Characterization of adhesin variants in Indian isolates of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) are causative agents of diarrhea, being characterized by aggregative adherence to cultured epithelial cells. In this study, phenotypic properties of EAEC were analyzed with respect to AA, hemagglutination, clump and biofilm formation, all of which are mediated by aggregative adherence fimbriae (AAF). The strains were also screened for AAF types, AAF adhesin variants and Dr adhesin by PCR. Of the three known AAF types, AAF/I and AAF/II adhesin variants were identified. An association between the AAF/adhesin genotypes and the subtypes/scores of phenotypic properties was sought and it was observed that strains harboring same adhesins displayed different subtypes/scores and vice versa.     

Characterization of an outer membrane protein associated with haemagglutination and adhesive properties of enteroaggregative Escherichia coli O111:H12

Diarrhoeagenic Escherichia coli strains of serotype O111:H12 are characterized by their aggregative pattern of adherence on cultured epithelial cells and thus are considered enteroaggregative E. coli (EAEC). We have previously shown that these EAEC strains lack the genes encoding the aggregative fimbriae I and II described in other heterologous EAEC strains. In this paper, we show compelling data suggesting that a plasmid-encoded outer membrane 58 kDa protein termed aggregative protein 58 (Ap58) produced by EAEC O111:H12 strains, is associated with the adherence capabilities and haemagglutination of animal red blood cells. This conclusion is supported by several lines of evidence: (i) adherent O111:H12 strains are able to produce Ap58; (ii) non-adherent O111:H12 strains are unable to produce Ap58; (iii) antibodies raised against Ap58 inhibited adherence and haemagglutination of epithelial and bovine red blood cells, respectively; (iv) a non-adherent E. coli K-12 host strain containing the ap58 gene determinant on plasmid pVM15 displayed abundant adherence to cultured HEp-2 cells; and (v) the purified Ap58 bound specifically to HEp-2 and bovine red blood cells. Our findings indicate that the aggregative adherence in the O111:H12 strains may be also mediated by non-fimbrial adhesins. We believe our data contribute to the understanding of the adherence mechanisms of these organisms.     

Enteroaggregative and cell-detaching<Emphasis Type="Italic">Escherichia coli</Emphasis> strains among polish children with and without diarrhea

To determine the association of enteroaggregative (EAEC) and cell-detaching (CDEC)Escherichia coli with diarrhea of unknown origin among children from Wroc?aw (Poland),E. coli strains isolated from stool specimens of children with diarrhea were examined for mannose-resistant adherence to HEp-2 cells. EAEC were isolated from 10 of 39 (26%) children examined with diarrhea and 4 of 20 (20%) age-matched controls. CDEC were present in 14 (36%) cases of diarrhea and 7 (35%) healthy subjects. Cell-detaching activity was distinctly associated with hemolysin production. Among hemolytic CDEC strains cytotoxic necrotizing factor 1 (CNF1) synthesis prevailed among isolates obtained from cases of diarrhea (57%) in comparison with isolates obtained from healthy controls (14.3%). Although neither EAEC nor CDECE. coli strains were associated with diarrhea of children in this setting, there were differences among EAEC and CDEC strains isolated from children with and without diarrhea.     

Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients

We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC.     

Two Novel EHEC/EAEC Hybrid Strains Isolated from Human Infections

The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC) of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC). Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC). After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H⁻, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF). The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H⁻, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized.     

Characterization of serotypes and outer membrane protein profiles in enteroaggregative Escherichia coli strains.

Twenty-four Escherichia coli strains mainly isolated from children with diarrhea in São Paulo, and showing characteristics of enteroaggregative E. coli (EAEC), were characterized by serotyping and outer membrane protein (OMP) profiles. The relationship between these characteristics was evaluated, as well as the usefulness of OMP profiles in the clonal analysis of EAEC strains. All strains presented aggregative adherence to HeLa cells and were classified in two groups based on their interaction with the EAEC DNA probe. A diversity of serotypes and OMP profiles was observed in both groups studied. Although no significant correlation between serotypes and OMP profiles was observed, unique OMP profiles were identified in 80% of the probe-positive strains which were distributed in only 4 OMP profiles. This result may indicate the presence of a few clones in the probe-positive group. On the other hand, probe-negative strains seem to constitute a more diverse group. In general, the observed heterogeneity in serotypes and OMP profiles described in the present study suggest a great genetic diversity in EAEC isolates of either the same or different serotypes and in strains presenting the same EAEC markers identified in our community.     

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new Y ehD f imbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.     

