Analysis of Escherichia coli colonization factor antigen I linear B-cell epitopes, as determined by primate responses, following protein sequence verification.

Colonization factor antigen I (CFA/I)-bearing strains of enterotoxigenic Escherichia coli (ETEC) are responsible for a significant percentage of ETEC diarrheal disease worldwide whether the disease presents as infant diarrhea with high mortality or as traveler's diarrhea. CFA/I pili (fimbriae) are virulence determinants that consist of repeating protein subunits (pilin), are found in several ETEC serogroups, and promote attachment to human intestinal mucosa. While CFA/I pili are highly immunogenic, the antigenic determinants of CFA/I have not been defined. We wished to identify the linear B-cell epitopes within the CFA/I molecule as determined by primate response to the immunizing protein. To do this, we (i) resolved the discrepancies in the literature on the complete amino acid sequence of CFA/I by N-terminal and internal protein sequencing of purified and selected proteolytic fragments of CFA/I, (ii) utilized this sequence to synthesize 140 overlapping octapeptides covalently attached to polyethylene pins which represented the entire CFA/I protein, (iii) immunized three rhesus monkeys with multiple intramuscular injections of purified CFA/I subunit in Freund's adjuvant, and (iv) tested serum from each monkey for its ability to recognize the octapeptides in a capture enzyme-linked immunosorbent assay. Eight linear B-cell epitopes were identified; the region containing an epitope at amino acids 11 to 21 was strongly recognized by all three individual rhesus monkeys, while the amino acid stretches 22 to 29, 66 to 74, 93 to 101, and 124 to 136 each contained an epitope that was recognized by two of the three rhesus monkeys. The three other regions containing epitopes were recognized by one of the three individuals. The monkey antiserum to pilus subunits recognized native intact pili by immunogold labeling of CFA/I pili present on whole H10407 cells. Therefore, immunization with pilus subunits induces antibody that clearly recognizes both synthetic linear epitopes and intact pili. We are currently studying the importance of these defined epitope-containing regions as vaccine candidates.     

Monoclonal antibodies against the different subcomponents of colonization factor antigen II of enterotoxigenic Escherichia coli.

Monoclonal antibodies (MAbs) against the different coli surface antigens CS1, CS2, and CS3 of colonization factor antigen II (CFA/II) of enterotoxigenic Escherichia coli (ETEC) were generated by fusing F/O myeloma cells with spleen cells from BALB/c mice immunized with different preparations of purified CFA/II. Five hybrids that produced antibodies specific for CS1, CS2, or CS3 in high titer were cloned and propagated. All the anti-CS MAbs were of the immunoglobulin G1 isotype, and all gave single precipitation lines in immunodiffusion tests when reacting with CFA/II-positive E. coli extracts containing the corresponding CS factor. The binding of all the MAbs to solid-phase-bound CFA/II could be completely inhibited by purified CFA/II containing the corresponding CS factor. However, whereas one MAb against CS3 was inhibited by all of 18 different CFA/II-positive strains tested, another anti-CS3 MAb was inhibited by bacteria expressing CS1 and CS3 (CS1 + CS3 strains) or CS3 alone but not by CS2 + CS3 strains, suggesting antigenic differences in CS3 when expressed by different strains. Use of the anti-CS MAbs in slide agglutination, immunodiffusion, or a CFA inhibition enzyme-linked immunosorbent assay revealed differences in the relative distribution of the various CS factors of CFA/II in clinical ETEC isolates from different geographic areas. By using the anti-CS MAbs in an enzyme-linked immunosorbent assay-nitrocellulose replica method, CFA/II-positive colonies could be detected in stool cultures from infected animals without prior isolation of the ETEC organisms.     

Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli

The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP. Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing the cof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.     

Characterization of monoclonal antibodies against putative colonization factors of enterotoxigenic Escherichia coli and their use in an epidemiological study.

