Evaluation of the immunogenicity and protective efficacy of a recombinant CS6-based ETEC vaccine in an Aotus nancymaae CS6 + ETEC challenge model.

Colonization factors or Coli surface antigens (CFs or CS) are important virulence factors of Enterotoxigenic E. coli (ETEC) that mediate intestinal colonization and accordingly are targets of vaccine development efforts. CS6 is a highly prevalent CF associated with symptomatic ETEC infection both in endemic populations and amongst travelers. In this study, we used an Aotus nancymaae non-human primate ETEC challenge model with a CS6 + ETEC strain, B7A, to test the immunogenicity and protective efficacy (PE) of a recombinant CS6-based subunit vaccine. Specifically, we determined the ability of dscCssBA, the donor strand complemented recombinant stabilized fusion of the two subunits of the CS6 fimbriae, CssA and CssB, to elicit protection against CS6 + ETEC mediated diarrhea when given intradermally (ID) with the genetically attenuated double mutant heat-labile enterotoxin LT(R192G/L211A) (dmLT). ID vaccination with dscCssBA + dmLT induced strong serum antibody responses against CS6 and LT. Importantly, vaccination with dscCssBA + dmLT resulted in no observed diarrheal disease (PE = 100%, p = 0.03) following B7A challenge as compared to PBS immunized animals, with an attack rate of 62.5%. These data demonstrate the potential role that CS6 may play in ETEC infection and that recombinant dscCssBA antigen can provide protection against challenge with the homologous CS6 + ETEC strain, B7A, in the Aotus nancymaae diarrheal challenge model. Combined, these data indicate that CS6, and more specifically, a recombinant engineered derivative should be considered for further clinical development.

A systematic review of ETEC epidemiology focusing on colonization factor and toxin expression.

INTRODUCTION: Vaccine development for enterotoxigenic Escherichia coli (ETEC) is dependent on in-depth understanding of toxin and colonization factor (CF) distribution. We sought to describe ETEC epidemiology across regions and populations, focusing on CF and toxin prevalence. METHODS: We conducted a systematic review of the published literature, including studies reporting data on ETEC CF and toxin distributions among those with ETEC infection. Point estimates and confidence intervals were calculated using random effects models. RESULTS: Data on 17,205 ETEC isolates were abstracted from 136 included studies. Approximately half of the studies (49%) involved endemic populations, and an additional 17% involved only travel populations. Globally, 60% of isolates expressed LT either alone (27%) or in combination with ST (33%). CFA/I-expressing strains were common in all regions (17%), as were ETEC expressing CFA/II (9%) and IV (18%). Marked variation in toxins and CFs across regions and populations was observed. DISCUSSION/CONCLUSIONS: These results demonstrate the relative importance of specific CFs in achieving target product profiles for a future ETEC vaccine. However, heterogeneity across time, population, and region, confounded by variability in CF and toxin detection methodologies, obfuscates rational estimates for valency requirements.

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.

Pathogenicity and convalescent excretion of Campylobacter in rural Egyptian children.

Campylobacter infection in developing countries has not received much public health attention because of the observation that infections are not associated with disease beyond the first 6 months of life. A cohort of 397 Egyptian children aged less than 3 years, who were observed twice weekly during 1995--1998, experienced an incidence of 0.6 episodes of Campylobacter diarrhea per child-year. A total of 13% of the Campylobacter diarrheal episodes were characterized by severe dehydration. Age-specific incidence rates (episodes per year) were 0.9 in infants aged less than 6 months, 1.5 in those 6--12 months, and 0.4 and 0.2 in the second and third years of life, respectively. Convalescent excretion of Campylobacter after a diarrheal episode might be enhancing transmission and contributing to this high incidence. Observed risk factors for Campylobacter diarrhea were poor hygienic conditions and the presence of animals in the house. Regardless of the child's age, a first infection by Campylobacter was associated with diarrhea (odds ratio = 2.45; 95% confidence interval: 1.61, 3.71); however, subsequent infections were associated with diarrhea only in children aged less than 6 months. This observation that natural infection did not confer protection during the first 6 months of life poses a challenge to vaccine development.

The interrelationship of malnutrition and diarrhea in a periurban area outside Alexandria, Egypt.

