EPEC Adherence Factor Research Articles

Escherichia coli O128 strains from infants with diarrhea commonly show localized adhesion and positivity in the fluorescent-actin staining test but do not hybridize with an enteropathogenic E. coli adherence factor probe

Twenty-nine strains of Escherichia coli O128 isolated from infants with diarrhea that did not produce heat-stable enterotoxin, heat-labile enterotoxins, or Vero cytotoxin showed localized attachment to HEp-2 cells (LA). Only four strains hybridized with the enteropathogenic E. coli adherence factor (EAF) probe. One of the 25 LA+ EAF- strains attached to 72% of cells, while a plasmid-negative variant attached to 0.5% of cells. LA+ EAF- and LA+ EAF+ strains gave a positive fluorescent-actin staining test that correlates with the ability to cause attaching and effacing lesions in the intestine. The use of the EAF probe alone to detect LA+ strains is inadequate for epidemiological studies.

Screening for enteropathogenic Escherichia coli in infants with diarrhea by the fluorescent-actin staining test

The attaching effacing (AE) adherence property is now recognized as an important virulence characteristic of enteropathogenic Escherichia coli (EPEC). The fluorescent-actin staining (FAS) test (S. Knutton, T. Baldwin, P. H. Williams, and A. S. McNeish, Infect. Immun. 57:1290-1298, 1989), which is diagnostic for the AE lesions produced by EPEC (and Vero cytotoxin-producing E. coli), has provided an additional tool with which to investigate this important class of enteric pathogens. In this study, we screened for the AE adherence property in two groups of E. coli isolated from infants with diarrhea by using the FAS test and compared the results with those from O:H serotyping, localized adhesion to HEp-2 cells (LA), and the EPEC adherence factor (EAF) probe. Only 16 of 41 (39%) E. coli strains previously diagnosed as EPEC by O antigen serogrouping were FAS test positive, and of these only 12 belonged to recognized EPEC O:H serotypes; 9 strains which did belong to EPEC O:H serotypes were FAS test negative. Of a second group of 297 untyped E. coli, 7 (2.3%) were FAS test positive, and of these only 2 belonged to EPEC serogroups; 5 belonged to serogroups not regarded as EPEC serogroups or were nontypeable. Of the 23 FAS-test-positive strains identified, 10 were EAF probe positive and showed good LA; 13 were EAF probe negative and showed a quantitatively distinctly poor LA. EAF-positive and EAF-negative strains, however, showed equally good adhesion to human small intestinal mucosa. None of the FAS-test-positive E. coli hybridized with probes for Vero toxins 1 or 2. We conclude that the FAS test is diagnostic not only for classical EPEC and Vero cytotoxin-producing E. coli but also for EPEC strains which are not currently being diagnosed because they belong to serotypes not generally regarded as EPEC serotypes.

Oligonucleotide probe for detection of the enteropathogenic Escherichia coli (EPEC) adherence factor of localized adherent EPEC

The enteropathogenic Escherichia coli adherence factor (EAF) probe detects isolates of enteropathogenic E. coli that exhibit localized adherence to HEp-2 cells. A 21-base oligonucleotide probe was constructed on the basis of a sequence from within the 1-kb EAF probe and was shown to have greater sensitivity and specificity than the EAF fragment probe in detecting localized adherent E. coli.

Clonal Diversity and Virulence Factors in Strains of Escherichia coli of the Classic Enteropathogenic Serogroup 0114

Eighty-eight Escherichia coli strains of the enteropathogenic (EPEC) group O114 that were isolated from humans and animals in geographically different places and over more than 30 years were examined for virulence markers, O:H serotypes, and for electrophoretic types by multilocus enzyme electrophoresis. Four major genetically tightly related clusters of strains showed close correlation between electrophoretic types and other phenotypic characters. Cluster I contained 35 EPEC class II strains of serotypes O114:H9 and O114:H- and 5 enterotoxigenic E. coli belonging to O114:H21 and O114:H49. Clusters II and III comprised 36 O114:H4, O114:H32, and O114:H- strains; most were of doubtful pathogenicity except one Verotoxin-positive O114:H4 strain isolated from a human with diarrhea. Cluster IV contained 9 classic EPEC strains of serogroup O114:H2 that were characterized by localized adherence to HEp-2 cells and by the EPEC adherence factor.

