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.     

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.     

Prevalence of enterotoxigenic Escherichia coli strains harboring the longus pilus gene in Brazil

The longus type IV pilus gene (lngA) was highly prevalent (32.8%) among Brazilian enterotoxigenic Escherichia coli strains producing both heat-labile and heat-stable enterotoxins and bearing the CFA/I, CS1CS3, or CS6 antigen. Furthermore, lngA was more often found in strains isolated from children with diarrhea than in strains isolated from children without diarrhea.     

Colonization factors of enterotoxigenic Escherichia coli isolated from children with diarrhea in Argentina.

A prospective study was performed to evaluate the presence of colonization factor antigens (CFAs) in enterotoxigenic Escherichia coli (ETEC) strains isolated from 1,211 children with diarrhea in Argentina. One hundred nine ETEC strains that were isolated from seven different laboratories in various regions of the country were tested for CFAs by using monoclonal antibodies against CFA/I and E. coli surface antigens CS1, CS2, and CS3 of CFA/II and CS4 and CS5 of CFA/IV; a polyclonal antiserum against CS6 was used. The CFAs searched for were found in 52% of the ETEC strains: 23% of the strains carried CFA/I, 17% carried CFA/IV, and 12% carried CFA/II. All of the CFA/I strains produced heat-stable enterotoxin, and several of them were of the prevalent serotypes O153:H45 and O78:H12. Among the 19 strains expressing CFA/IV, 16 expressed CS5 and CS6 and produced the heat-stable enterotoxin and most were of serotype O128:H21; the remaining 3 strains produced CS6 only. No ETEC strains expressing CS4 were found. Most (11 of 13) of the CFA/II-carrying ETEC strains expressed CS1 and CS3, and 10 of them were of the O6:K15:H16 serotype and produced both heat-labile and heat-stable toxins. As many as 24 of the 109 CFA-negative ETEC strains gave mannose-resistant hemagglutination with erythrocytes from different species; 4 strains had high surface hydrophobicity, suggesting the presence of additional, as yet undefined, colonization factors in up to 25% of the ETEC isolates.     

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.     

Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II,and CFA/IV

Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.     

Prospective Cohort Study of Enterotoxigenic Escherichia coli Infections in Argentinean Children

In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P = 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P = 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P = 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P = 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P = 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P = 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.     

Prevalence of the enterotoxigenic Escherichia coli colonization factors CFA/I, CFA/II and E8775 isolated in the South Pacific (New Caledonia, Republic of Vanuatu and Wallis and Futuna)

We tested the expression of adherence properties of enterotoxigenic Escherichia coli (ETEC) strains isolated in New-Caledonia, Vanuatu and Wallis and Futuna by examining for the presence of colonization factor E8775 using an agglutination test and an immuno-diffusion technique with specific antisera. Approximately 19% of ETEC strains possessed CFA/I and 21% a CFA/II. The E8775 antigen was found on 1.8% of the strains. This last factor was found on strains of the serogroup 025 from Vanuatu. Two strains 078 usually CFA/I+ possessed a CFA/II and three strains of the serogroup 0126 possessed a CFA/I. The results of this study emphasis the need to continue the search for other mechanisms of adhesion used by ETEC strains without any of the three factors of colonization.     

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.     

Plasmids coding for colonization factor antigens I and II, heat-labile enterotoxin, and heat-stable enterotoxin A2 in Escherichia coli.