Monoclonal antibodies (MAbs) against five putative colonization factors (PCFs), i.e., colonization factor antigen (CFA)/III, coli surface antigen (CS)7 and CS17, PCFO159, and PCFO166 of enterotoxigenic Escherichia coli (ETEC), were produced. Hybridomas (one each) producing specific antibodies against the respective PCFs were selected. All the MAbs reacted with the corresponding fimbriae but not with CFA/I, CFA/II, or CFA/IV or the heterologous PCFs in bacterial agglutination and enzyme-linked immunosorbent assays (ELISAs). In immunoelectron microscopy these MAbs bound along the fimbriae, and they also reacted with the corresponding subunits in immunoblots. The five MAbs were used to evaluate the prevalence of CFA/III, CS7, CS17, PCFO159, and PCFO166 in ETEC strains isolated from children with diarrhea in Argentina. One hundred five ETEC isolates negative for CFA/I, CFA/II, and CFA/IV were tested in slide agglutination or in a dot blot test for spontaneously agglutinating strains; positive results were confirmed by inhibition ELISAs. It was found that 27% of the CFA-negative ETEC strains carried one of the PCFs. The sensitivity of slide agglutination with these MAbs was similar to that with specific polyclonal antisera; however, the specificity was higher. PCFO166 was found in 9.5% of the strains tested, mainly in ETEC of serogroup O78 producing heat-stable toxin alone. CS17 and CS7 were identified in 6.7 and 5.7%, respectively, of strains producing heat-labile toxin only, most of which belonged to serogroup O114. PCFO159 was found in 3.8% of the isolates tested, whereas CFA/III was detected in only one ETEC strain.     

Use of a new oligonucleotide probe for detection of colonization factor antigen III gene in enterotoxigenicEscherichia coli

An alkaline phosphatase-labeled 30-mer oligonucleotide probe was designed to detect the gene for pilus colonization factor antigen III (CFA/III) of the human type of enterotoxigenicEscherichia coli (ETEC). The CFA/III probe was used to identify CFA/III-producing ETEC among 303Escherichia coli obtained from subjects with traveler's diarrhea. Six isolates positive for the CFA/III gene were found. This result was confirmed immunologically by using a specific monoclonal antibody developed against CFA/III. These six isolates, isolated from travelers returning from India, Pakistan and China, were all positive for the gene of heat-labile enterotoxin and possessed an identical serotype (025:H^–).     

Prevalence and association of the longus pilus structural gene (lngA) with colonization factor antigens, enterotoxin types, and serotypes of enterotoxigenic Escherichia coli.

Human enterotoxigenic Escherichia coli (ETEC) produces a plasmid-encoded type IV pilus termed longus (for long pilus). Regardless of the geographic origins of ETEC strains, the longus structural gene lngA was found to have the highest level of association with ETEC producing colonization factor antigen (CFA) CFA/II, followed by ETEC producing CFA/I and CFA/IV. ETEC bearing the less prevalent CFA/III and putative colonization factors and ETEC negative for CFA and putative colonization factor also contained lngA-related sequences. lngA was found in a considerable number of ETEC serotypes and was more often associated with ETEC producing heat-stable enterotoxins than with ETEC producing both heat-labile and heat-stable enterotoxins or heat-labile enterotoxin alone. lngA was found more often in strains isolated from children with diarrhea than in strains from healthy children, suggesting an association with intestinal disease. We conclude that longus is a widely distributed antigenic determinant in ETEC that is highly associated with known plasmid-encoded virulence factors, namely, CFAs and enterotoxins. A longus-specific probe may be a helpful epidemiological tool to assist in the identification of ETEC.     

Detection of colonization factor antigen I-positive enterotoxigenic Escherichia coli with a cloned polynucleotide probe.

We compared a new colony hybridization assay with an established enzyme-linked immunosorbent assay for detection of enterotoxigenic Escherichia coli (ETEC) expressing colonization factor antigen I (CFA/I). The tests were applied to 135 human ETEC strains. Of these isolates, 30 had previously been characterized for CFAs. A strain harboring the plasmid vector of the polynucleotide gene probe, nine non-ETEC strains from healthy infants, and eight ETEC strains of animal origin were included for further evaluation of probe specificity. The two assays showed a high level of concordance in the specific detection of ETEC strains expressing CFA/I. A total of 24 strains tested positive in the CFA/I hybridization assay, while 23 of those strains were positive in the CFA/I enzyme-linked immunosorbent assay. The single discrepant result could be explained by the loss of a regulatory gene. The strain harboring the plasmid vector of the probe, the non-ETEC E. coli strains, and the ETEC strains of animal origin were all negative in the CFA/I probe assay.     