Comparison of two assay methods for patterns of adherence to HEp-2 cells of Escherichia coli from patients with diarrhea

To determine whether methodological differences in the HEp-2 adherence assay could explain conflicting results of field studies, 244 strains of Escherichia coli from Mexican children with diarrhea were tested for patterns of adherence by the method used at the Center for Vaccine Development, University of Maryland (CVD), and at the Center for Infectious Diseases, University of Texas Medical School and School of Public Health (UTH). The CVD assay differentiated three phenotypes of adherent E. coli, including localized, diffuse, or aggregative adherence (LA, DA, or AA, respectively). There was agreement on pattern of adherence in 241 of the 244 strains (98.8%) tested by the CVD method in both Baltimore and Houston, and AA+ was the most common phenotype (28.5% of isolates). Among these isolates, the UTH assay detected only two adherent phenotypes (LA and DA), since it did not distinguish the AA pattern. The LA+ strains detected by each assay were compared for positivity with the enteropathogenic E. coli adherence factor (EAF) gene probe. Of the 16 strains LA+ by the CVD method, 100% were EAF+; in contrast, only 11 of 22 strains LA+ by the UTH method were EAF+ (P = 0.00074). These results help explain why in pediatric field studies in Mexico where isolates were tested by the UTH method (J. J. Mathewson, R. A. Oberhelman, H. L. Dupont, F. J. de la Cabada, and E. V. Garibay, J. Clin. Microbiol. 25:1917-1919, 1987) LA+ strains often did not belong to enteropathogenic E. coli O serogroups and why the AA pattern was not observed; the opposite was found in studies of pediatric diarrhea in Chile in which the CVD assay was used (M. M. Levine, V. Prado, R. M. Robins-Browne, H. Lior, J. B. Kaper, S. Moseley, K. Gicquelais, J. P. Nataro, P. Vial, and B. Tall, J. Infect. Dis. 158:224-228, 1988). Since it appears that both assays identify E. coli strains associated with diarrheal illness, the genetic relationships among these strains should be examined in future studies.

Attaching effacement of the rabbit enterocyte brush border is encoded on a single 96.5-kilobase-pair plasmid in an enteropathogenic Escherichia coli O111 strain

An enteropathogenic Escherichia coli (EPE) O111 serotype a,b,H- strain carried the following four plasmids: pLV501 (96.5 kilobase pairs [kbp]) specifying resistance to chloramphenicol, tetracycline, and kanamycin; pLV502 (8 kbp) specifying ampicillin resistance; pLV503 (1.9 kbp) specifying streptomycin resistance; and pLV504 (80 kbp) with no resistance markers. This EPEC attached to HEp-2 cells to produce localized clumps of bacteria (localized adhesion) and attached intimately to the enterocyte surface, leading to loss of the brush border (attaching effacement). Plasmid pLV501 was also found to specify the ability to produce localized adhesion on HEp-2 cells and attaching effacement in a rabbit ileal explant model system. Restriction maps showed considerable dissimilarities between pLV501 and pMAR-2, an EPEC plasmid carrying the EPEC adherence factor (EAF) genes. Furthermore, pLV501 did not hybridize with the EAF probe, whereas pLV504 did. There was sequence homology between pLV501 and large plasmids in all seven other well-characterized EPEC, only five of which hybridized with the EAF probe. These findings indicate that pLV501 carries at least one of the genes responsible for production of the brush border damage characteristic of EPEC.

Examination of strains belonging to enteropathogenic Escherichia coli serogroups for genes encoding EPEC adherence factor and Vero cytotoxins

Four hundred and forty-nine strains isolated from patients with diarrhoea and belonging to 13 enteropathogenic Escherichia coli (EPEC) O serogroups were tested with a DNA probe for the EPEC adherence factor (EAF). Positive results were obtained with only 36 strains; they belonged to 10 O serogroups and flagellar typing showed they were usually of the "classical" EPEC serotypes. Thirty-four of the 36 EAF-positive strains showed localised adhesion to HEp-2 cells. The two remaining strains, of serotypes O114:H2 and O127:H4, showed low level or no adhesion to HEp-2 cells. No colonies hybridising with the EAF probe were identified in cultures from 115 faecal specimens from healthy children. Sixteen of the 449 strains hybridised with one or both probes for the Vero cytotoxin genes VT1 and VT2; 15 of the 16 strains belonged to serogroups O26 and O128. None of the strains hybridised with both the EAF and VT gene probes. These studies show that the great majority of strains belonging to EPEC O serogroups do not possess the EPEC adherence factor or carry VT genes.