Colonization factor antigens I and II (CFA/I and CFA/II) are important in the pathogenesis of diarrhea in humans caused by some enterotoxigenic Escherichia coli (ETEC). Plasmid DNA from 16 CFA/I+ and five CFA/II+ ETEC were examined by Southern blot analysis with enterotoxin gene probes and were compared with plasmid DNA from derivatives of the same ETEC that had lost the ability to produce these colonization factors. Among the 16 CFA/I+ ETEC strains, the loss of CFA/I was accompanied by the loss of a plasmid of between 34 and 68 megadaltons (MDa) coding for heat-stable enterotoxin A2 (ST-A2) in 12 strains, by the loss of a 60-MDa plasmid coding for heat-labile enterotoxin (LT) and ST-A2 in one strain, or by deletions of a segment of DNA encoding for ST-A2 in three strains. Among five CFA/II+ ETEC strains, the loss of CFA/II was associated with the loss of a plasmid of 75 MDa coding for LT and ST-A2 in three strains, with the loss of genes coding for LT and ST-A2 from a 68-MDa plasmid in one strain, or with no discernible loss of a plasmid or DNA sequences coding for enterotoxins in the remaining strain. The loss of CFA/I and CFA/II production was associated with the loss of DNA sequences encoding for ST-A2 in 20 of 21 ETEC examined.     

New Surface-Associated Heat-Labile Colonization Factor Antigen (CFA/II) Produced by Enterotoxigenic Escherichia coli of Serogroups O6 and O8

Enterotoxigenic Escherichia coli (ETEC) belonging to serogroups O6 and O8 do not possess the H-10407-type colonization factor antigen (CFA/I). However, these frequently isolated ETEC were found to possess a second and distinct heat-labile surface-associated colonization factor antigen, termed CFA/II. Whereas CFA/I mediates mannose-resistant hemagglutination of human group A erythrocytes, CFA/II does not. CFA/II mediates mannose-resistant hemagglutination of bovine erythrocytes, and mannose-resistant hemagglutination is rapid only at reduced temperature (4 degrees C). Because CFA/II, like CFA/I, is spontaneously lost by many ETEC isolates in the laboratory, it was possible to produce specific anti-CFA/II serum by preparing antiserum against living cells of a prototype strain (PB-176) and adsorbing this serum with living and heat-treated cells of its CFA/II-negative derivative strain PB-176-P. This serum, which neutralized the colonization factor activity of CFA/II-positive strains in infant rabbits, was employed to confirm the presence of CFA/II on ETEC which exhibited mannose-resistant hemagglutination of bovine but not human erythrocytes. CFA/II, like CFA/I, mediates adherence of the bacteria to the mucosal surface of the small intestine, as demonstrated by indirect immunofluorescence. CFA/II appears to be an important virulence factor for humans since CFA/II-positive ETEC are frequently isolated from diarrhea cases, particularly travelers' diarrhea, in Mexico; these ETEC were not uncommon in a collection of isolates from Bangladesh. The O6:H16 strain of ETEC responsible for an outbreak of diarrhea in the United States was also shown to be CFA/II positive. CFA/I and CFA/II were never found on the same serotypes of ETEC, but 98% of the heat-stable and heat-labile enterotoxin-producing ETEC belonging to the frequently isolated serogroups O6, O8, O15, O25, O63, and O78 were positive for either CFA/I or CFA/II.     

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.     

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.     

Adhesion of enterotoxigenic Escherichia coli to human small intestinal enterocytes.

An improved enterocyte adhesion assay has been used to examine a collection of 44 strains of enterotoxigenic Escherichia coli (ETEC) for their ability to adhere to the brush border of isolated human duodenal enterocytes. Fourteen strains showed good adhesion; in each case the ability to adhere correlated with the production of colonization factor antigen I or II (CFA/I or CFA/II) fimbriae. CFA/II-positive producing coli surface antigens 1 and 3 (CS1 and CS3), coli surface antigens 2 and 3 (CS2 and CS3), and only coli surface antigen 3 (CS3) each showed good adhesion. CS3-mediated brush border attachment of CFA/II-positive ETEC was demonstrated by electron microscopy with monospecific antibody and an immunogold labeling technique. One CFA/I-positive ETEC strain was nonadherent in the assay, as were ETEC producing type 1 somatic fimbriae. Five animal ETEC strains producing K88, K99, F41, and 987P fimbriae were slightly more adhesive than control strains, but adhesion was significantly less than that of CFA-positive ETEC. Twenty five human ETEC strains that lacked CFA/I and CFA/II were nonadherent, suggesting either that the surface antigens responsible for adhesion to human intestinal mucosa in these strains were not being produced or that mucosal receptors for these strains are present in regions of the small intestine other than the duodenum.     