Multi-locus sequence typing of enteroaggregative Escherichia coli isolates from Nigerian children uncovers multiple lineages

Background

Enteroaggregative Escherichia coli (EAEC) are defined by their stacked-brick adherence pattern to human epithelial cells. There is no all-encompassing genetic marker for EAEC. The category is commonly implicated in diarrhea but research is hampered by perplexing heterogeneity.

Methodology/Principal Findings

To identify key EAEC lineages, we applied multilocus sequence typing to 126 E. coli isolates from a Nigerian case-control study that showed aggregative adherence in the HEp-2 adherence assay, and 24 other EAEC strains from diverse locations. EAEC largely belonged to the A, B1 and D phylogenetic groups and only 7 (4.6%) isolates were in the B2 cluster. As many as 96 sequence types (STs) were identified but 60 (40%) of the EAEC strains belong to or are double locus variants of STs 10, 31, and 394. The remainder did not belong to predominant complexes. The most common ST complex, with predicted ancestor ST10, included 32 (21.3%) of the isolates. Significant age-related distribution suggests that weaned children in Nigeria are at risk for diarrhea from of ST10-complex EAEC. Phylogenetic group D EAEC strains, predominantly from ST31- and ST394 complexes, represented 38 (25.3%) of all isolates, include genome-sequenced strain 042, and possessed conserved chromosomal loci.

Conclusions/Significance

We have developed a molecular phylogenetic framework, which demonstrates that although grouped by a shared phenotype, the category of ‘EAEC’ encompasses multiple pathogenic lineages. Principal among isolates from Nigeria were ST10-complex EAEC that were associated with diarrhea in children over one year and ECOR D strains that share horizontally acquired loci.     

Pathogenesis of enteroaggregative Escherichia coli infection

Enteroaggregative Escherichia coli (EAEC) is emerging as a significant diarrheal pathogen in multiple population groups. Although most commonly associated with pediatric diarrhea in developing countries, EAEC is also linked to diarrhea in adults including HIV-positive patients and travelers and has been a cause of food-borne outbreaks in the industrialized world. Current data suggest that one set of virulence elements is not associated with all EAEC strains, but that combinations of multiple factors prevail. Pathogenesis is believed to be initiated with adherence to the terminal ileum and colon in an aggregative, stacked-brick-type pattern by means of one of several different hydrophobic aggregative adherence fimbriae. Some strains of EAEC may then elaborate cytotoxins including the plasmid-encoded toxin and the enterotoxins, EAST1 and ShET1. An AraC homolog termed AggR regulates several genes contributing to fimbrial biogenesis in 'typical EAEC strains'. AggR has now also been shown to regulate genes on a chromosomal island. Sequencing of the EAEC type strain 042 completed at the Sanger Center has revealed two other chromosomal islands that are being explored for their pathogenetic potential. This article reviews these virulence elements and presents on-going areas of research in EAEC pathogenesis.     

Structural Insight into Host Recognition by Aggregative Adherence Fimbriae of Enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.     

Solution structure of the novel dispersin protein of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked-brick pattern of adherence to epithelial cells. Adherence is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid. We recently described a highly prevalent pAA plasmid-borne gene, aap, which encodes a protein (nicknamed dispersin) that is secreted to the bacterial cell surface. Dispersin-null mutants display a unique hyper-aggregating phenotype, accompanied by collapse of AAF pili onto the bacterial cell surface. To study the mechanism of this effect, we solved the structure of dispersin from EAEC strain 042 using solution NMR, revealing a stable beta-sandwich with a conserved net positive surface charge of +3 to +4 among 23 dispersin alleles. Experimental data suggest that dispersin binds non-covalently to lipopolysaccharide on the surface of the bacterium. We also show that the AAF organelles contribute positive charge to the bacterial surface, suggesting that dispersin's role in fimbrial function is to overcome electrostatic attraction between AAF and the bacterial surface.     

Heat-Resistant Agglutinin 1 Is an Accessory Enteroaggregative Escherichia coli Colonization Factor

