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Operation Bright Star (OBS) is a biennial, multinational exercise in Egypt involving 15000 US troops. Consistent with past observations in deployed troops, diarrhea is the most significant cause of morbidity. Focused efforts are ongoing to develop vaccines against the most common pathogens affecting our troops. As part of these efforts, diarrhea surveillance was conducted during OBS to monitor pathogens associated with illness and to identify new vaccine targets. A retrospective review was conducted of prior studies with similar methods. Soldiers with diarrhea presenting to the OBS clinic provided a stool sample that was inoculated into Carey-Blair transport media. Within 3 days, the Cary-Blair tubes were transported to the Naval Medical Research Unit no. 3 in Cairo where bacterial culture was performed. As part of the evaluation, 5 Escherichia coli-like colonies were collected and tested for toxin production using the GM1-ELISA. Toxin-positive isolates were further tested for colonization factors (CF) by a dot-blot assay using a standardized panel of monoclonal antibodies against CFA/I, CS1-CS7, CS17, CS8 (CFA/III), CS12 (PCFO159), and CS14 (PCFO166). Enterotoxigenic E. coli (ETEC) was the most frequently isolated pathogen during each OBS from which data were collected. The rate of ETEC-associated diarrhea ranged from 22% to 58%. Over time, there were dramatic shifts in the frequency and distribution of CFs. Over the 5 years of study, an increasing number of ETEC isolates had no known CF identified, and in 2001, only 40% of ETEC was associated with known CFs. The most commonly identified CF was CS6. Diarrheal disease, particularly ETEC, continues to be a common malady among US military personnel deployed to Egypt. We have identified ETEC CF types, especially CS6, which should be considered potential vaccine candidates. However, despite intensive testing, CFs could not be identified in most of the ETEC isolated, highlighting the need for further studies to identify novel CFs and alternative vaccine targets.

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.

Background: The aim of this study was to characterize phenotypically enterotoxins, colonization factors (CFs) and the antibiotic susceptibility of enterotoxigenic Escherichia coli (ETEC) strains isolated from cases of acute diarrhea that occurred in Europeans traveling to resorts in Mombasa, Kenya; this information is critical for the development of vaccines and empirical treatment., Methods: Over a 1-year period from 1996 to 1997, five E. coli-like colonies were obtained from each of 463 cases with acute diarrhea. These strains were characterized for enterotoxins using GM-1 ELISA, for CFs using a dot-blot assay, and for antibiotic susceptibility using antibiotic disks., Results: Of 164 strains characterized for ETEC phenotype, 30 (18%) expressed heat-labile toxin (LT) only, 83 (51%) heat-stable toxin (ST) only, and 51 (31%) both LT and ST. Analysis for CF expression demonstrated that 107 (65%) of the strains were positive for CFs, including CFA/IV (46%), CFA/II (35%), and CFA/I (5%), while less than 4% expressed less common CFs. All ETEC strains tested were resistant to erythromycin and sensitive to ceftriaxone. Over one-third of the strains were resistant to sulfamethoxazole-trimethoprim or tetracycline. Six strains were resistant to nalidixic acid; none of these were resistant to ciprofloxacin., Conclusions: Cumulatively, our findings indicate that ETEC in this region comprises a highly diverse group of bacterial enteropathogens, and that the development of prophylactic agents against ETEC faces major challenges because of this diversity.

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.

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.

Three oligonucleotide primers were used in a polymerase chain reaction (PCR) assay for the simultaneous amplification of regions of the invasive plasmid antigen (ipaH) of Shigella spp., flagellin gene (flaA) of Campylobacter spp., and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC). The multiplex assay was performed using DNA extracted by a chaotropic method directly from diarrhoeal stools. The diagnostic efficacy of the assay was analyzed by agarose gel electrophoresis. This assay shows a novel approach for the diagnosis of diarrhoea caused by Shigella spp., ETEC, and Campylobacter spp.

The Escherichia coli heat shock regulon consists of approximately twenty polypeptides that are coordinately and transiently induced upon a temperature upshift under the control of an alternative sigma factor, designated sigma-32 or HtpR. Preliminary observations on one of the proteins of the heat shock response, protein D 48.5, suggested that its induction by sigma-32 during heat shock may be modulated by catabolite repression. In this study, a disk diffusion assay was used to screen the effect of several compounds on the expression of a lacZ fusion in the gene encoding protein D 48.5. This assay indicated that the expression of this protein was controlled, at least in part, by the catabolite repression response. A more indepth analysis of the expression of protein D 48.5 under both steady-state and heat shock conditions was conducted in both a wild type and cya crp background. This analysis revealed that in the cya crp background, the steady-state level of protein D 48.5 was elevated relative to the wildtype, but that the heat shock induction of the protein was reduced in magnitude relative to the wild type strain, suggesting a direct link between these two global responses. The lacZ fusion in the structural gene for protein D 48.5 should prove useful as a reporter mechanism to probe the physiology and regulation of the heat shock response.

Protein D48.5 was recognized as a heat-inducible protein of Escherichia coli during the screening of a group of random, temperature-inducible Mud-Lac fusion mutants. Physiological and genetic analysis demonstrated that (i) the structural gene for this protein, designated htpI, is a member of the sigma 32-dependent heat shock regulon, (ii) at 37 degrees C the synthesis of protein D48.5 is nearly constitutive, increasing slightly with growth rate in media of different composition, and (iii) this protein is essential for growth at high temperature.