentration and GC content of the primers were evaluated and an optimal reaction procedure was established. Application of the RAPD analysis to ten ETEC strains belonging to five different serotypes showed that strains of the same serotype shared identical or almost identical band profiles, suggesting a similar genetic composition. The use of RAPD profiles as a tool in epidemiological analysis of ETEC is discussed.

Relationship between outer membrane protein and lipopolysaccharide profiles and serotypes of enterotoxigenic Escherichia coli isolated in Brazil.

The electrophoretic profiles of outer membrane proteins and lipopolysaccharide of sixty-five enterotoxigenic Escherichia coli of different serotypes and virulence-associated factors, toxin and colonization factors were determined. A close relationship between serotype and outer membrane protein and lipopolysaccharide patterns could be observed. No correlation could be found between the electrophoretic profiles and the expression of virulence-associated factors. The observed homogeneity of outer membrane protein and lipopolysaccharide profiles suggested the presence of only a few clones in the samples studied, and supported the use of outer membrane protein and lipopolysaccharide analysis as a useful epidemiological tool in the characterization of enterotoxigenic Escherichia coli isolates.

Clonal relationships among Escherichia coli serogroup O6 isolates based on RAPD.

The genetic diversity in a group of Escherichia coli strains belonging to serogroup O6 but expressing different H antigens was investigated by random amplification of polymorphic DNA (RAPD). Isolates of serotypes H16, H1, H31, and non-motile (NM) strains were typed using a set of 3 primers with different G + C contents. The amplified band arrays allowed the identification of 3 main clonal clusters corresponding to each O:H serotype analyzed. Based on their RAPD profiles NM strains could be assigned to either H1 or H31 serotypes. The results indicate that the flagellar antigen and the RAPD fingerprint represent reliable clonal markers in this E. coli group.

Diversity of surface structures and virulence genetic markers among enteroaggregative Escherichia coli (EAEC) strains with and without the EAEC DNA probe sequence.

The expression of surface structures and the presence of DNA sequences related to putative virulence factors were investigated in 22 enteroaggregative Escherichia coli strains (EAEC). Fimbria was the most frequent (72.7%) structure identified. Only strains hybridising with the EAEC DNA probe carried aggA, but one strain produced a similar but unrelated bundle-like structure. All probe-positive and 62.5% of the probe-negative strains carried the virulence genes tested; aspU and irp2 prevailed among the former strains. The EAEC probe-positive strains were more diverse, and some of these strains, which promoted cell detachment, also carried the hly and pap sequences, thus suggesting they might represent uropathogenic E. coli.

Prevalence of invasive ability and other virulence-associated characteristics in Providencia alcalifaciens strains isolated in São Paulo, Brazil.

Providencia alcalifaciens is an invasive enteric pathogen. The present study determined the prevalence of invasive ability in P. alcalifaciens strains isolated in São Paulo, Brazil, mainly from patients with diarrhoea. Invasion of HeLa cells was found in 17 (42%) of 41 strains studied. Most (88%) of the invasive strains were isolated from diarrhoeal stools. The invasive property was identified in 50% of P. alcalifaciens strains isolated as pure cultures or from stool samples where no other enteropathogen was identified. All the invasive strains caused actin condensation in infected cells. Plasmid profile analysis showed the presence of plasmids of 35.8-180 kb in 70% of the strains regardless of their invasive ability, suggesting that invasiveness in P. alcalifaciens is not plasmid related. No homology with a probe for gene sequences for invasion of enteroinvasive Escherichia coli and Shigella strains was identified in colony hybridisation assays. The invasive property of P. alcalifaciens was confirmed in the present study, but this characteristic did not predominate among strains isolated from patients with diarrhoea in São Paulo City. The presence of other virulence mechanisms and the role of non-invasive P. alcalifaciens strains as a cause of diarrhoea remain to be established.

Virulence properties of Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups isolated from calves with diarrhea.

Nineteen Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups were isolated from calves with diarrhea in Paraná State. Brazil, and studied for virulence markers associated with EPEC or enterohemorrhagic E. coli (EHEC). The 19 isolates belonged to 12 serotypes with isolates of O26:H11, O119:H25 and O114:H- being the most prevalent Localized adherence (LA) was demonstrated for 37% of the isolates, consisting of all four O26:H11, both O114:H- and one O114:H40 isolates. All the LA strains were positive in the fluorescent-actin staining (FAS) test and possessed attaching-effacing E. coli (eae) sequences, but only O114 strains hybridized with the EPEC adherence factor (EAF) probe. None of the strains produced Shiga-like toxins (Verotoxin). Only the O26:H11 strains hybridized with the EHEC plasmid specific (CVD419) probe and were enterohemolytic, properties associated with EHEC strains. This investigation demonstrates that among the bovine strains isolated only those of serogroup O114 behaved as typical EPEC.

Properties of Shiga toxin-producing Escherichia coli (STEC) isolates from sheep in the State of São Paulo, Brazil.