Enteroaggregative Escherichia coli (EAEC) is an important cause of acute and persistent diarrhea. The defining stacked brick adherence pattern of Peruvian EAEC isolate 042 has previously been attributed to aggregative adherence fimbriae II (AAF/II), which confer aggregative adherence on laboratory E. coli strains. EAEC strains also show exceptional autoaggregation and biofilm formation, other phenotypes that have hitherto been ascribed to AAF/II. We report that EAEC 042 carries the heat-resistant agglutinin (hra1) gene, also known as hek, which encodes an outer membrane protein. Like AAF/II, the cloned EAEC 042 hra1 gene product is sufficient to confer autoaggregation, biofilm formation, and aggregative adherence on nonadherent and nonpathogenic laboratory E. coli strains. However, an 042 hra1 deletion mutant is not deficient in these phenotypes compared to the wild type. EAEC strain 042 produces a classic honeycomb or stacked brick pattern of adherence to epithelial cells. Unlike wild-type 042, the hra1 mutant typically does not form a tidy stacked brick pattern on HEp-2 cells in culture, which is definitive for EAEC. Moreover, the hra1 mutant is significantly impaired in the Caenorhabditis elegans slow kill colonization model. Our data suggest that the exceptional colonization of strain 042 is due to multiple factors and that Hra1 is an accessory EAEC colonization factor.Enteroaggregative Escherichia coli (EAEC) was originally identified as the etiologic agent of persistent diarrhea in developing countries but is gaining increasing prominence for its role in a wider spectrum of diarrheal syndromes. EAEC strains have been implicated in acute as well as persistent diarrhea among adults and children (reviewed in references 25 and 40). A recent meta-analysis found that EAEC is significantly associated with disease in every group at high risk for diarrhea, including young children, human immunodeficiency virus-positive individuals, and visitors to developing countries (24). In addition to its association with disease in epidemiological studies in developing countries, EAEC has also been identified as a principal cause of diarrheal disease in Germany, the United Kingdom, and the United States (11, 26, 51).Aggregative adherence is the defining characteristic of EAEC (38). EAEC strains adhere to the intestinal epithelium, and to epithelial cells in culture, in a characteristic two-dimensional “stacked brick” fashion. The pattern features bacteria adhering to the eukaryotic surface, other bacteria, and the solid substratum. Four types of fimbriae have so far been documented as conferring aggregative adherence (4, 14, 17, 37). Two noncontiguous plasmid loci containing the complete complement of genes encoding aggregative adherence fimbriae I (AAF/I) or AAF/II are sufficient to confer aggregative adherence on nonadherent E. coli (14, 49). The plasmid bearing type IV pili found in Serbian EAEC outbreak strain C1096 are also sufficient to confer a weak aggregative adherence phenotype on E. coli K-12 (17). AAF additionally play an essential role in production of a superfluous EAEC-associated biofilm, which could account for the association of these strains with persistent diarrhea in epidemiological studies (46).Some categories of diarrheagenic pathogens have a conserved set of adhesins which allow them to overcome flushing across the intestinal epithelium. Typical enteropathogenic E. coli isolates, for example, all possess bundle-forming pili and the outer membrane adhesin intimin, whereas atypical enteropathogenic E. coli isolates possess intimin but not bundle-forming pili (reviewed in reference 10). EAEC strains, by contrast, are considerably heterogeneous. While many EAEC strains carry genes encoding one of the known aggregative adherence fimbriae, some EAEC do not harbor any known AAF even though they do demonstrate aggregative adherence (4, 7, 13, 14). This, and the presence of multiple adhesins in most mucosal colonizers (53), points to the likelihood of other EAEC adhesins. Imuta et al. recently implicated a TolC secreted factor in adherence (27), and Montiero-Neto et al. (33) described a 58-kDa nonstructural adhesin in O111:H12 EAEC. However, the former factor is only a contributor to aggregative adherence and the latter adhesin is not found in other EAEC. Overall, nonstructural EAEC adhesins have received little attention.The outer membrane protein Tia was originally characterized as an invasin and later shown to confer adhesive properties on enterotoxigenic E. coli (ETEC) (20, 21). Fleckenstein et al. (21) observed that a tia gene probe hybridized to DNA from non-ETEC strains, one of which was EAEC strain 042. As the Southern blot data published by Fleckenstein et al. showed bands of different intensities, as well as size, between ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, which carries tia, and EAEC strain 042, we hypothesized that the probe was recognizing a similar, rather than identical, gene (21).We have determined that EAEC strain 042 harbors a gene encoding the heat-resistant agglutinin 1 (hra1), a hemagglutinin originally reported from an O9:H10:K99 porcine ETEC strain. Hra1 has also been reported from uropathogenic E. coli strains and neonatal meningitis E. coli strain RS218, in which context it is otherwise known as Hek (19, 48). (The hek nomenclature was introduced after hra1, to delineate the form of the gene found in invasive human pathogens from that of a porcine isolate [19].) A role for the outer membrane protein Hra1/Hek in adherence by neonatal meningitis E. coli has recently been defined (19).Although hra1/hek has been reported from multiple pathogens, its role in colonization and virulence has only been conclusively studied in the neonatal meningitis E. coli strain RS218 (19). In this paper, we demonstrate that the EAEC hra1 gene is sufficient to confer colonization-associated phenotypes, including aggregative adherence and biofilm formation, on laboratory E. coli strains. Intriguingly, we find that although it confers these phenotypes on K-12 and is expressed in 042, hra1 is not required for in vitro colonization-associated phenotypes demonstrated by 042. The hra1 gene is, however, essential for the formation of a true stacked brick pattern in EAEC and for optimal in vivo colonization in a Caenorhabditis elegans model.     