Adhesive properties of enteropathogenicEscherichia coli isolated from infants with acute diarrhea in Africa

The adhesive properties of 69 enteropathogenic Escherichia coli (EPEC) strains were studied. The strains were isolated from diarrheal stools of infants in Burundi, Africa, and identified by serotyping with 12 classical EPEC O serogroup antisera. A test for adhesion to HEp-2 cells revealed that 52% of the strains showed localized adherence and 13% diffuse adherence. Localized adherence phenotype was found in previously described serogroups O86, O111, O125, O127, O128 and O142; strains belonging to another serogroup, O126, also exhibited localized adherence. Use of an EPEC adherence factor DNA probe in colony hybridization and in Southern blot techniques revealed that all strains exhibiting localized adherence and no strain exhibiting diffuse adherence produced a positive reaction; the genes were localized on high molecular weight plasmids (50-70 megadaltons). In vitro adhesion tests with human enterocytes performed concurrently with all 69 strains showed that only six of them adhered. These strains belonged to the O26, O117, O125, O128 and O142 serogroups. The adhesin CFA/I was detected only in the O128 Escherichia coli. The strain of serogroup O142 exhibited both adhesion to human enterocytes and localized adherence to HEp-2 cells, which suggests that the adhesive systems of enterotoxigenic and enteropathogenic Escherichia coli may coexist.

Serotype, antimicrobial resistance, and adherence properties of Escherichia coli strains associated with outbreaks of diarrheal illness in children in the United States.

Since most recorded outbreaks of diarrhea in U.S. infants attributed to Escherichia coli occurred before currently available pathogenicity assays existed, we examined the characteristics of nonenterotoxigenic E. coli strains isolated from 50 outbreaks of diarrheal disease in U.S. infants between 1934 and 1987. We assayed the strains for enteropathogenic E. coli (EPEC) serotype, localized adherence (LA) and diffuse adherence to tissue cultures, the presence of EPEC adherence factor genes, Shiga-like (Vero) toxin production, and antimicrobial resistance. EPEC serotypes were identified in 28 outbreaks (56%). LA to HeLa cells was found in 23 outbreak strains and correlated 100% with the EPEC adherence factor probe. LA was observed in 21 of 28 EPEC and 2 of 22 non-EPEC strains; however, 5 of 23 strains that were LA positive for HeLa cells did not adhere to HEp-2 or HL cells. One strain was diffuse adherence positive, and none was Shiga-like toxin positive. Multiple resistance was common in EPEC (64%), LA-positive (74%), and LA-positive EPEC (76%) strains but not in others (10%). EPEC serotypes or LA was found in 60% (n = 30) of the outbreak strains. The remaining E. coli strains may represent nonpathogenic normal flora, as-yet-undefined pathogens, or pathogens that have lost virulence-associated traits during storage or subculturing.

Epithelial cell invasion: an overlooked property of enteropathogenic Escherichia coli (EPEC) associated with the EPEC adherence factor.

In order to investigate the ability of enteropathogenic Escherichia coli (EPEC) to invade epithelial cells, 24 strains of diarrhea-causing E. coli were studied with a HEp-2 cell-gentamicin invasion assay. Invasive ability was expressed as the percentage of the inoculum surviving gentamicin after incubation of bacteria with HEp-2 cells. Geometric mean survival of EPEC strains possessing the EPEC adherence factor (EAF+ EPEC) was 5.177%, which was significantly greater than survival of enteroinvasive E. coli (EIEC) strains (1.871%). EPEC strains lacking EAF (EAF-EPEC), enterotoxigenic E. coli (ETEC), and enterohemorrhagic E. coli (EHEC) were significantly less invasive (geometric mean survival, 0.032%, 0.013%, and 0.009%, respectively). The variation in bacterial recovery was not due to differences in the number of HEp-2 cells remaining attached to the plates, as measured by the retention of crystal violet stain in parallel assays. Transmission electron microscopy confirmed the presence of many intracellular EAF+ EPEC and EIEC, whereas EAF- EPEC, EHEC, and ETEC were found primarily outside the cells. Epithelial cell invasion is an overlooked property of EAF+ EPEC of potential relevance in disease pathogenesis.

Escherichia coli O114:nonmotile as a pathogen in an outbreak of severe diarrhea associated with a day care center.

