[Emerging Infectious Diseases * Volume 3 * Number 3 * July-September 1997] [Dispatches] Multidrug-Resistant Enteroaggregative Escherichia coli Associated with Persistent Diarrhea in Kenyan Children --------------------------------------------------------------------------- To study the association of multidrug-resistant enteroaggregative Escherichia coli with persistent diarrhea in Kenyan children, stool specimens were obtained from 862 outpatients under 5 years of age from July 1991 to June 1993. E. coli O44 was identified as the sole bacterial pathogen in four patients experiencing at least 14 days of fever, vomiting, and diarrhea. Disk diffusion testing showed E. coli O44 resistance to tetracycline, ampicillin, erythromycin, trimethoprim-sulphamethoxazole, and amoxicillin/clavulanate and sensitivity to chloramphenicol, nalidixic acid, azithromycin, and cefuroxime. Further studies are needed to clarify the epidemiology, clinical spectrum, and pathogenesis of enteroaggregative E. coli infection. Escherichia coli infection is an important cause of illness and death in infants in developing countries (1). On the basis of patterns of adherence to tissue culture cells (HEp-2 or HeLa), E. coli strains can be classified into three groups: localized, diffuse, and aggregative (2). Much remains unknown about these strains. Enteroaggregative E. coli (EAggEC), which exhibits aggregative adherence, has been associated with diarrhea in children in Chile (3) and with persistent diarrhea in children in India (4). We report the first evidence of multidrug-resistant EAggEC associated with persistent diarrhea in Kenyan children. From July 1991 to June 1993, stool specimens from 862 outpatients under 5 years of age at Malindi Hospital were examined for pathogenic organisms. Standard methods for isolating enteric pathogens were used. Laboratory tests to detect pathogenic factors, e.g., verotoxins (VT) in cultures of all E. coli isolates, were done by applying the conventional tissue culture method (which uses the Vero cell line [5] and the VT1 or VT2 genes [6]) and polymerase chain reaction (7). The genes for heat-labile enterotoxin and heat-stable enterotoxin, VT, and invasiveness were tested by DNA probes on all E. coli strains. The strains were further examined for adherence to HEp-2 cells (8) and tested by the disk diffusion method (9) for susceptibility to the antibiotics chloramphenicol, erythromycin, ampicillin, nalidixic acid, cefuroxime, trimethoprim-sulphamethoxazole, amoxicillin/clavulanate, tetracycline, and azithromycin. Bacterial pathogens were found in 27.7% of the samples; 119 E. coli isolates were obtained. The results indicated that many of the bacteria, e.g., pathogenic E. coli, Salmonella spp., and Shigella spp. (Table), had been transmitted by the fecal-oral route. E. coli O44 was isolated from four patients; the isolates occurred in an aggregative adherence pattern as chains and nearly random aggregates on HEp-2 cells. Table. Identification of enteric pathogens in children with diarrhea Pathogens (number tested) Number Percentage (%) Bacteria (862) 239 28.0 Escherichia coli enteropathogenic E. coli (EPEC) 71 8.0 EAgg E. coli (ETEC) 4 0.5 enterotoxigenic E. coli 43 5.0 enterohemorrhagic E. coli 1 0.1 Salmonella spp. 63 7.3 Shigella spp. 56 6.5 Campylobacter spp. 42 4.9 Vibrio parahaemolyticus 4 0.5 Parasites (862) 109 12.6 Entamoeba histolytica 67 7.8 Giardia lamblia 42 4.9 Viruses (427) Rotavirus 69 16.2 ------------------------------------------------- Mixed infections: ETEC/EPEC = 12, EPEC/Campylobacter = 8, Salmonella/ETEC = 7, Salmonella/EPEC = 3, Shigella/Campylobacter = 2, Vibrio/Shigella = 2, Shigella/Salmonella = 1, EPEC /Shigella = 6, ETEC/Shigella = 4. ------------------------------------------------- The case descriptions of the four patients from whom the O44 strains were isolated are as follows: Patient 1, age 28 months, had fever, gross blood in stool, vomiting, and diarrhea lasting 14 days; Patient 2, age 35 months, had fever, abdominal pain, nausea, vomiting, and diarrhea lasting 15 days; Patient 