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.     

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.     

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.     

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.     

Experimental enterotoxin-induced Escherichia coli diarrhea and protection induced by previous infection with bacteria of the same adhesin or enterotoxin type.

The diarrheal response to an initial and a second infection with Escherichia coli expressing various enterotoxins (the heat-stable toxin [ST] alone or in combination with the heat-labile toxin [LT]) and colonization factor antigens (CFA/I, CFA/II, or E8775-type) was studied in the reversible tie adult rabbit diarrhea model. An initial infection with high doses (1 X 10(10) to 5 X 10(11) bacteria) of the various strains regularly induced diarrhea which was usually self-limiting (only 7 of 85 animals died). The diarrheal response to equally effective doses of different strains producing both ST and LT (ST/LT) did not differ significantly with serotype or colonization factor antigen. ST/LT-producing strains appeared to induce severe disease more regularly than ST-producing strains carrying the same adhesin. Previous infection with CFA/I-carrying, ST/LT-producing E. coli protected all animals reinfected with an otherwise highly diarrheogenic dose of the same strain as well as against challenge with a CFA/I-carrying, ST/LT-producing strain with different O-, K-, and H-antigens. Fecal excretion of bacteria was also significantly reduced in the protected animals, although not completely eliminated. When only one of the two antigens, CFA/I and LT, was shared by the immunizing and rechallenge strains, partial protection was evident consistent with independent antibacterial (anti-CFA) and antitoxic (anti-LT) immune mechanisms. Oral immunization with purified CFA/I significantly reduced fluid secretion in intestinal loops infected with CFA/I-carrying enterotoxigenic bacteria.     

Enterotoxigenic Escherichia coli in a population of infants with diarrhea in Chile.

The incidence of enterotoxigenic Escherichia coli (ETEC) was investigated in 95 E. coli strains isolated from 48 infants with diarrhea in Santiago, Chile. By using standard biological assays and DNA-DNA hybridization procedures, ETEC was found in 31.2% of the cases: 14 strains produced heat-stable enterotoxin (ST) only, three strains produced heat-labile enterotoxin (LT) and ST, and two strains produced LT only. DNA probes detected all enterotoxin producers except one ST-producing strain. The ST strains hybridized with one or both of the human ST probes (ST Ib and ST A2). Two of the LT-ST strains hybridized with the ST Ia and ST Ib probes, and the third strain did not hybridize with any of the ST probes. Only the ST group expressed multiple resistance (85.7%) and colonization factor antigen I (CFA I) (92.8%); CFA II was found in two of three LT-ST strains. The O153:H45 serotype was found in 10 of 14 ST strains, and O6:K15:H16 was found in one LT strain and in two LT-ST strains. These findings suggest that ETEC, especially strains that produce ST, may be an important cause of diarrhea among Chilean infants.     

Attachment Factors Among Enterotoxigenic Escherichia coli from Patients with Acute Diarrhea from Diverse Geographic Areas

To cause diarrhea, enterotoxigenic Escherichia coli (ETEC) must initially colonize the small bowel. Different surface structures have been implicated in this initial attachment. Recognized attachment factors include colonization factor antigens I and II (CFA/I and CFA/II) and type I pili. Several methods of detection for each of these factors have been reported. In this study, we screened for the presence of these attachment factors among enterotoxigenic E. coli isolated from 40 patients with acute diarrhea and 40 asymptomatic control individuals and examined their ability to attach to ATCC 407 human intestinal cells in vitro. Of 40 patients with diarrhea, 16 (40%) had enterotoxigenic E. coli isolates which exhibited an attachment trait. Fourteen (35%) of these isolates demonstrated the ability to attach to ATCC 407 cells, whereas only four isolates from asymptomatic controls attached (P < 0.02). A total of 20% of the patient isolates and 7.5% of the control isolates possessed CFA/I. Only one patient isolate demonstrated CFA/II. Evidence for type I pili was found on 10% of the patient isolates and 12.5% of the control isolates. Attachment to ATCC 407 cells allowed the detection of 87.5% (14 of 16) of enterotoxigenic E. coli isolates with any type of attachment trait. Of the 14 cases demonstrating attachment ability to ATCC 407 cells, 7 did not attach in the presence of mannose. Three of these showed evidence for both CFA/I and type I pili, one showed only CFA/I, and one showed only type I pili. Two of those mannose-sensitive attaching isolates showed no other demonstrable trait. Seven patient isolates showed mannose-resistant attachment. Of these, two were classified as possessing CFA/I, and one was classified as possessing CFA/II. The four remaining isolates could not be classified into any recognized attachment factor category, suggesting that other attachment factors remain to be identified.     

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.     

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.     

Phenotypic Diversity of Enterotoxigenic Escherichia coli Strains from a Community-Based Study of Pediatric Diarrhea in Periurban Egypt

No past studies of diarrhea in children of the Middle East have examined in detail the phenotypes of enterotoxigenic Escherichia coli (ETEC) strains, which are important pathogens in this setting. During a prospective study conducted from November 1993 to September 1995 with 242 children under 3 years of age with diarrhea living near Alexandria, Egypt, 125 episodes of diarrhea were positive for ETEC. ETEC strains were available for 98 of these episodes, from which 100 ETEC strains were selected and characterized on the basis of enterotoxins, colonization factors (CFs), and O:H serotypes. Of these representative isolates, 57 produced heat-stable toxin (ST) only, 34 produced heat-labile toxin (LT) only, and 9 produced both LT and ST. Twenty-three ETEC strains expressed a CF, with the specific factors being CF antigen IV (CFA/IV; 10 of 23; 43%), CFA/II (5 of 23; 22%), CFA/I (3 of 23; 13%), PCFO166 (3 of 23; 13%), and CS7 (2 of 23; 9%). No ETEC strains appeared to express CFA/III, CS17, or PCFO159. Among the 100 ETEC strains, 47 O groups and 20 H groups were represented, with 59 O:H serotypes. The most common O serogroups were O159 (13 strains) and O43 (10 strains). O148 and O21 were each detected in five individual strains, O7 and O56 were each detected in four individual strains, O73, O20, O86, and O114 were each detected in three individual strains, and O23, O78, O91, O103, O128, and O132 were each detected in two individual strains. The most common H serogroups were H4 (16 strains), 12 of which were of serogroup O159; H2 (9 strains), all of which were O43; H18 (6 strains); H30 (6 strains); and H28 (5 strains); strains of the last three H serogroups were all O148. Cumulatively, our results suggest a high degree of clonal diversity of disease-associated ETEC strains in this region. As a low percentage of these strains expressed a CF, it remains possible that other adhesins for which we either did not assay or that are as yet undiscovered are prevalent in this region. Our findings point out some potential barriers to effective immunization against ETEC diarrhea in this population and emphasize the need to identify additional protective antigens commonly expressed by ETEC for inclusion in future vaccine candidates.     

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.     

Hemagglutination patterns of enterotoxigenic and enteropathogenic Escherichia coli determined with human, bovine, chicken, and guinea pig erythrocytes in the presence and absence of mannose.

A hemagglutination (HA)-typing system has been developed for the presumptive identification of enterotoxigenic Escherichia coli (ETEC) possessing the colonization factor antigens (CFA) CFA/I or CFA/II. E. coli isolates are grown on CFA agar and tested for mannose-sensitive (MS) or mannose-resistant (MR) HA of human, bovine, chicken, and guinea pig erythrocytes. CFA/I-positive ETEC exhibit MRHA with human, bovine, and chicken erythrocytes, but no HA with guinea pig erythrocytes. CFA/II-positive ETEC produce HA (MRHA) only with bovine and chicken erythrocytes. Common pili appear to be the primary MS-hemagglutinin of E. coli because the prototype strain K-12 exhibits HA (MSHA) with all but bovine erythrocytes. However, only 6.6% (23 of 351) of E. coli belonging to the classical enteropathogenic E. coli serogroups (EPEC) possessed the same HA pattern as strain K-12; 42% of the EPEC cultures (146 of 351) were similar to K-12 in producing MSHA with chicken and guinea pig erythrocytes and no HA with bovine erythrocytes, but different in that these produced either no HA or MRHA with human erythrocytes. These EPEC-associated HA patterns were assigned to a separate category, termed HA type III. Non-EPEC serogroups associated with sporadic diarrhea (i.e., the facultatively enteropathogenic E. coli, or FEEC) and 41% (19 of 46) of available Salmonella isolates also produced HA type III patterns. This observation is of considerable interest because many FEEC possess somatic antigens cross-reactive with Salmonella. Although the biochemical basis for this result has not been established, the data reported herein suggest a relationship between the HA type III phenotype and virulence (enteropathogenicity) in both the EPEC and FEEC serogroups. We propose that HA typing be used in conjunction with serotyping of E. coli to determine the degree of association of HA type III E. coli with sporadic diarrhea in infants and young children.     

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.     

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.     

Adjuvant activity of a nontoxic mutant of Escherichia coli heat-labile enterotoxin on systemic and mucosal immune responses elicited against a heterologous antigen carried by a live Salmonella enterica serovar Typhimurium vaccine strain

Systemic and mucosal antibody responses against both the major subunit of colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) and the somatic lipopolysaccharide expressed by recombinant bivalent Salmonella vaccine strains were significantly enhanced by coadministration of a detoxified derivative with preserved adjuvant effects of the ETEC heat-labile toxin, LT((R192G)). The results further support the adjuvant effects of LT((R192G)) and represent a simple alternative to improve responses against passenger antigens expressed by orally delivered Salmonella vaccine strains.     