BACKGROUND: In the developing world, children are often observed to have both diarrhea and malnutrition. This observation has led many researchers to speculate that diarrhea may produce malnutrition and that malnutrition may predispose to diarrhea. In this study, the interrelationship between diarrhea and malnutrition was investigated among 143 Egyptian children less than 3 years of age. METHODS: For 22 months, children were followed for diarrhea at twice weekly home visits and measured for nutritional status at approximately 3-month intervals. Nutritional measurements were converted to z-scores based on the National Center for Health Statistics/World Health Organization (NCHS/WHO) reference population. RESULTS: Three hundred fifty-eight diarrheal episodes were reported with only 1% of episodes lasting 14 days or more. Stunting, wasting, and low weight-for-age were found in 19%, 3%, and 7%, of these children, respectively. When testing whether malnutrition predisposes to diarrhea, a weight-for-age z-score of <-2 standard deviations was associated with increased incidence of diarrhea (RR = 1.7, P < 0.01) but not height-for-age or weight-for-height. Diarrhea itself was associated with a subsequent attack of diarrhea (RR = 2.1, P < 0.001). During short intervals of follow-up (approximately 3 months), an association was detected between diarrhea episodes and growth faltering for height-for-age z-score (-0.16, P < 0.05). This association was reduced, however, when analyzed during 6-month intervals, if no diarrhea was reported in either the first or second half of this interval. CONCLUSIONS: In a population with moderate malnutrition, both low weight-for-age and diarrhea itself are associated with increased diarrhea risk. Diarrhea alone does not appear to contribute substantially to malnutrition when children have diarrhea-free time for catch-up growth.

Induction of systemic antifimbria and antitoxin antibody responses in Egyptian children and adults by an oral, killed enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine.

We assessed serologic responses to an oral, killed whole-cell enterotoxigenic Escherichia coli plus cholera toxin B-subunit (ETEC-rCTB) vaccine in 73 Egyptian adults, 105 schoolchildren, and 93 preschool children. Each subject received two doses of vaccine or placebo 2 weeks apart, giving blood before immunization and 7 days after each dose. Plasma antibodies to rCTB and four vaccine-shared colonization factors (CFs) were measured by enzyme-linked immunosorbent assay. Immunoglobulin A (IgA) antibodies to rCTB and CFA/I were measured in all subjects, and those against CS1, CS2, and CS4 were measured in all children plus a subset of 33 adults. IgG antibodies to these five antigens were measured in a subset of 30 to 33 subjects in each cohort. Seroconversion was defined as a >2-fold increase in titer after vaccination. IgA and IgG seroconversion to rCTB was observed in 94 to 95% of adult vaccinees, with titer increases as robust as those previously reported for these two pediatric cohorts. The proportion showing IgA seroconversion to each CF antigen among vaccinated children (range, 70 to 96%) and adults (31 to 69%), as well as IgG seroconversion in children (44 to 75%) and adults (25 to 81%), was significantly higher than the corresponding proportion in placebo recipients, except for IgA responses to CS2 in adults. IgA anti-CF titers peaked after one dose in children, whereas in all age groups IgG antibodies rose incrementally after each dose. Independently, both preimmunization IgA titer and age were inversely related to the magnitude of IgA responses. In conclusion, serologic responses to the ETEC-rCTB vaccine may serve as practical immune outcome measures in future pediatric trials in areas where ETEC is endemic.

Seroepidemiology of Helicobacter pylori infection in a population of Egyptian children.

BACKGROUND: To describe the seroepidemiology of Helicobacter pylori infection in a population of Egyptian children under 3 years. METHODS: A cohort of children under 36 months, residing in Abu Homos, Egypt, were visited at home twice weekly. Information regarding the child's breastfeeding status was obtained, and periodic anthropometric and household hygiene surveys were performed. In June 1997, a serosurvey was conducted on 187 study participants over 6 months old. The serosurvey was repeated in October 1997. All sera were tested for IgG antibodies to H. pylori. RESULTS: The June prevalence of H. pylori infection was 10%, and the incidence from June to October was 15%. Between June and October, 8 (42%) of 19 children that were positive for H. pylori infection seroreverted to negative. All seroreversions occurred in children 6-17 months. Other than age, no sociodemographic or environmental factor was significantly associated with incident H. pylori infection. There was no significant differences in the weight-for-age, weight-for-height, and height-for-age z-scores between children with and without prevalent H. pylori infection. CONCLUSIONS: Infection with H. pylori is common in Egyptian children under 3 years old and is not associated with malnutrition. No predictors for H. pylori infection were found. Our preliminary evidence for transient H. pylori infections in young children needs to be confirmed in a prospective cohort study, and predictors for persistent infection should be sought, since only these may be relevant to the known sequellae of infection.

IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene.

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene. In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.

Astrovirus diarrhea in Egyptian children.

