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.     

A combination vaccine consisting of three live attenuated enterotoxigenic Escherichia coli strains expressing a range of colonization factors and heat-labile toxin subunit B is well tolerated and immunogenic in a placebo-controlled double-blind phase I trial in healthy adults

Immune responses against colonization factors (CFs) and the nontoxic B component of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LTB) are considered to be important for immunity against diarrhea caused by ETEC. Individual live attenuated ETEC derivatives that have had their toxin genes removed and whose aroC, ompC, and ompF genes are deleted have shown promise as vaccines against ETEC. The development of such strains has culminated in the testing of a three-strain-combination live attenuated vaccine known as ACE527, comprised of strains ACAM2025 expressing colonization factor antigen I (CFA/I) and LTB; ACAM2022, expressing CS5, CS6, and LTB; and ACAM2027, expressing CS1, CS2, CS3, and LTB. The recombinant CF and LTB genes expressed in the three strains were inserted into the bacterial chromosome to ensure their stable inheritance and expression without the requirement for any selection. ACE527 has been tested in a randomized placebo-controlled, double-blind, phase I safety and immunogenicity study in healthy adult volunteers and proved to be well tolerated and immunogenic at dose levels of 10(10) and 10(11) total CFU. There was no indication of strain interference on the basis of fecal shedding patterns, with all three being detected in the feces of 50% and 83% of low- and high-dose vaccine recipients, respectively. Similarly, strong immune responses to LTB and to CFs expressed on all three constituent strains were induced, with at least 50% of subjects in the high-dose group responding to LTB, CFA/I, CS3, and CS6.     

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.     

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.     

Homologous and cross-reactive immune responses to enterotoxigenic Escherichia coli colonization factors in Bangladeshi children

We have studied homologous (HoM) and cross-reacting (CR) immunoglobulin A (IgA) antibody responses to colonization factors (CFs) in Bangladeshi children with diarrhea due to enterotoxigenic E. coli (ETEC) strains of the CF antigen I (CFA/I) group (CFA/I, n = 25; coli surface antigen 4 [CS4], n = 8; CS14, n = 11) and the CS5 group (CS5, n = 15; CS7, n = 8), respectively. The responses to the HoM, CR, and heterologous (HeT) CF antigens in each group of patient were studied and compared to that seen in healthy children (n = 20). In the CFA/I group (CFA/I and CS14), patients responded with antibody-secreting cell (ASC) responses to HoM CFs (geometric mean, 156 to 329 ASCs/10(6) peripheral blood mononuclear cells [PBMCs]) and to CR CFs ( approximately 15 to 38 ASCs/10(6) PBMCs) but least of all to the HeT CS5 antigen (2 to 4 ASCs/10(6) PBMCs). For the CS5 group of patients with ETEC (CS5 and CS7), likewise, responses to HoM CFs (230 to 372 ASCs/10(6) PBMCs) and CR CFs (27 to 676 ASCs/10(6) PBMCs) were seen, along with lower responses to the HeT CFA/I antigen (9 to 38 ASCs/10(6) PBMCs). Both groups of patients responded with CF-specific IgA antibodies to HoM and CR antigens in plasma but responded less to the HeT CFs. The responses in patients were seen very soon after the onset of diarrhea and peaked around 1 week after onset. Vaccinees who had received two doses of the oral, killed whole-cell ETEC vaccine (CF-BS-ETEC) responded with plasma IgA antibodies to CFA/I, a component of the vaccine, but also to the CR CS14 antigen, which was not included in the vaccine, showing that antibody responses can be stimulated by a CFA/I-containing ETEC vaccine to a CR-reacting antigen in individuals in countries where ETEC is endemic.     

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.     

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.     

Mutations in the periplasmic chaperone leading to loss of surface expression of the colonization factor CS6 in enterotoxigenic Escherichia coli (ETEC) clinical isolates

Enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by adhesion to human enterocytes by one or more colonization factors (CFs) and secretion of heat-labile (LT) and/or heat-stable (ST) enterotoxins. Expression of coli surface antigen 6 (CS6) on the bacterial surface, usually associated with ETEC strains that produce ST alone or in combination with LT, is rarely found in strains expressing only LT. However, a number of LT-only strains which are genotypically positive but phenotypically negative for CS6 have been identified. In this study, eight such strains from India and Guinea-Bissau belonging to different clones were analysed. The CS6 operon cssABCD was transcribed but protein analyses suggested that the structural subunits CssA and CssB of CS6 were absent in the periplasm. Most strains contained truncating mutations within the periplasmic chaperone-encoding gene cssC and protein modelling indicated that this severely affected the substrate-binding capacity of the chaperone. A single-nucleotide polymorphism (SNP) (A-->T) in the 5'-untranslated region of cssC distinguished the eight strains from ETEC strains that do express CS6 on the surface and may be a potential marker for ETEC strains containing phenotypically silent cssABCD. The study emphasizes the importance of using both genotypic and phenotypic methods in epidemiological studies of ETEC, e.g. for vaccine development.     

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.     

Genotypic and phenotypic identification of coli surface antigen 6-positive enterotoxigenic Escherichia coli.

A polynucleotide probe comprising the gene encoding a major structural subunit protein of coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) was developed. Eighty-nine ETEC isolates were examined in parallel with the probe in a colony hybridization assay and in a recently developed polyclonal-antibody-based inhibition enzyme-linked immunosorbent assay (ELISA). The two assays showed a high level of concordance in the detection of CS6-positive ETEC (kappa = 0.84, P < 0.00001). Thus, 36 of the 89 ETEC isolates were identified as CS6-positive by both assays. Six strains that were negative for other colonization factor antigens were positive with the CS6 probe but negative in the ELISA, suggesting lack of surface CS6 expression in these strains. One strain was probe negative but positive in the ELISA, while the remaining 46 strains were negative in both assays. The phenotypic and genotypic assays will prove useful in vaccine-oriented studies of ETEC disease.     

Gene encoding the major subunit of CS1 pili of human enterotoxigenic Escherichia coli.

Some enterotoxigenic strains of Escherichia coli (ETEC) utilize the CS1 pilus for colonization of human intestinal epithelium. We have cloned the gene which encodes the major CS1 subunit and called it cooA (for coli surface antigen one). Hybridization showed that the ETEC strain from which it was cloned carried cooA on a plasmid different from the one encoding its positive regulator, rns. Based on the cooA DNA sequence, cleavage with signal peptidase would be expected to produce a mature protein of 15.2 kDa; a 16-kDa polypeptide that reacted with CS1-specific antiserum was observed on electrophoresis. At the protein level, there was 92% similarity and 55% identity between cooA and cfaB, the major colonization factor antigen I (CFA/I) antigen. However, CS1-specific antisera did not react with CfaB. No hybridization was seen between either of two different cooA probes and total DNA from ETEC strains expressing AFA-1, CFA/I, CS2, CS3, CS4, CS5, or CS6.     

Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children from developing countries and in adult travelers from industrialized countries to the developing world (16, 21). According to the World Health Organization (WHO), ETEC is the second most common cause of diarrhea after rotavirus in children less than 5 years of age and is therefore an important target for vaccine development (11). Diarrhea due to ETEC develops between 8 and 72 h after initial infection, usually due to the ingestion of contaminated food and water (21). The disease varies from a mild illness to one of great severity, usually without leukocytes or fecal blood but often with vomiting and, potentially, dehydration (10).The ability of ETEC to adhere to and colonize the human intestinal mucosa has been correlated with the presence of specific antigenic fimbriae called colonization factors (CFs), which have been designated colonization factor antigens (CFAs), coli surface antigens (CSs), or putative colonization factors (PCFs), followed by a numeric designation. The CFs are mainly fimbrial or fibrillar proteins, although some are not fimbrial in structure (21). To date, over 25 human ETEC CFs have been described. In turn, these CFs have been divided into different families: (i) a CFA/I-like group including CFA/I, CS1, CS2, CS4, CS14, and CS17; (ii) a CS5-like group including CS5, CS7, CS18, and CS20; and (iii) a unique group including CS3, CS6, and CS10 to CS12 (8, 21, 33).Following CF-mediated mucosal adhesion, ETEC elaborates one or both of two enterotoxins: heat-labile toxin (LT), a protein multimer which shares many features with cholera toxin and which binds to intracellular adenylylcyclase, leading to increased cyclic AMP levels, and/or heat-stable toxin (ST), a small-peptide molecule that similarly activates guanylylcyclase and which produces increased intracellular cyclic GMP. For both toxins, the increased chloride secretion resulting from these toxins produces a watery diarrhea (10, 16). Both of these virulence factors are plasmid encoded. ST is encoded by two different genes: estA and st1, which produce STh (originally isolated from ETEC in humans) and STp (originally from a pig isolate), respectively. LT toxin is encoded by the eltA and eltB genes (12). The diagnosis of ETEC infection relies upon the detection of either the genes themselves or their gene products in clinical specimens.Currently, derivatives of LT and the CFs are targets for the development of vaccines against ETEC. However, the great variability of ETEC CFs requires determination of the CF types prevalent in different geographic locations (21, 33). The aims of this study were (i) to determine the clinical and epidemiological characteristics of ETEC diarrhea in Peruvian children, (ii) to determine the presence of ST and LT, (iii) to determine the presence and distribution of colonization factors in these strains, and (iv) to determine the antibiotic susceptibilities of these strains.     

Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of childhood diarrhea in developing countries and in travelers. However, this pathogen has often not been reported in surveys of diarrheal pathogens, due to lack of simple standardized methods to detect ETEC in many laboratories. ETEC expresses one or both of two different enterotoxin subtypes: heat-stable toxins, a heat-labile toxin (LT), and more than 22 different colonization factors (CFs) that mediate adherence to the intestinal cell wall. Here we compare established phenotypic and genotypic detection methods and newly developed PCR detection methods with respect to sensitivity, specificity, positive predictive value, and ease of performance. The methods include GM1-enzyme-linked immunosorbent assay and dot blot techniques using specific monoclonal antibodies (MAbs) for phenotypic detection of the toxins and CFs, respectively, as well as different PCR and DNA/DNA hybridization techniques, including new PCR assays, for genotypic identification of the toxin and CF genes, respectively. We found very good general agreement in results derived from genotypic and phenotypic methods. In a few strains, LT and CFs were identified genetically but not phenotypically. Based on our analyses, we recommend initial screening for ETEC in clinical samples by multiplex toxin gene PCR. Toxin-positive strains may then be analyzed by dot blot tests for detection of the CFs expressed on the bacterial surface and by PCR for determination of additional CFs for which MAbs are currently lacking as well as for strains that harbor silent CF genes.     

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.     

Attenuated Shigella flexneri 2a Delta guaBA strain CVD 1204 expressing enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 fimbriae as a live mucosal vaccine against Shigella and ETEC infection

To construct a prototype hybrid vaccine against Shigella and enterotoxigenic Escherichia coli (ETEC), the genes encoding the production of ETEC CS2 and CS3 fimbriae were isolated and expressed in attenuated Shigella flexneri 2a guaBA strain CVD 1204. The CS2 cotA to -D genes, isolated from ETEC strain C91F, and the CS3 cstA to -H genes, subcloned from plasmid pCS100, were cloned into ~15-copy-number-stabilized pGA1 behind the osmotically regulated ompC promoter, resulting in high expression of both fimbriae. Under nonselective in vitro growth conditions, pGA1-CS2 and pGA1-CS3 were stable in CVD 1204, exhibiting a plasmid loss of only approximately 1% per duplication. Expression of CS2 and CS3 reduced the invasiveness of Shigella for HeLa cells and slowed the intracellular growth rate. Guinea pigs immunized intranasally with CVD 1204(pGA1-CS2) or CVD 1204(pGA1-CS3), or with a mixture of these strains, developed secretory immunoglobulin A (IgA) in tears and serum IgG antibodies against Shigella lipopolysaccharide, CS2, and CS3 antigens. Moreover, the animals were protected against keratoconjunctivitis following conjunctival challenge with virulent S. flexneri 2a strain 2457T. Animals immunized with Shigella expressing CS2 or CS3 developed serum antibodies that agglutinated Shigella as well as an ETEC strain bearing the homologous fimbriae, whereas animals immunized with combined CVD 1204(pGA1-CS2) and CVD 1204(pGA1-CS3) developed antibodies that agglutinated all three test strains. These observations support the feasibility of a multivalent vaccine against shigellosis and ETEC diarrhea consisting of multiple Shigella live vectors expressing relevant ETEC antigens.     

CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15

Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H- strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the nfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.     

Disease burden due to enterotoxigenic Escherichia coli in the first 2 years of life in an urban community in Bangladesh

A cohort of 321 children was followed from birth up to 2 years of age to determine the incidence of enterotoxigenic Escherichia coli (ETEC) in Bangladesh. The average number of diarrheal days and incidence rates were 6.6 and 2.3/child/year, respectively. ETEC was the most common pathogen and was isolated in 19.5% cases, with an incidence of 0.5 episode/child/year. The prevalence of rotavirus diarrhea was lower (10%). ETEC expressing the heat-stable enterotoxin (ST) was predominant. Strains isolated from diarrheal cases were positive for colonization factors (CFs) in higher frequency (66%) than from healthy children (33%) (P < 0.001). The heat-labile toxin (LT)-positive strains from healthy children were more often CF negative (92%) than those isolated from children with diarrhea (73%) (P < 0.001). In children with symptomatic or asymptomatic infections by CFA/I, CS1 plus CS3, CS2 plus CS3, or CS5 plus CS6 strains, a repeat episode of diarrhea or infection by the homologous CF type was uncommon. Repeat symptomatic infections were noted mostly for LT- and ST-expressing ETEC. ETEC diarrhea was more prevalent in children in the A and AB groups than in those in the O blood group (P = 0.032 to 0.023). Children with ETEC diarrhea were underweight and growth stunted at the 2-year follow-up period, showing the importance of strategies to prevent and decrease ETEC diarrheal morbidity in children.     

Mucosal and systemic immune responses in patients with diarrhea due to CS6-expressing enterotoxigenic Escherichia coli

Colonization factor CS6 expressed by enterotoxigenic Escherichia coli (ETEC) is a nonfimbrial polymeric protein. A substantial proportion of ETEC strains isolated from patients in endemic settings and in people who travel to regions where ETEC is endemic are ETEC strains expressing CS6, either alone or in combination with fimbrial colonization factor CS5 or CS4. However, relatively little is known about the natural immune responses elicited against CS6 expressed by ETEC strains causing disease. We studied patients who were hospitalized with diarrhea (n = 46) caused by CS6-expressing ETEC (ETEC expressing CS6 or CS5 plus CS6) and had a disease spectrum ranging from severe dehydration (27%) to moderate or mild dehydration (73%). Using recombinant CS6 antigen, we found that more than 90% of the patients had mucosal immune responses to CS6 expressed as immunoglobulin (IgA) antibody-secreting cells (ASC) or antibody in lymphocyte supernatant (ALS) and that about 57% responded with CS6-specific IgA antibodies in feces. More than 80% of the patients showed IgA seroconversion to CS6. Significant increases in the levels of anti-CS6 antibodies of the IgG isotype were also observed in assays for ASC (75%), ALS (100%), and serum (70%). These studies demonstrated that patients hospitalized with the noninvasive enteric pathogen CS6-expressing ETEC responded with both mucosal and systemic antibodies against CS6. Studies are needed to determine if the anti-CS6 responses protect against reinfection and if protective levels of CS6 immunity are induced by vaccination.     

Evolutionary and functional relationships of colonization factor antigen i and other class 5 adhesive fimbriae of enterotoxigenic Escherichia coli

Colonization factor antigen I (CFA/I) is the archetype of eight genetically related fimbriae of enterotoxigenic Escherichia coli (ETEC) designated class 5 fimbriae. Assembled by the alternate chaperone pathway, these organelles comprise a rigid stalk of polymerized major subunits and an apparently tip-localized minor adhesive subunit. We examined the evolutionary relationships of class 5-specific structural proteins and correlated these with functional properties. We sequenced the gene clusters encoding coli surface antigen 4 (CS4), CS14, CS17, CS19, and putative colonization factor antigen O71 (PCFO71) and analyzed the deduced proteins and the published homologs of CFA/I, CS1, and CS2. Multiple alignment and phylogenetic analysis of the proteins encoded by each operon define three subclasses, 5a (CFA/I, CS4, and CS14), 5b (CS1, CS17, CS19, and PCFO71), and 5c (CS2). These share distant evolutionary relatedness to fimbrial systems of three other genera. Subclass divisions generally correlate with distinguishing in vitro adherence phenotypes of strains bearing the ETEC fimbriae. Phylogenetic comparisons of the individual structural proteins demonstrated greater intrasubclass conservation among the minor subunits than the major subunits. To correlate this with functional attributes, we made antibodies against CFA/I and CS17 whole fimbriae and maltose-binding protein fusions with the amino-terminal half of the corresponding minor subunits. Anti-minor subunit Fab preparations showed hemagglutination inhibition (HAI) of ETEC expressing homologous and intrasubclass heterologous colonization factors while anti-fimbrial Fab fractions showed HAI activity limited to colonization factor-homologous ETEC. These results were corroborated with similar results from the Caco-2 cell adherence assay. Our findings suggest that the minor subunits of class 5 fimbriae may be superior to whole fimbriae in inducing antiadhesive immunity.     

Discovery and phylogenetic analysis of novel members of class b enterotoxigenic Escherichia coli adhesive fimbriae

Enterotoxigenic Escherichia coli (ETEC) is recognized to be a common cause of acute watery diarrhea in children from developing countries. Colonization factors (CFAs) have been identified predominantly in ETEC isolates secreting heat-stable enterotoxin (ST) or cosecreting ST with a heat-labile toxin (LT). We hypothesized that LT-only-secreting ETEC produces unique colonization factors not previously described in ST and LTST-secreting ETEC. A set of degenerate primers based on nucleotide sequence similarities between the major structural genes of CS20 (csnA), CS18 (fotA), CS12 (cswA), and porcine antigen 987 (fasA) was developed and used to screen a collection of 266 LT-secreting ETEC isolates in which no known CFA was detected. PCR-amplified products of different molecular masses were obtained from 49 (18.4%) isolates. Nucleotide sequence analysis of the PCR amplicons followed by GenBank nucleotide BLASTn analysis revealed five novel DNA sequences; translated amino acid BLASTx analysis confirmed sequence similarity to class 1b major structural proteins encoded by csnA, fotA, and fasA. Strains expressing the novel CFAs were phylotyped and analyzed using multilocus sequence typing (MLST; Achtman scheme), and the types detected were compared to those of a collection of archived global E. coli strains. In conclusion, application of the degenerate primer sets to ETEC isolates from surveillance studies increased the total number of ETEC isolates with detectable CFAs by almost 20%. Additionally, MLST analysis suggests that for many CFAs, there may be a requirement for certain genetic backgrounds to acquire and maintain plasmids carrying genes encoding CFAs.