3, age 24 months, had fever, gross blood in stool, vomiting, and diarrhea lasting 14 days; and Patient 4, age 26 months, had fever, abdominal pain, vomiting, and diarrhea lasting 14 days. The patients were not related and lived in different communities. These particular strains of E. coli O44 had similar patterns of resistance to tetracycline, ampicillin, erythromycin, trimethoprim-sulphamethoxazole, and amoxicillin/clavulanate; they were all sensitive to chloramphenicol, nalidixic acid, azithromycin, and cefuroxime. Persistent diarrhea is increasingly recognized as an important public health problem among children in developing countries (10) and is a research priority of the Diarrhoeal Diseases Control Programme of the World Health Organization (11). In the four patients from whom it was isolated, EAggEC was the sole bacterial pathogen recovered. However, tests for parasitic causes of persistent diarrhea, such as Cyclospora and Cryptosporidium, were not available at the time of our study and were not performed. The association of EAggEC with persistent diarrhea will be strengthened by extending this kind of study to other areas in Kenya and identifying all causes of persistent diarrhea. Tetracycline, ampillicin, and trimethoprim-sulphamethoxazole are recommended by the Kenyan Ministry of Health for the empiric treatment of diarrhea. These drugs were largely ineffective against Shigella spp. and EAggEC. Our results are consistent with the findings of Yamamoto et al., who found multidrug resistance in EAggEC strains from Thailand, Mexico, Chile, and Peru (12), and suggest that monitoring sensitivity to antibiotics in Kenya is necessary for optimum selection of effective antibiotics and elimination of antibiotics with little therapeutic value. Similarly, the epidemiology, clinical spectrum, and pathogenesis of EAggEC infection and the reservoir of the putative etiologic agent are still poorly understood or unknown. Further clinical, epidemiologic, and laboratory studies are needed to clarify these issues. References 1. Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic and enteroadherent. J Infect Dis 1987;15:377-89. 2. Scaletsky ICA, Silva MLM, Trabulsi LR. Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect Immun 1984;45:534-6. 3. Nataro JP, Kaper JB. Patterns of adherence of diarrhaegenic Escherichia coli to HEp-2 cells. Pediatr Infec Dis J 1987;6:829-31. 4. Bhan MK, Raj P, Levine MM. Enteroaggregative Escherichia coli association with persistent diarrhea in a cohort of rural children in India. J Infect Dis 1989;159:1061-4. 5. Konowalchuk J, Speirs JI, Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun 1977;18:775-9. 6. Tomatsukuri S, Yamamoto K, Shibata S, Leaneo F, Honda T. Detection of a heat-labile enterotoxin gene in enterotoxigenic Escherichia coli by densitometric evaluation using highly specific enzyme-linked oligonucleotide probes. Eur J Clin Microbiol Infect Dis 1991;10:1048-55. 7. Pollard DR, Johnson WM, Lior H, Tyler SD, Rozee KR. Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction. J Clin Microbiol 1990;28:540-5. 8. Cravioto AR, Gross J, Scotland SM, et al. An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenic serotype. Current Microbiology 1979;3:95-9. 9. Bauer AW, Kirby WMM, Sherris JC, Turk M. Antibiotic susceptibility testing by a standardized single disc method. Am J Clin Pathol 1966;4:493-6. 10. Bhan MK, Arora NK, Ghai OP, Ramachandran K. Major factors in diarrhoea related mortality among rural children. Indian J Med Res 1986;83:9-12. 11. World Health Organization: Diarrhoeal Disease Control Programme: persistent diarrhoea in children - research priorities. Geneva: World Health Organization; 1985; CDD/DDM/85.1. 12. Yamamoto T, Echeverria P, Yokota T. Drug resistance and adherence to human intestines of enteroaggregative Escherichia coli. J Infect Dis 1992;165:744-9. --------------------------------------------------------------------------- Emerging Infectious Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA URL: ftp://ftp.cdc.gov/pub/EID/vol3no3/ascii/sang.txt