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.     

Characterization of enterotoxigenic Escherichia coli isolated from U.S. troops deployed to the Middle East.

Enterotoxigenic Escherichia coli (ETEC) was a common cause of traveler's diarrhea in U.S. soldiers in the Middle East in 1989 and 1990. To determine which bacterial components would be useful in a vaccine, potential protective antigens (toxin, colonization factor antigen [CFA], and serotype) from 189 ETEC isolates were examined. Nearly half of the isolates expressed both ETEC toxins, 39% had only heat-stable enterotoxin (ST), and 17% had heat-labile enterotoxin (LT). CFA/I was the least common colonization factor antigen (11%), CFA/II was common (34%), as was CFA/IV (31%), and 24% expressed none of these CFAs. Fifty-seven O:H serotypes were found. Serotype O6:H16 was the most common, occurring in 29% of the ETEC isolates, usually with LT-ST and CFA/II. Generally, CFA/II was associated with expression of both toxins, CFA/IV was associated with expression of ST, and none of the CFAs was routinely found with LT. We conclude that ETEC from soldiers in the Middle East expressed a variety of antigens and that an effective vaccine will require multiple protective antigens.     

Beyond Serotypes and Virulence-Associated Factors: Detection of Genetic Diversity among O153:H45 CFA/I Heat-Stable Enterotoxigenic Escherichia coli Strains

Characterization of enterotoxigenic Escherichia coli (ETEC) has been based almost exclusively on the detection of phenotypic traits such as serotypes and virulence-associated factors: heat-labile (LT) and heat-stable (ST) toxins and colonization factors (CFs). In the present work we show that the analysis of band patterns generated by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of digested chromosomal DNA can be used to detect genetic diversity among ETEC strains expressing identical phenotypic traits. The study included 29 ETEC isolates from Latin America and Spain expressing the phenotype O153:H45 CFA/I ST plus 1 rough derivative, 2 nonmotile derivatives, and 1 O78:H12 CFA/I ST isolate, and a representative of a genetically distinct ETEC group. The results showed that the O153:H45 CFA/I ST ETEC isolates belong to a single clonal cluster whose isolates share on average, 84% of the RAPD bands and 77% of the PFGE restriction fragments, while the O78:H12 isolate shared only 44 and 4% of the RAPD bands and PFGE fragments, respectively, with the isolates of the O153:H45 group. More relevantly, RAPD and PFGE fingerprints disclosed the presence of different clonal lineages among the isolates of the O153:H45 cluster. Some of the genetic variants were isolated from defined geographic areas, while places like S?o Paulo City in Brazil and the middle-eastern part of Argentina were populated by several genetic variants of related, but not identical, ETEC strains. These results show that molecular biology-based typing methods can disclose strain diversity, which is usually missed in studies restricted to phenotypic typing of ETEC.     

Presence of cfaD-homologous sequences and expression of coli surface antigen 4 on enterotoxigenic Escherichia coli; relevance for diagnostic procedures.

We examined the ability of a colonization factor antigen I (CFA/I) polynucleotide probe to identify coli-surface antigen 4 producing (CS4+) strains of enterotoxigenic Escherichia coli (ETEC). At low stringency (LS) the probe hybridized to colony lysates of strains previously shown to produce CS4 or CFA/I fimbriae. Only DNA from CFA/I+ strains maintained a stable probe-target hybrid under high stringency (HS) conditions. On examination of several clones from three previous CS4 producers, identified as positive in LS and negative in HS colony hybridization, spontaneous loss of nucleotide sequences homologous to a gene encoding a positive CFA/I regulator, CfaD, was found to be associated with lacking expression of CS4. Our findings indicate that, on stored or subcultured isolates of ETEC, identification of CS4 strains may benefit from applying gene probe technology.     

Phenotypic profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in rural Egypt

Enterotoxigenic Escherichia coli (ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir.