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Escherichia coli is a common member of the normal flora of the human intestine. Strains that acquire bacteriophage or plasmid DNA encoding enterotoxins or invasion factors become virulent and can cause either a plain, watery diarrhea or inflammatory dysentery. Diarrheagenic E. coli is now classified into five groups: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and Shiga toxin-producing E. coli (STEC). The STEC is more important than the others because infections may result in life-threatening sequelae, such as hemolytic uremic-syndrome (HUS) in humans. Among STEC, seropathotype A (STEC O157:H7, O157:NM) is the most important because of its incidence, involvement in outbreaks, and association with severe disease. Seropatho-type A STEC is widely distributed in the world, but in some countries, the presence of the organisms and human infection have not been reported. The objective of this study was to elucidate the level of STEC contamination in animals in Vietnam where seropathotype A STEC has not yet been reported.
Fecal samples from 100 cows at a stock farm in a suburb of Hanoi were collected in sterilized containers, and laboratory analysis started within one hour after sampling. A gram of stool was inoculated into 10 mL of modified EC broth (Difco Laboratories, Detroit, MI) containing 20 mg/L of novobiocin and incubated at 37°C for 15 hours. This enriched culture was streaked on a sorbitol-MacConkey (Difco Laboratories) agar plates for isolation of colonies. After overnight incubation at 37°C, five sorbitol-fermenting and colonies and five sorbitol non-fermenting colonies were examined. The organisms identified as E. coli by standard biochemical tests were again inoculated into Luria Bertani (LB) broth and incubated at overnight at 37°C. The organisms cultured in LB broth were examined by polymerase chain reaction (PCR) to detect diarrhea-associated genes. A multiplex PCR was used as previously reported.1 The target genes analyzed were stx for STEC, eae for EPEC, elt/est for ETEC, ipaH for EIEC, and aggR for EAEC.
Among the 100 stool samples, no amplicons for elt, est, ipaH, and aggR were obtained. An amplicon for stx was obtained in eight samples, of which two were also positive for eae. The serotype of the two strains with both stx and eae was O157:H7. The serotypes of the other six strains carrying only stx could not be determined with 43 commercially available anti-sera (Denka, Seiken, Japan). Flagellar gene fliC types were determined by restriction fragment length polymorphism analysis of PCR-amplified fliC gene according to the method of Machado and others.2 The analysis of fliC identified types 8b, 39, 16, 25b, and 19a, which correspond to H8, H39, H16, H25, and H19, respectively. Further characterization of the eight stx-positive strains was performed by investigating the presence of stx1, stx2 variant types, eae, ehxA, sfpA, saa, iha, toxB, and efa1. The primers described by Toma and others3 were used to detect these genes. Two O157:H7 strains had the stx2c variant, eae, ehxA, iha, toxB, and efa1 genes, but did not have saa and sfpA (Table 1
). Among the six isolates of non-O157:H7 STEC, three had saa and iha, one had iha, and two had no putative adhesin gene. All five categories of human diarrheagenic E. coli have been isolated from mammals and birds.4–6 However, in this study, although 1,000 E. coli isolates from 100 stool samples were examined, no diarrheagenic E. coli was detected, except for eight isolates of STEC. This finding is consistent with the fact that there are few reports of diarrheagenic E. coli, other than STEC, isolated from cows.
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Received March 11, 2005. Accepted for publication March 30, 2005.
* Address correspondence to Noboru Nakasone, Division of Bacterial Pathogenesis, Graduate School of Medicine, University of Ryukyus, 207 Uehara, Nishihara, Okinawa, 903-0215, Japan. E-mail: noboru{at}med.u-ryukyu.ac.jp 
Authors’ addresses: Noboru Nakasone, Naomi Higa, Claudia Toma, Tianyan Song, and Masaaki Iwanaga, Division of Bacterial Pathogenesis, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa, 903–0215, Japan, Telephone: 81-98-895-1124, Fax: 81-98-895-1408, E-mails: noboru{at}med.u-ryukyu.ac.jp, higabac{at}med.u-ryukyu.ac.jp, k950417{at}med.u-ryukyu.ac.jp, k018763{at}med.u-ryukyu.ac.jp, and iwanaga{at}med.u-ryukyu.ac.jp. Hoang Huy Tran and Minh Bine Nguyen, National Institute of Hygiene and Epidemiology, 1 Yersin Street, Hanoi, Vietnam, Telephone: 84–4–8212417, Fax: 84–4–9715470, E-mail: binhminh{at}fpt.vn. Yoshio Ichinose, Seihi Public Health Center, 1-9-5, Nameshi, Nagasaki, 852-8061, Japan, Telephone: 81-95-856-0691, Fax: 81-95-856-0692, E-mail: yichinose{at}pref.nagasaki.lg.jp.
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