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.

Enterotoxigenic Escherichia coli colonization factor types collected from 1997 to 2001 in US military personnel during operation Bright Star in northern Egypt.

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.

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.

Phenotypic diversity of enterotoxigenic Escherichia coli (ETEC) isolated from cases of travelers' diarrhea in Kenya.

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.