This study describes the epidemiology of astrovirus diarrhea among a population-based cohort of 397 children aged <3 years residing in rural Egypt from 1995 to 1998. The age-specific incidence rates of astrovirus diarrheal episodes per person-year were 0.38 for infants aged <6 months, 0.40 for those aged 6-11 months, 0.16 for those aged 12-23 months, and 0.05 for those aged 24-35 months. The overall incidence rate of astrovirus diarrhea was the same as that of rotavirus diarrhea, 0.19 episodes per person-year. Astrovirus infection was pathogenic and associated with severe dehydration in 17% of the cases. The most frequent serotype was HAstV-1, and, in order of decreasing frequency, HAstV-5, HAstV-8 and HAstV-3, HAstV-6, HAstV-4, and HAstV-2. In determining whether astrovirus diarrhea was associated with a reduced incidence of subsequent disease, there was evidence to suggest HAstV-1 homotypic immunity but not heterotypic immunity. Because we observed 38% of the incidence of astrovirus diarrhea to occur in infants aged <6 months, a candidate astrovirus vaccine would have to confer immunity very early in life.

Epidemiology of rotavirus diarrhea in Egyptian children and implications for disease control.

Reliable epidemiologic data are essential for formulating effective policy to control rotavirus disease through immunization. The objective of this study was to describe the epidemiology of rotavirus diarrhea in a population-based cohort of children under 3 years of age residing in Abu Homos, Egypt, in 1995-1996. Rotavirus diarrhea incidence rates (episodes per person-year) were 0.13 for infants aged <6 months, 0.61 for those aged 6-11 months, 0.17 for those aged 12-23 months, and 0.15 for those aged 24-35 months. Fifty-six percent of children with rotavirus diarrhea had clinical dehydration; 90% of rotavirus diarrheal episodes occurred between July and November. In infants under 1 year of age, receipt of breast milk was associated with a lower incidence of rotavirus diarrhea. No other sociodemographic or environmental factor was found to be significantly associated with rotavirus diarrhea. Of 46 rotavirus isolates with strains identified, 41 (89%) were G serotypes 1 and 2. Rotavirus diarrhea was a major cause of morbidity in this cohort. Promotion of breastfeeding may exert a protective effect in young infants in this setting, but improvements in water and sanitation are unlikely to be effective preventive measures. The use of effective immunization against rotavirus in early infancy should be considered a public health priority.

PIP: This study describes the epidemiology of rotavirus diarrhea in a population-based cohort of children under 3 years of age residing in Abu Homos, Egypt, during 1995-96. Samples consisted of a cohort of children under the age of 24 months assembled from two villages in the vicinity of Abu Homos. The age-specific incidence rates of rotavirus diarrheal episodes per person-year were 0.13 for infants aged 6 months, 0.61 for those aged 6-11 months, 0.17 for those aged 12-23 months, and 0.15 for those aged 24-35 months. No rotavirus diarrheal incidence occurred in infants under 20 weeks of age. The monthly incidence rates of rotavirus diarrhea demonstrate that 90% of the disease episodes occurred during the warmer months of July-November, with a peak incidence in August. In infants under 1 year of age, breast-feeding was associated with a lower incidence of rotavirus diarrhea. Promotion of breast-feeding may employ a protective effect in young infants in this setting, but improvements in water and sanitation are unlikely to be effective preventive measures.

Characterization of unusual G8 rotavirus strains isolated from Egyptian children.

We report the first detection of P[14], G8 rotaviruses isolated in Egypt from the stool of children participating in a 3 year study of rotavirus epidemiology. Two strains, EGY1850 and EGY2295, were characterized by a serotyping enzyme immunoassay (EIA), virus neutralization, and sequence analysis of the genes encoding VP7 and the VP8* portion of the VP4 gene. These two strains shared a high level of homology of their VP7s (87.8% nucleotide [nt], 97.2% amino acid [aa]) and VP4s (89.6% nt, 97.1% aa) and had the highest VP7 identity to serotype G8 (> 82% nt, > 92% aa) and VP4 identity to genotype P[14] (> or = 81% nt, > 91% aa) strains. Serological results with a VP7 G8-specific and VP4 P[14]-specific neutralizing monoclonal antibodies supported the genetic classification of EGY1850 and EGY2295 as P[14], G8. Genogroup analysis supports earlier findings that human G8 rotaviruses may be genetically related to bovine rotaviruses. These findings demonstrate that our understanding of the geographic distribution of rotavirus strains is incomplete, emphasize the need to monitor rotavirus serotypes, and extend the known distribution of serotype G8 and genotype P[14] strains in Africa.

