chi-square test was used. ... EAggEC isolates were tested forantimicrobial suscepti- bility byagar .... Comparison of DNA probes and the Sereny test for identifica-.
Vol. 31, No. 2
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1993, p. 351-353
0095-1137/93/020351-03$02.00/0 Copyright X 1993, American Society for Microbiology
Colonization by Enteroaggregative Escherichia coli in Travelers with and without Diarrhea MITCHELL B. COHEN,l,2* JENNIFER A. HAWKINS,1 LANA S. WECKBACH,3 JOSEPH L. STANECK,3 MYRON M. LEVINE,4 AND JEFFERY E. HECK5 Division of Gastroenterology and Nutrition, Department of Pediatrics, Children's Hospital Medical Center, Cincinnati, Ohio 452291; Division of Digestive Diseases, Department of Medicine,2 and Departments of Pathology and Laboratory Medicine3 and Family Medicine,S University of Cincinnati Cincinnati, Ohio 45267, and Center for Vaccine Development, University of Maryland, Baltimore, Maryland 212012 Received 27 August 1992/Accepted 16 November 1992
Enteroaggregative Escherichia coli (EAggEC) has been found to be associated with pediatric diarrhea in developing countries. In order to determine the role of EAggEC as an agent of traveler's diarrhea, we used a sensitive and specific DNA probe for EAggEC to screen bacterial colony blots from 278 volunteers before and after travel. Colonization with EAggEC was infrequent (2.5%) prior to travel but rose to 27 to 33% after travel in volunteers who took either placebo or trimethoprim-sulfamethoxazole. Travelers who took trimethoprimsulfamethoxazole were colonized with organisms that were uniformly resistant to that antimicrobial agent; when volunteers received ciprofloxacin, colonization with EAggEC was prevented (2.0%o). Although colonization rates were high in the placebo and trimethoprim-sulfamethoxazole groups, only a minority of travelers who were colonized with EAggEC experienced diarrhea. On the basis of our data, we suggest that colonization with EAggEC alone is not sufficient to cause traveler's diarrhea. dose of trimethoprim-sulfamethoxazole (TMP-SMZ; 160/800 mg), ciprofloxacin (500 mg), or placebo beginning on the day of arrival at their destination and continuing for up to 2 weeks thereafter. Stool samples were obtained prior to travel and during the trip if diarrhea occurred. If diarrhea did not occur, a stool sample was obtained within 10 days of the subject's return. Traveler's diarrhea was strictly defined as three or more unformed stools in 8 h accompanied by one or more of the following signs and symptoms of enteric infection: nausea, vomiting, abdominal cramps, or fever of >1000F (37.80C). Travelers who experienced either (i) two loose stools in 8 h and one or more signs or symptoms of enteric infection or (ii) three or more loose stools in 8 to 24 h with or without other signs or symptoms of enteric infection were termed to have "equivocal" diarrhea. Of 344 subjects who enrolled in the study, 278 (81%) completed the study and were included in the data analysis. Preparation of the DNA probes and colony blots. Five E. coli isolates from each patient were fixed on Whatman 541 filters and were hybridized under stringent conditions with a 32P-labeled 1.0-kb EcoRI-PstI DNA fragment from pCVD432 as described previously (2). In each hybridization reaction, E. coli strains were included as positive (strain 17-2) and negative (ATCC 25922) controls. The sensitivity of this EAggEC probe has previously been reported to be 89% and the specificity has been reported to be 99% (2). These same isolates were also examined by gene probe assays for EPEC, ETEC, EIEC, and EHEC. The EPEC adherence factor probe was a 1-kb SalI-BamHI fragment of pJPN16 derived from strain E2348/69 (20). For detection of ETEC, three separate probes were used. (i) The LT probe was a 1.2-kb HincII fragment removed from pEWD299 and cloned on BamHI linkers into pACYC184 (pCVD403) (7, 9, 19). (ii) The STp probe was a 157-bp PstI fragment of pRIT10036 cloned into pUC13 (pCVD426) (19). (iii) The STh probe was a 216-bp EcoRI fragment of pSLM004 cloned into pUC13 (pCVD427) (19). The EIEC probe was a 2.5-kb HindIII
Four categories of diarrheagenic Escherichia coli are now clearly recognized: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC) (16). In addition, by using a particular technique, certain isolates of E. coli from patients with diarrhea have been shown to adhere to HEp-2 or HeLa cells with a localized, diffuse, or aggregative pattern (21). Localized adherence is highly associated with EPEC strains. Organisms that demonstrate diffuse adherence (10, 13) and aggregative adherence (enteroaggregative E. coli [EAggEC]) (3, 4, 6, 17, 24) have both been associated with diarrheal disease. However, the relationship between these non-EPEC adherent organisms and the development of diarrheal disease remains uncertain. Although EAggEC has been epidemiologically associated with infantile diarrhea in rural Chile (17) and with persistent diarrhea in children in India (4), Mexico (6), and Brazil (24), EAggEC was isolated with a similar frequency from patients with acute diarrhea and controls in studies in Brazil (11), Mexico (10), and Thailand (8). In order to determine the possible significance of EAggEC as an agent of traveler's diarrhea, we used a sensitive and specific DNA probe (2) to screen bacterial colony blots from travelers with and without diarrhea before and after travel.
MATERIALS AND METHODS Patient population. Patients were recruited from healthy adult volunteers planning travel of .2 weeks in duration to Central or South America, the Caribbean, or Mexico. The present study was part of a double-blind, randomized, placebo-controlled study on the efficacy of antibiotic prophylaxis for traveler's diarrhea (14). The study protocol was approved by the Institutional Review Board of the University of Cincinnati. Patients were treated with a single daily *
Corresponding author. 351
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COHEN ET AL. TABLE 1. Isolation of EAggEC No. (%) of EAggEC:
Antimicrobial agent
Placebo Ciprofloxacin TMP-SMZ aP< bp