Identification of enterotoxigenic Escherichia coli harboring longus type IV pilus gene by DNA amplification

DNA amplification of lngA, the structural gene of longus type IV pilus produced by human enterotoxigenic Escherichia coli (ETEC) was achieved by the use of specific oligonucleotide primers designed from the nucleotide sequence of lngA. A 630-bp fragment representing the entire lngA gene was amplified in eight prototype strains previously characterized as longus positive. Five ETEC strains producing colonization factor antigen III (CFA III) (also a type IV pilus) were also positive by PCR, confirming the DNA homology between CFA III and longus. None of the non-ETEC and non-E. coli enteropathogens studied showed the 0.63-kbp amplicon. The procedure thus detected only ETEC strains harboring type IV pili genes with or without other colonization factors. Except for five lngA PCR-positive, probe-positive strains, all lngA PCR-positive strains produced the pilin as demonstrated by immunoblotting. To test the amplification procedure in a clinical setting, a collection of 264 fresh clinical E. coli strains isolated from 88 Mexican children with diarrhea was screened by PCR. Among 82 ETEC isolates found, 30 (36.5%) were lngA PCR-positive. Twenty-seven percent of the children shed ETEC that possessed lngA. In parallel with DNA probes or PCR protocols to detect enterotoxin genes, the lngA PCR method may prove useful for detection of ETEC harboring type IV pilus genes in epidemiological studies.     

Monoclonal antibodies against different epitopes on colonization factor antigen I of enterotoxin-producing Escherichia coli.

Hybridoma-secreting monoclonal antibodies (MAbs) against colonization factor antigen I (CFA/I) were produced by the fusion of spleen cells from immunized BALB/c mice with F/O myeloma cells. The 25 MAbs with the highest antibody titer against CFA/I, as determined by an enzyme-linked immunosorbent assay, were studied for specificity and the capacity to agglutinate CFA/I-carrying bacteria. Most of the MAbs agglutinated a majority of 16 CFA/I-positive strains tested but not any of a number of CFA-deficient mutants or strains carrying other colonization factors (CFA/II and CFA/IV). One MAb that agglutinated all the CFA/I-positive strains and one MAb that did not agglutinate any of these strains were compared for their reactivities with different preparations of CFA/I. Whereas both MAbs bound to or could be inhibited by isolated CFA/I fimbriae as well as the subunit protein, only the agglutinating MAb bound to CFA/I, as expressed on whole bacteria. The nonagglutinating MAb, on the other hand, bound considerably better than the agglutinating MAb to a peptide corresponding to the 46 N-terminal amino acid residues of the CFA/I subunit protein. These results suggest that the two MAbs are directed against different epitopes on CFA/I.     

Type IV longus pilus of enterotoxigenic Escherichia coli: occurrence and association with toxin types and colonization factors among strains isolated in Argentina

The longus type IV pilus structural gene (lngA) was sought among 217 clinical enterotoxigenic Escherichia coli (ETEC) strains isolated in Argentina. lngA was present in 20.7% of the isolates and was highly associated with ETEC producing heat-stable toxin and the most common colonization factors. The prevalence of longus among ETEC strains in Argentina was comparable to that of colonization factor antigen I (CFA/I), CFA/II, and CFA/IV in other regions of the world.     

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types.     

Monoclonal antibodies against fimbrial subunits of colonization factor antigen I (CFA/I) inhibit binding to human enterocytes and protect against enterotoxigenicEscherichia coliexpressing heterologous colonization factors

EnterotoxigenicEscherichia coli(ETEC) bind to enterocytes in the small intestine by means of antigenically distinct colonization factors (CFs). By immunizing with isolated subunits of CFA/I fimbriae we have previously produced monoclonal antibodies (MAbs) that cross-react immunologicallyin vitrowith several CFs. Two of these MAbs [S(subunit)-CFA/I 17:8 and S-CFA/I 5:6] were found to significantly inhibit the binding of ETEC strains expressing either homologous or heterologous CFs, i.e. CFA/I and CS4, to isolated human jejunal enterocytes. The two MAbs also conferred passive protection against fluid accumulation in rabbit ileal loops caused by CFA/I- as well as CS4-expressing ETEC strains. Immunoelectron microscopy studies showed that both MAbs bound specifically to CFA/I as well as to CS4 fimbriae expressed on bacteria. These results indicate the possibility to induce anti-CF antibodies that can protect against ETEC infection caused by bacteria expressing not only homologous but also heterologous CFs, by immunizing with fimbrial subunits.     

Use of nonradioactive DNA hybridization for identification of enterotoxigenic Escherichia coli harboring genes for colonization factor antigen I, coli surface antigen 4, or putative colonization factor O166.

We developed an accurate nonradioactive colony hybridization assay (NCHA) using a digoxigenin-labeled polynucleotide probe and an antidigoxigenin alkaline phosphatase conjugate for the identification of enterotoxigenic Escherichia coli (ETEC) harboring genes for colonization factor antigen I (CFA/I), coli surface antigen 4 (CS4), or putative colonization factor O166 (PCFO166). In this 2-day assay, visual registration of color intensity could be used to distinguish between CFA/I-positive strains and strains with the genetic potential to express CS4 or PCFO166. A rapid NCHA was developed by which the results could be read visually 7 h and 45 min after inoculation of the bacteria. In the rapid NCHA, densitometry verified the visual discrimination between four groups of E. coli; ETEC with the CFA/I gene, ETEC with the CS4 gene, ETEC with the PCFO166 gene, and E. coli strains that lack such genes. As a confirmatory test, plasmids from ETEC with the CFA/I, CS4, or PCFO166 gene were differentiated by their characteristic restriction fragment patterns in nonradioactive Southern blot hybridization.     

Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of infectious diarrhea worldwide. Four categories of antigens have been commonly studied: O serogroup, H serogroup, colonization factor antigens (CFA), and toxins. A database has been complied from published reports of nearly 1,000 ETEC isolates from 18 locations and analyzed to determine the occurrence, distribution, and associations of O serogroup, H serogroup, CFA, and toxin type. Tables listing the associations of antigens are presented. This analysis documents the widespread nature and variety of ETEC. Even the most common combination of antigens, O6:H16 CFA/II LTST, accounted for only 11% of the ETEC isolates in the database. It was isolated from 12 locations. Many phenotypes occurred only once. CFA detection based on enzyme-linked antibodies with polyclonal sera is suggested as the preferred assay. A combination of CFA and toxin-based antigens is suggested as the most practical vaccine.     

Colonization factor antigens I and II and type 1 somatic pili in enterotoxigenic Escherichia coli: relation to enterotoxin type.

Enterotoxigenic Escherichia coli (ETEC) isolates from 36 persons with acute traveler's diarrhea from whom no other pathogens were recovered were tested (after no more than three subcultures) for the presence of colonization factor antigens I and II (CFA/I and CFA/II) and type 1 somatic pili. CFA/I or CFA/II was identified in 7 of 10 strains with heat-labile and heat-stable enterotoxins (LT+/ST+), but in only 2 of 12 LT-/ST+ (P less than 0.05) and 0 of 14 LT+/ST- (P less than 0.02) strains. CFA pili were not found among 74 non-enterotoxigenic E. coli strains. Type 1 somatic pili were demonstrable in 42% of the 36 ETEC and in 49% of the 74 non-enterotoxigenic E. coli isolates. The nine ETEC isolates bearing a CFA were serially subcultured on 10 consecutive days and retested for CFA and toxin. After five subcultures only one strain had lost a CFA, but after 10 passages three strains were negative: two lost CFA/I and one lost CFA/II. The strain that lost CFA/II became negative for both LT and ST as well and was found to lack a 48- and a 60-megadalton plasmid. The two strains that lost CFA/I also became negative for ST, but plasmid analysis revealed no plasmid loss. Disappearance of the CFA/I phenotype without loss of a plasmid can be explained by phase variation, as exhibited by type 1 somatic pili, or by rearrangement of base sequences in the CFA/I plasmid genome. If purified pili vaccines are to provide broad-spectrum protection against ETEC diarrhea, the search must be intensified to identify the antigens responsible for adhesion to intestinal mucosa in the many ETEC strains that lack CFA/I and CFA/II.     

Distribution of colonization factor antigens among enterotoxigenic Escherichia coli strains isolated from patients with diarrhea in Nepal, Indonesia, Peru, and Thailand.

Samples (1,318) of enterotoxigenic Escherichia coli (ETEC) isolated in 1994-1995 from children with diarrhea from Nepal, Indonesia, Peru, and Thailand were examined for colonization factor antigen (CFA) and coli surface (CS) antigens. Fifty-five percent of 361 heat-labile and heat-stable (LT-ST), 14% of 620 LT-only, and 48% of 337 ST-only ETEC had CFA/CS antigens. LT-ST ETEC strains were predominantly in the CFA II group, and ST only strains were in the CFA IV group. Additional studies are needed to identify ETEC strains that do not have CFA/CS antigens.     

Roles of different putative colonization factor antigens in colonization of human enterotoxigenic Escherichia coli in rabbits.

The role of some well-characterized putative colonization factors (PCFs) in enterotoxigenic Escherichia coli (ETEC), i.e. PCFO159, PCFO166, CS7, CS17 and CFA/III, for colonization of the bacteria in the intestine was studied in a non-ligated rabbit intestine model (RITARD). Intestinal administration of 10(11) organisms of the various strains only resulted in very mild symptoms with loose stools during a few days in most of the animals. Strains expressing PCFO159, CS7, CS17 and CFA/III were shed in the stool for a significantly longer period than PCF/CS-negative ETEC. However, the mean time of shedding PCFO166 positive organisms did not significantly exceed that of non-fimbriated E. coli. All strains that colonized rabbit intestine, as assessed by prolonged fecal excretion, also gave rise to high serum antibody responses against the homologous fimbriae whereas non-colonizing strains failed to induce such responses. This study strongly suggests that several of the recently described PCFs, e.g. PCFO159, CS7, CS17 and CFA/III are colonizing factors and strong immunogens.     

Detection and characterization of colonization factor of enterotoxigenic Escherichia coli isolated from adults with diarrhea.

The fimbriate colonization factor antigen (CEA) of Escherichia coli strain H-1047 was isolated and used to prepare anti-CFA antiserum. Enterotoxigenic E. coli (ETEC) isolated from 29 adults with diarrhea acquired in Mexico were examined for CFA by using this serum. Retrospectively, it was found that ETEC possessing the H-10407-type CFA were isolated from 25 (86%) of these diarrhea cases as compared with 2 of 11 (18%) from asymptomatic controls from whom ETEC had been isolated. CFA was found onE. coli of various serotypes, as demonstrated by bacterial agglutination by the anti-CFA serum. Heat treating the cells at 65 degress C for 1 h prevented the agglutination. CFA-positive strains did not react with anti-CFA serum when the cultures were grown at a low incubation temperature (18 degrees C). E. coli isolates identified serologically as CFA positive were shown to adhere to the intestinal villous surfaces of infant rabbits. By the indirect immunofluorescence technique, it was found that adhesion occurred preferentially in the upper 20 cm of the small intestine. Also, the ability or inability of various isolates to adhere to intestinal mucosa in vivo correlated with the presence or absence of fimbriae on the cells when grown in vitro. Agglutinability with anti-CFA serum, fimbriae, and adhesiveness were spontaneously lost by many isolates after laboratory passage in a manner previously described with E. coli H-10407. These observations suggest that the H-10407-type CFA plays a role in the virulence of ETEC possessing this antigen.     