Fecal samples from 48 sheep from two farms in São Paulo, SP, Brazil, were examined to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC). Forty-two STEC strains were isolated from 25 (52.1%) of 48 sheep feces, and were examined for the presence of genes encoding STEC-related virulence factors. Twenty-one (50.0%) of the 42 STEC isolates were positive for stx(1) and stx(2), 16 isolates (38.1%) were stx(1), and five (11.9%) were stx(2). Expression of Shiga toxins was demonstrated by the Vero cell toxicity test for all the strains carrying stx. Fourteen of the STEC strains (33.3%) carried the enterohemolysin gene (ehly) and presented the enterohemolytic phenotype, and five (11.9%) were positive for the plasmid encoded katP gene. The eae gene was not present in any of the isolates. STEC strains presenting stx(1), stx(2) and ehly were most commonly (23.8%) recovered from these sheep. The predominant STEC serotype found was ONT:H8, and others included O5:H-, O16:H-, O75:H-, O75:H8, O87:H16, O91:H-, O146:H21, O172:H-, OR:H-, ONT:H- and ONT:H16. This is the first report on ovine STEC in South America, and identifies a number of ovine non-O157 STEC that belong to serotypes implicated in human disease.

High occurrence of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle in Rio de Janeiro State, Brazil.

In order to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains, 197 fecal samples of healthy cattle from 10 dairy farms, four beef farms and one slaughterhouse at Rio de Janeiro State, Brazil, were examined for Shiga toxin (Stx) gene sequences by polymerase chain reaction (PCR). For presumptive isolation of O157:H7 E. coli, the Cefixime-potassium tellurite-sorbitol MacConkey Agar (CT-SMAC) was used. A high occurrence (71%) of Stx was detected, and was more frequently found among dairy cattle (82% vs. 53% in beef cattle), in which no differences were observed regarding the age of the animals. Dot blot hybridization with stx1 and stx2 probes revealed that the predominant STEC type was one that had the genes for both stx1 and stx2 in dairy cattle and one that had only the stx1 gene for beef cattle. Three (1.5%) O157:H7 E. coli strains were isolated from one beef and two dairy animals by the use of CT-SMAC. To our knowledge, this is the first report of O157:H7 isolation in Brazil. A PCR-based STEC detection protocol led to the isolation of STEC in 12 of 16 randomly selected PCR-positive stool samples. A total of 15 STEC strains belonging to 11 serotypes were isolated, and most of them (60%) had both stx1 and stx2 gene sequences. Cytotoxicity assays with HeLa and Vero cells revealed that all strains except two of serotype O157:H7 expressed Stx. The data point to the high prevalence of STEC in our environment and suggest the need for good control strategies for the prevention of contamination of animal products.

Frequency of Shiga toxin-producing Escherichia coli (STEC) isolates among diarrheic and non-diarrheic calves in Brazil.

The occurrence of Shiga toxin (Stx) gene sequences was examined in 344 fecal samples from diarrheic (n=139) and non-diarrheic (n=205) calves from 12 beef farms in São Paulo State, Brazil to study the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains. Forty-four (12.7%) animals were found to be positive for stx. The frequency of carriage of stx was higher in diarrheic calves (28/139, 20%) than in non-diarrheic animals (16/205, 7.8%) (P<0.001). Among the 24 STEC strains recovered from the animals, 12 isolates carried stx1, four stx2, and 8 carried both stx1 and stx2 genes. The eae and the enterohaemolysin (Ehly) gene sequences occurred at high frequencies in these STEC strains (41.6 and 50.0%, respectively). A total of 16 serotypes were identified. The serotypes O111:NM (four isolates), O111:H8 (two) and O118:H16 (one), currently described as enterohaemorrhagic E. coli (EHEC), were isolated from cattle in Brazil for the first time. These findings reinforce the importance of cattle as a reservoir of EHEC strains in Brazil.

Presence of colonization factor antigen IV (CS5CS6) in O29:H21 enterotoxigenic Escherichia coli isolated from children with diarrhea in Brazil.

1. Enterotoxigenic Escherichia coli (ETEC) strains belonging to serotype O29:H21 were analyzed for the presence of colonization factor antigens (CFA). 2. CFA/IV was detected in all twelve strains studied, and immunological analysis demonstrated that it was composed of coli surface antigens 5 (CS5) and 6 (CS6) presenting molecular weights of approximately 21 kDa and approximately 16 kDa, respectively. 3. CS6 antigenic homology was observed between O29:H21 and reference strains E11881C and E17018A. 4. CS6 of the O29:H21 strains was composed of two peptide bands, similar to the reference strain E17018A.

Diffuse adherence, ST-I enterotoxin and CFA/IV colonization factor are encoded by the same plasmid in the Escherichia coli O29:H21 strain.

Escherichia coli O29:H21 is a human enterotoxigenic serotype that produces heat-stable (ST-I) enterotoxin, adheres diffusely to HeLa cells, and presents colonization factor antigen IV (CFA/IV) composed of CS5CS6 surface antigens. In one strain studied the genes for diffuse adherence and CFA/IV (CS5CS6) production were found to be present in the same plasmid encoding ST-I. The virulence plasmid (Ent) presented two unrelated basic replicons homologous to repFIC and repW. Gene(s) encoding diffuse adherence did not share homology with the probe for F1845 fimbrial adhesin which is responsible for this phenotype in other E. coli strains. Ent plasmids containing genes for diffuse adherence have not been described previously.