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.

Characterization of an enterotoxigenic Escherichia coli strain from Africa expressing a putative colonization factor.

An enterotoxigenic Escherichia coli (ETEC) strain of serotype O114:H- that expressed both heat-labile and heat-stable enterotoxins and tested negative for colonization factors (CF) was isolated from a child with diarrhea in Egypt. This strain, WS0115A, induced hemagglutination of bovine erythrocytes and adhered to the enterocyte-like cell line Caco-2, suggesting that it may elaborate novel fimbriae. Surface-expressed antigen purified by differential ammonium sulfate precipitation and column chromatography yielded a single protein band with M(r) 14,800 when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (16% polyacrylamide). A monoclonal antibody against this putative fimbrial antigen was generated and reacted with strain WS0115A and also with CS1-, CS17-, and CS19-positive strains in a dot blot assay. Reactivity was temperature dependent, with cells displaying reactivity when grown at 37 degrees C but not when grown at 22 degrees C. Immunoblot analysis of a fimbrial preparation from strain WS0115A showed that the monoclonal antibody reacted with a single protein band. Electron microscopy and immunoelectron microscopy revealed fimbria-like structures on the surface of strain WS0115A. These structures were rigid and measured 6.8 to 7.4 nm in diameter. Electrospray mass-spectrometric analysis showed that the mass of the purified fimbria was 14,965 Da. The N-terminal sequence of the fimbria established that it was a member of the CFA/I family, with sequence identity to the amino terminus of CS19, a new CF recently identified in India. Cumulatively, our results suggest that this fimbria is CS19. Screening of a collection of ETEC strains isolated from children with diarrhea in Egypt found that 4.2% of strains originally reported as CF negative were positive for this CF, suggesting that it is biologically relevant in the pathogenesis of ETEC.

Identification of Shigella flexneri subserotype 1c in rural Egypt.

In a population-based study of diarrhea in rural, northern Egypt, 60 Shigella flexneri strains were identified, of which 10 could not be definitively serotyped. Serological analysis with commercial reagents suggested that they were serotype 1, but the strains failed to react with subserotype 1a- or 1b-specific antibodies. All 10 strains reacted with MASF 1c, a monoclonal antibody specific for a provisional S. flexneri subserotype, 1c, first identified in Bangladesh and not previously detected outside of that region. Our results show that S. flexneri subserotype 1c is not unique to Bangladesh and that the inability to detect it may reflect both the limited use of suitable screening methods and the rarity of this subserotype.

Epidemiology of enterotoxigenic Escherichia coli diarrhea in a pediatric cohort in a periurban area of lower Egypt.

Enterotoxigenic Escherichia coli (ETEC) are diverse pathogens that express heat-labile (LT) and/or heat-stable (ST) enterotoxins, yet little is known about whether epidemiologic patterns of pediatric ETEC diarrhea vary by the expressed ETEC toxin phenotype. In total, 242 Egyptian children aged <3 years were prospectively followed in 1993-1995. ETEC episodes were detected during twice-weekly home visits, and asymptomatic ETEC excretion was identified from monthly cross-sectional surveys. ETEC episodes were 0.6 per child-year. ST-only ETEC was 2.6 times (P<.001) more common in warmer than cooler months, while LT-only ETEC showed no seasonal variation. Ownership of a household sanitary latrine, but not breast-feeding, was associated with a lower risk of both enterotoxin phenotypes. Coexpression of a colonization factor by LT- or ST-only ETEC strengthened the association with diarrhea. These findings indicate that the epidemiologic patterns of LT-only and ST-only ETEC are not identical and that disease interventions should include improved household sanitation.

Oral, inactivated, whole cell enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine: results of the initial evaluation in children. PRIDE Study Group.

Two randomized, double-blinded trials assessed the safety and immunogenicity of an oral, killed enterotoxigenic Escherichia coli (ETEC) plus cholera toxin B subunit vaccine in Egyptian children. Two doses of vaccine or E. coli K-12 were given 2 weeks apart to 105 6- to 12-year-olds and 97 2- to 5-year-olds. Safety was monitored for 3 days after each dose. Blood was collected before immunization and 7 days after each dose to measure immune responses. Few children reported postdosing symptoms, with no differences in the frequency of symptoms between treatment groups. Most vaccinees had an IgA antibody-secreting cell response against colonization factor antigen I (100%, 6-12 years; 95%, 2-5 years), coli surface antigen 2 (92%, 6-12 years; 83%, 2-5 years), and coli surface antigen 4 (93%, 6-12 years). Vaccination evoked a >/=4-fold rise in antitoxic IgA and IgG titers in 93% and 81% of children, respectively. In conclusion, the oral ETEC vaccine was safe and immunogenic in 2- to 12-year-old children, justifying further evaluation in infants.

Safety and immunogenicity of an oral, killed enterotoxigenic Escherichia coli-cholera toxin B subunit vaccine in Egyptian adults.

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of bacterial diarrhea in young children in developing countries. The safety and immunogenicity of a killed, oral ETEC vaccine consisting of whole cells plus recombinantly produced cholera toxin B subunit (rCTB) was evaluated in Egypt, which is endemic for ETEC diarrhea. Seventy-four healthy Egyptian adults (21-45 years old) were randomized and received two doses of the ETEC/rCTB vaccine (E003) or placebo 2 weeks apart. The frequency of adverse events after either dose did not differ by treatment group, and no severe adverse events were reported. After vaccination, peripheral blood IgA B cell responses to CTB (100%) and to vaccine colonization factor antigens CFA/I (94%), CS4 (100%), CS2 (81%), and CS1 (69%) were significantly higher than response rates for the placebo group. These favorable results in Egyptian adults indicate that the ETEC/rCTB vaccine is a promising candidate for evaluation in younger age groups in this setting.

An outbreak of foodborne illness caused by Escherichia coli O39:NM, an agent not fitting into the existing scheme for classifying diarrheogenic E. coli.

An outbreak of gastrointestinal illness with clinical and epidemiologic features of enterotoxigenic Escherichia coli (ETEC) occurred among patrons of a restaurant during April 1991. Illnesses among several groups of patrons were characterized by diarrhea (100%) and cramps (79%-88%) lasting a median of 3-5 days. Median incubation periods ranged from 50 to 56 h. A nonmotile strain of E. coli (E. coli O39), which was negative for heat-labile (LT) and heat-stable (STa, STb) ETEC toxins, was isolated only from ill patrons. This organism produced enteroaggregative E. coli heat-stable enterotoxin 1 and contained the enteropathogenic E. coli gene locus for enterocyte effacement; it did not display mannose-resistant adherence, but produced attaching and effacing lesions in the absence of mannose on cultured HEp-2 cells. E. coli that are not part of highly characterized but narrowly defined groups may be important causes of foodborne illness.

Enteroaggregative Escherichia coli heat-stable enterotoxin is not restricted to enteroaggregative E. coli.

Enteroaggregative Escherichia coli (EAggEC) have been implicated as diarrheal pathogens in several settings. Some EAggEC produce a distinct heat-stable enterotoxin named EAST1. The distribution and prevalence of the EAST1 gene in selected groups of bacterial enteropathogens were determined by colony hybridization. One hundred percent of 75 O157:H7 enterohemorrhagic E. coli (EHEC), 41% of 227 EAggEC, 41% of 149 enterotoxigenic E. coli, 22% of 65 enteropathogenic E. coli (EPEC), and 38% of 47 E. coli stool isolates from asymptomatic children hybridized with an EAST1 DNA probe. None of 55 enteroinvasive E. coli, 12 Yersinia enterocolitica, or 20 Vibrio cholerae non-O1 strains were EAST1 probe-positive. Concordance between EAST1 genotype and enterotoxicity was shown in examined strains of EAggEC, EHEC, and EPEC. The gene encoding EAST1 is more broadly distributed among diarrheogenic E. coli than previously known and may represent an additional determinant in the pathogenesis of E. coli diarrhea.

Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers.

Enteroaggregative Escherichia coli (EAggEC) are diarrheal pathogens defined by aggregative adherence to HEp-2 cells. In an effort to identify pathogenic EAggEC isolates, four groups of 5 volunteers were fed 1 of 4 different EAggEC strains, each at a dose of 10(10) cfu. Strain 042 caused diarrhea in 3 of 5 adults; 3 other EAggEC isolates (17-2, 34b, and JM221) failed to elicit diarrhea. A gene encoding enterotoxin EAST1 was found in strains 042 and 17-2 but not 34b or JM221; a 108-kDa cytotoxin was expressed in all 4 isolates. All 4 isolates showed a modest degree of gentamicin protection in HEp-2 cells. 17-2, 34b, and JM221 expressed the fimbrial antigen AAF/I; 042 did not express this fimbria as determined by immunogold electron microscopy and genetic probe hybridization.