Five infants from a day care center developed severe diarrhea associated with enteropathogenic Escherichia coli O114:nonmotile (EPEC O114:NM) and required hospitalization. Five additional cases of diarrhea associated with EPEC O114:NM subsequently occurred, four in hospital contacts of the patients and one in a household contact. Biochemically, all EPEC O114:NM isolates were sorbitol nonfermenters. All isolates produced low concentrations of cytotoxin with a mean of 10(1.23) CD50/mg of protein. Cytotoxin was not neutralized with antibody to Shiga-like toxin I or II. Heat-labile and heat-stable enterotoxins were not present by gene probe analysis. Stool isolates from 9 of 10 hospitalized infants were positive for EPEC adherence factor by colony blot DNA probe analysis. The severity of the disease, sorbitol nonfermentation, and presence of enteroadherence are unusual features of this organism.

Serotype-Specific Prevalence of Escherichia coli Strains with EPEC Adherence Factor Genes in Infants with and without Diarrhea in Sao Paulo, Brazil

To examine interrelationships of classic enteropathogenic Escherichia coli (EPEC) serotypes, EPEC adherence factor (EAF) genes, and diarrheal disease, E. coli were studied from stools of 500 infants less than 1 y of age with acute diarrhea and 500 age-matched controls. EAF-containing (EAF+) E. coli of three common classic EPEC serotypes (O111:H-, odds ratio [OR] 36.0; O111:H2, OR 55.0; O119:H6, OR 3.7) were individually strongly associated with diarrhea, as were EAF+ strains of less common classic serotypes combined (OR 5.3). Among EPEC serogroups, neither EAF+ strains of nonclassic serotypes (OR 1.8) nor EAF-strains of classic (OR 2.2) or nonclassic (OR 1.4) serotypes were significantly associated with diarrhea. At least one EAF+ non-EPEC serogroup serotype (O88:H25) may represent an unrecognized EPEC serotype. Serotype-specific variation in the association of EAF+ E. coli with diarrhea suggests that other factors are also important in determining virulence; thus, both EAF detection and E. coli serotyping are desirable in studying the etiology of diarrheal disease.

Properties of adherence factor plasmids of enteropathogenic Escherichia coli and the effect of host strain on expression of adherence to HEp-2 cells.

EPEC adherence factor (EAF) plasmids from three strains of enteropathogenic Escherichia coli (EPEC) - E2347/69 (O127:H6), E20517 (O111:H2) and E24582 (O142:H6) - were examined. The EAF plasmids were all marked with ampicillin resistance by transposition of Tn801 to give pDEP1, pDEP2 and pDEP11, respectively. All three plasmids showed incompatibility with an FIme and an FIV plasmid and had some similarity in restriction enzyme digest patterns. Plasmid pDEP1 differed from pDEP2 and pDEP11 in being autotransferring and fertility-inhibition positive. An EAF probe consisting of a 1 kb BamHI-SalI restriction endonuclease fragment of the prototype EAF-associated plasmid pMAR2 hybridized to similar-sized SalI-BamHI fragments of pDEP1 and pDEP11 but to a different-sized fragment of plasmid pDEP2. Loss of the EAF plasmids from EPEC strains resulted in a marked reduction in the ability of these strains to adhere to HEp-2 cells. The EAF-plasmid-negative variants did not express a 94 kDa outer-membrane protein (OMP). When these EAF plasmids were reintroduced into EAF-plasmid-negative EPEC strains a high level of adherence equivalent to that of the parent EPEC strains was restored and a 94 kDa OMP was usually expressed. However, when EAF plasmids were transferred into E. coli K12 or non-EPEC E. coli the host strains either did not adhere or adhered poorly to the HEp-2 cells. These transconjugants did not express a 94 kDa OMP.

Nature and distribution of mucosal lesions associated with enteropathogenic and enterohemorrhagic Escherichia coli in piglets and the role of plasmid-mediated factors.

Bacterial attachment-effacement (att-eff) is emerging as an important virulence characteristic common to both enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). The contribution of the plasmid-encoded EPEC adherence factor to the production of mucosal lesions and diarrhea was investigated in gnotobiotic piglets. Bacterial att-aff in the intestinal mucosa of piglets infected with plasmid-cured EPEC strain E2348/69 (O127) was indistinguishable from that in piglets infected with the parent strain, but the distribution of lesions was different; it occurred in the small intestines of 6 of 7 piglets infected with the parent strain compared with only 2 of 11 (P = 0.006) infected with the plasmid-cured strain. Plasmid-encoded factors in EPEC and EHEC strains did not appear to contribute to bacterial competition with normal gut microflora. Of 13 strains belonging to five EPEC serogroups, O55, O142, O26, O119, and O111, 3 fulfilled the criteria for EHEC (2 O26 and 1 O111). There were three distinct patterns of bacterial association with the intestinal mucosa of infected piglets. (i) EHEC strains caused bacterial att-eff associated with extensive destruction of surface and glandular epithelia in the large intestines with little or no inflammatory response. (ii) Some EPEC strains caused severe diarrhea which correlated with the extent of bacterial att-eff in the proximal small intestine, disruption of the epithelial cell membrane, and inflammation. It is suggested that, with respect to virulent strains, this degree of involvement determines the clinical outcome. Mildly pathogenic strains (O127 and O119), in which bacterial att-eff was restricted to the distal halves of the small and large intestines, caused little or no diarrhea. In such strains, nonimmune host factors (smaller, poorly feeding, and lethargic piglets) tended to play a determining role with regard to the degree of involvement of the small intestine and hence the clinical outcome. (iii) One strain (O55) caused illness and mucosal damage which could not be accounted for by the sparse bacterial att-eff observed in the gut. Instead, bacteria penetrated into and proliferated in the lamina propria, undermining the villous tips in the small intestine. Bacterial att-eff was the most important virulence factor in most of the strains examined, but plasmid-mediated factors facilitated bacterial adhesion in the small intestine, which may explain the reduced pathogenicity of the plasmid-cured variant of strain E2348/69 for human volunteers.

HEp-2 adhesion and the expression of a 94 kDa outer-membrane protein by strains of Escherichia coli belonging to enteropathogenic serogroups.

Sixty strains of Escherichia coli belonging to enteropathogenic serogroups (EPEC) were examined for the ability to adhere to HEp-2 cells, the possession of the genes encoding EPEC adherence factor (EAF) and the ability to express an outer-membrane protein (OMP) of 94 kDa thought to be involved in bacterial adhesion to eukaryotic cells. An absolute correlation was found between HEp-2 adhesion and the possession of the genes encoding EAF. An OMP of 94 kDa was observed in the SDS-PAGE profile of most adhesive strains. In some strains this protein was prone to proteolytic degradation. An antiserum raised to a HEp-2 adhesive strain of EPEC did not react with the 94 kDa OMP of all EPEC which were EAF-positive and HEp-2 adhesive, indicating some interstrain antigenic variation of this protein. Although this 94 kDa protein was surface-exposed, specific antibodies binding to the 94 kDa protein in situ in the outer membrane did not interfere with adhesion of EPEC to HEp-2 cells. Therefore, these studies question the value of this protein as a potential vaccine component.

Detection of enteric pathotypes of eEcherichia coli by hybridization using six DNA probes

A set of 6 DNA probes was tested to evaluate the incidence of various Escherichia coli pathotypes among 540 strains isolated in France from diarrhoeal stools of infants, children and adults. Enterotoxigenic E. coli were detected using 3 gene probes for enterotoxins LT, STaH and STaP. Enteroinvasive E. coli were detected using one DNA probe which specifically hybridizes with bacteria expressing the cell invasion phenotype ⪡INV⪢. They represented 1.5 % and 1.1 % of the total, respectively. An SLTI probe which contains the structural gene for the A subunit of Shiga-like toxin I was constructed to detect enterohaemorrhagic E. coli. Among the 5 strains detected, only 1 belonged to serotype O157:H7. An attempt was made to detect enteropathogenic E. coli (EPEC) using both an EPEC-adherence factor and the above mentioned SLTI probes. Under the experimental conditions, they did not appear to be efficient at detecting this pathotype.

Characterization of plasmids encoding the adherence factor of enteropathogenic Escherichia coli.

Volunteer studies have shown that a 60-megadalton plasmid is required for full virulence of the human enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6). The plasmid, designated pMAR2, encodes localized adherence to HEp-2 cells in tissue culture via the adhesin known as the EPEC adherence factor (EAF). Using a DNA probe for the EAF, we have previously shown that these genes are specific for EPEC and are usually encoded on plasmids ranging from 55 to 65 megadaltons. In this study, Southern blot analysis and S1 nuclease homology determination reveal a high degree of sequence conservation among these plasmids, despite some variation in restriction maps. Phenotypic characterization of the prototype EAF plasmid pMAR2 reveals that the plasmid belongs to the group IncFII and is negative for alpha-hemolysin, colicin, and aerobactin synthesis, as well as biochemical markers and antibiotic resistance. Regions encoding adherence to HEp-2 cells were localized by Tn801 insertion mutagenesis. Adherence genes were then cloned as two distinct plasmid regions which confer the adherence phenotype only when complementing each other in trans.

Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells.

A total of 516 Escherichia coli strains randomly isolated from coprocultures of 154 Chilean children with diarrhea and 66 controls were examined with DNA probes and tested for adherence to HEp-2 cells. Three adherence patterns were distinguished, localized, true diffuse and "aggregative." Enteropathogenic E. coli (EPEC) were detected by EPEC adherence factor probe among 86 of the 372 isolates (23%) from patients with diarrhea vs. 14 of 144 (10%) strains from controls (P less than 0.0002). Of 95 strains that manifested localized adherence, 97% were EPEC adherence factor probe-positive; thus the HEp-2 assay may serve as an alternative to the probe in identifying EPEC adherence factor-positive EPEC. True diffuse adherence was not associated with diarrhea. In contrast the aggregative pattern appears to signify a new, distinct class of diarrheagenic E. coli (enteroadherent-aggregative E. coli). The aggregative pattern was found in only 3 of 27 enterotoxigenic, 0 of 4 enteroinvasive, 0 of 2 enterohemorrhagic and 2 of 86 EPEC strains but in 84 of 253 probe-negative strains (P less than 0.00001) from patients with diarrhea; in comparison only 20 of 134 probe-negative strains from controls were aggregative E. coli (P less than 0.00001 vs. probe-negative strains from diarrhea patients).

HeLa cell adherence and cytotoxin production by enteropathogenic Escherichia coli isolated from infants with diarrhea in Thailand.

Enteropathogenic Escherichia coli (EPEC) strains isolated from hospitalized infants with diarrhea in Thailand were examined for HeLa cell adherence and cytotoxin production. Of 101 strains examined, 56 adhered to HeLa cells in a localized pattern (LA), 27 adhered in a diffuse pattern (DA), and 18 did not adhere. All 56 LA EPEC strains were O:K serotype O119:K69. A total of 20 (83%) of 24 EPEC O86:K61 strains and 7 (38%) of 19 EPEC strains belonging to six other O:K serotypes exhibited DA. All LA EPEC strains hybridized with a DNA probe for genes encoding EPEC adherence factor, whereas none of the 27 DA or 18 nonadherent EPEC strains hybridized with EPEC adherence factor probe. Sonic extracts of 57 (58%) of 98 EPEC strains tested at a dilution of 1:100 caused greater than 25% mortality of HeLa cell monolayers. A total of 50 (88%) of 57 cytotoxic sonic extracts were inhibited to various degrees by a 1:500 dilution of polyclonal rabbit antisera to purified Shiga toxin. The mean percent inhibition of cytotoxic sonic extracts by anti-Shiga toxin was 67% (range, 29 to 89%). Fifty percent (38 of 56) of LA EPEC strains, fifty-two percent (14 of 27) of DA EPEC strains, and fifty-three percent (8 of 15) of nonadherent EPEC strains produced Shiga-like toxins. Both adherence and low levels of cell-associated cytotoxins were identified in EPEC strains from Thailand, but there did not appear to be an association between these two factors.

HeLa cell-adherent enteropathogenic Escherichia coli in children under 1 year of age in Thailand.

Enteropathogenic Escherichia coli (EPEC) was isolated from 11% of 148 Hmong children under 1 year old with diarrhea at a refugee camp in northern Thailand. Of 16 children with EPEC-associated diarrhea, 11 were infected with EPEC that adhered to HeLa cells in a diffuse pattern, 3 were infected with EPEC that adhered to HeLa cells in a localized adherence (LA) pattern, and 2 were infected with EPEC that were nonadherent. In Bangkok, EPEC was isolated from 6% of 64 children under 1 year old with diarrhea and 7% of 56 children of the same age without diarrhea. Of four children with diarrhea, two were infected with EPEC with an LA pattern, and two were infected with nonadherent EPEC. Of four children without diarrhea, one was infected with EPEC with an LA pattern, one was infected with EPEC that adhered in a diffuse pattern, and two were infected with nonadherent EPEC. The 21 EPEC isolates with an LA pattern hybridized with the EPEC adherence factor DNA probe. EPEC was the only enteric pathogen identified in 16 (80%) of 20 children with EPEC-associated diarrhea. EPEC was as frequently isolated from children under 1 year old as were other bacterial enteric pathogens. The problem of identifying EPEC with pools of polyvalent antisera are described, and the need to identify additional enteropathogenic determinants of EPEC is discussed.