Phenotypic and genotypic analysis of enterotoxigenic Escherichia coli in samples obtained from Egyptian children presenting to referral hospitals

Hospital surveillance was established in the Nile River Delta to increase the understanding of the epidemiology of diarrheal disease among Egyptian children. Between September 2000 and August 2003, samples obtained from children less than 5 years of age who had diarrhea and who were seeking hospital care were cultured for enteric bacteria. Colonies from each culture with a morphology typical of that of Escherichia coli were tested for the heat-labile (LT) and heat-stable (ST) toxins by a GM-1-specific enzyme-linked immunosorbent assay and colonization factor (CF) antigens by an immunodot blot assay. Enterotoxigenic E. coli (ETEC) isolates were recovered from 320/1,540 (20.7%) children, and ETEC isolates expressing a known CF were identified in 151/320 (47%) samples. ST CFA/I, ST CS6, ST CS14, and LT and ST CS5 plus CS6 represented 75% of the CFs expressed by ETEC isolates expressing a detectable CF. Year-to-year variability in the proportion of ETEC isolates that expressed a detectable CF was observed (e.g., the proportion that expressed CFA/I ranged from 10% in year 1 to 21% in year 3); however, the relative proportions of ETEC isolates expressing a CF were similar over the reporting period. The proportion of CF-positive ETEC isolates was higher among isolates that expressed ST. ETEC isolates expressing CS6 were isolated significantly less often (P < 0.001) than isolates expressing CFA/I in children less than 1 year of age. Macrorestriction profiling of CFA/I-expressing ETEC isolates by using the restriction enzyme XbaI and pulsed-field gel electrophoresis demonstrated a wide genetic diversity among the isolates that did not directly correlate with the virulence of the pathogen. The genome plasticity demonstrated in the ETEC isolates collected in this work suggests an additional challenge to the development of a globally effective vaccine for ETEC.     

Prevalence of toxin types and colonization factors in enterotoxigenic Escherichia coli isolated during a 2-year period from diarrheal patients in Bangladesh

The prevalence of toxin types and colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) was prospectively studied with fresh samples (n = 4,662) obtained from a 2% routine surveillance of diarrheal stool samples over 2 years, from September 1996 to August 1998. Stool samples were tested by enzyme-linked immunoassay techniques and with specific monoclonal antibodies for the toxins and CFs. The prevalence of ETEC was 14% (n = 662), with over 70% of the strains isolated from children 0 to 5 years of age, of whom 93% were in the 0- to 3-year-old age range. Of the total ETEC isolates, 49.4% were positive for the heat-stable toxin (ST), 25.4% were positive for the heat-labile toxin (LT) only, and 25.2% were positive for both LT and ST. The rate of ETEC isolation peaked in the hot summer months of May to September and decreased in winter. About 56% of the samples were positive for 1 or more of the 12 CFs that were screened for. The coli surface antigens CS4, CS5, and/or CS6 of the colonization factor antigen (CFA)/IV complex were most prevalent (incidence, 31%), followed by CFA/I (23.5%) and coli surface antigens CS1, CS2, and CS3 of CFA/II (21%). In addition, other CFs detected in decreasing order were CS7 (8%), CS14 (PCFO166) (7%), CS12 (PCFO159) (4%), CS17 (3%), and CS8 (CFA/III) (2.7%). The ST- or LT- and ST-positive ETEC isolates expressed the CFs known to be the most prevalent (i.e., CFA/I, CFA/II, and CFA/IV), while the strains positive for LT only did not. Among children who were infected with ETEC as the single pathogen, a trend of relatively more severe disease in children infected with ST-positive (P < 0.001) or LT- and ST-positive (P < 0.001) ETEC isolates compared to the severity of the disease in children infected with LT only-positive ETEC isolates was seen. This study supports the fact that ETEC is still a major cause of childhood diarrhea in Bangladesh, especially in children up to 3 years of age, and that measures to prevent such infections are needed in developing countries.