Molecular Characterization of Enterotoxigenic Escherichia coli Isolates Recovered from Children with Diarrhea during a 4-Year Period (2007 to 2010) in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea. This study aimed to characterize ETEC strains isolated from Bolivian children aged <5 years according to enterotoxin profile, colonization factors (CFs), suggested virulence genes, and severity of disease. A total of 299 ETEC isolates recovered from children with diarrhea and 55 ETEC isolates from children without diarrhea (controls) were isolated over a period of 4 years. Strains expressing heat-labile toxin (LT) or heat-stable toxin (ST) alone were about equally common and twice as common as ETEC producing both toxins (20%). ETEC strains expressing human ST (STh) were more common in children aged <2 years, while ETEC strains expressing LT plus STh (LT/STh) were more frequent in 2- to 5-year-old children. Severity of disease was not related to the toxin profile of the strains. CF-positive isolates were more frequently identified in diarrheal samples than in control samples (P = 0.02). The most common CFs were CFA/I and CS14. CFA/I ETEC strains were more frequent in children aged <2 years than CS1+CS3 isolates and CS14 isolates, which were more prevalent in 2- to 5-year-old children. The presence of suggested ETEC virulence genes (clyA, eatA, tia, tibC, leoA, and east-1) was not associated with disease. However, east-1 was associated with LT/STh strains (P < 0.001), eatA with STh strains (P < 0.001), and tia with LT/STh strains (P < 0.001). A minor seasonal peak of ETEC infections was identified in May during the cold-dry season and coincided with the peak of rotavirus infections; this pattern is unusual for ETEC and may be important for vaccination strategies in Bolivia.     

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.     

Identification of enterotoxigenic Escherichia coli in patients with diarrhea in Asia with three enterotoxin gene probes.

A total of 984 enterotoxigenic Escherichia coli (ETEC) and 733 non-ETEC isolated from patients with diarrhea in Asia (one isolate per patient) were examined for homology with radiolabeled fragments of DNA encoding heat-labile toxin (LT) or heat-stable toxin of porcine (ST-P) or human (ST-H) origin. A total of 246 ETEC that produced LT and ST as determined by the Y-1 adrenal and suckling mouse assays were homologous with the LT probe. Of these 246 LTST ETEC, 156 (63%) were homologous with the ST-H, 46 (19%) were homologous with the ST-P, and 44 (18%) were homologous with both probes. A total of 401 LT ETEC were homologous with the LT probe. Of 337 ST ETEC identified by the suckling mouse assay, 244 (72%) were homologous with the ST-H, 84 (25%) were homologous with the ST-P, and 9 (3%) were homologous with both probes. None of the 733 isolates that were non-enterotoxigenic as determined by the Y-1 adrenal and suckling mouse assays was homologous with genes coding for enterotoxin. Four isolates (not included among the 984 ETEC examined) that were initially considered to produce LT because sterile culture supernatants produced rounding of Y-1 adrenal cells were not homologous with the LT probe. The sterile culture supernatants of these four isolates caused rounding after 8 h and subsequent destruction after 24 h of Y-1 adrenal tissue cultures. This effect was not inhibited by convalescent human cholera antiserum, Swiss Serum Institute cholera antitoxin, or antiserum to purified LT. These isolates were also negative in the Biken test previously used to identify LT-producing E. coli. The DNA hybridization technique with three enterotoxin gene probes was developed as a specific technique to identify ETEC in large numbers of specimens in Asia.     

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.     

Relationship among enterotoxigenic phenotypes, serotypes, and sources of strains in enterotoxigenic Escherichia coli.

The relationship among O groups, O:H serotypes and enterotoxigenic phenotypes was examined in 76 Escherichia coli strains isolated in Brazil from different sources. Of the 17 heat-labile and -stable enterotoxin (LT/ST)-producing strains whose O antigens were identified, 15 belonged to serotypes O6:H16 (7 strains), O63:H- (5 strains), and O139:H28 (3 strains). All 11 ST strains were in group OO128PAC, which was represented by four O:H serotypes. The 23 LT strains with the O antigen identified were distributed among serotypes of 14 O groups. Colonization factor CFA/I was not found in any of the LT strains, but it was found in six LT/ST and three ST strains. On the whole, each E. coli O:H serotype had a particular fermentation pattern. LT/ST as well as ST strains were all isolated from patients with diarrhea, whereas LT strains were isolated from patients with diarrhea, normal children, food, and river water.