Prevalence of HEp-2 cell-adherent Escherichia coli and characterisation of enteroaggregative E. coli and chain-like adherent E. coli isolated from children with and without diarrhoea, in Londrina, Brazil

A total of 919 Escherichia coli isolates from 125 children with diarrhoea (cases) and 98 controls were assayed for adherence to HEp-2 cells. Localised adherence was found only in isolates from cases. Diffuse, aggregative (AA), chain-like adherence (CLA) and variants of the AA pattern were found in both cases and controls. The AA isolates were tested for gene sequences associated with enteroaggregative E. coli (EAEC). Only 25% of the isolates hybridised with the EAEC probe, and the aafA, astA and pet gene sequences were found in 7.9%, 44.7% and 7.9% of the isolates, respectively. The aggA gene was not found, although 7.9% were positive for aggC. The CLA isolates reacted with the EAEC probe (55.6%), and the aggC, astA and pet gene sequences were found in 66.7%, 33.3% and 11.1%, respectively. The aggR (55.6%), aspU (55.6%), shf (33.3%) and she (22.2%) genes were also found in CLA isolates.     

Effect of lactoferrin on enteroaggregative E. coli (EAEC).

We previously demonstrated that lactoferrin inhibits adherence of enteropathogenic Escherichia coli to HEp-2 cells and decreases invasiveness of Shigella flexneri in HeLa cells by disruption of the type III secretory system (TTSS) of both enteropathogens. To determine whether these effects were specific to the TTSS, we assessed the activity of bovine lactoferrin on enteroaggregative E. coli (EAEC), enteropathogens whose virulence is not TTSS dependent. Bovine lactoferrin at a concentration of 1.0 and 0.1 mg/mL inhibited EAEC growth. Saturation with iron reversed the bacteriostatic effect. Lactoferrin under nonbacteriostatic conditions decreased EAEC adherence to HEp-2 cells as evaluated by microscopy and CFUs; this effect was not iron dependent. Lactoferrin inhibited EAEC biofilm formation and increased autoagglutination. Lactoferrin blocks EAEC adherence by inducing release and degradation of aggregative adherence fimbria, a key element of EAEC pathogenesis. We hypothesized that lactoferrin binding to lipid A of lipopolysaccharide disrupts the virulence proteins anchored to the bacterial outermembrane. These data suggest that the effect of lactoferrin on surface proteins is not restricted to organisms having a TTSS.     

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.     

Identification and characterization of a gene cluster mediating enteroaggregative Escherichia coli aggregative adherence fimbria I biogenesis.

The aggregative pattern of adherence (AA) exhibited by enteroaggregative Escherichia coli upon HEp-2 cells is a plasmid-associated property which correlates with aggregative adherence fimbria I (AAF/I) expression and human erythrocyte hemagglutination. By using cloning and mutagenesis strategies, two noncontiguous plasmid segments (designated regions 1 and 2) required for AA expression have previously been identified in enteroaggregative E. coli 17-2. TnphoA mutagenesis was performed on clones containing region 1, and 16 TnphoA mutants which were negative for the AA phenotype were analyzed. The TnphoA insertion site for each mutant was determined by junctional DNA sequencing. All 16 mutations occurred within a 4.6-kb span in region 1. Nucleotide sequence analysis of the region revealed four contiguous open reading frames, designated aggDCBA, in the same span. AA-negative TnphoA insertions into all open reading frames except aggB were obtained. On the basis of mutational analysis and protein homology data, it is inferred that aggA, aggC, and aggD are involved in biogenesis of AAF/I, encoding a major fimbrial subunit, outer membrane usher, and periplasmic fimbrial chaperone, respectively. By immunogold electron microscopy, polyclonal antiserum raised against the aggA gene product decorated AAF/I fimbriae, affirming that AggA encodes an AAF/I subunit.     

Studies on diarrheagenic Escherichia coli isolated from children with diarrhea in Myanmar

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. coli strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified into five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients.