Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.

Escherichia coli 025 strains that produce heat-stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST-producing and non-producing strains of E. coli 025 was investigated. All eight ST-producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladderbanding of LPS in SDS-polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsedfield gel electrophoresis (PFGE). In contrast, ST-non-producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli 025.Key words: Escherichia coli 025, Heat-stable toxin, LPS, Pulsed-field gel electrophoresis Enterotoxigenic Escherichia coli (ETEC) which produces heat-labile toxin (LT) and/or heat-stable toxin (ST) is one of the most common enteropathogens for humans, especially for children in developing countries (4,5,14). ETEC was formerly recognized as a pathogen causing diarrhea among travelers from developed countries where they have better hygienic conditions. However, ETEC strains have been disseminated recently in many countries including developed countries by transport of people and import of foods from endemic areas (3, 18). Many cases of ETEC infections have been reported in Japan (10,13,19), and it is speculated that this pathogen stably persists and is reproduced in the environment in Japan.One type of ETEC is E. coli which produces only ST. The outbreak of such E. coli infection in the primary schools in Yokohama in 1996 was recently reported by Mitsuda et al (12). The serotype of the strain was 025:NM, and it produced STIb (STIh). In this case the food polluted with the pathogen was found to be tuna paste served in the school lunch. There were other outbreaks caused by E. coli 025:H20 in Saitama prefecture in 1997 (11). More recently, we have encoun-*Address correspondence to Dr. Kazuyoshi Kawahara, Department of Bacteriology, The Kitasato Institute, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8642, Japan. Tel/Fax: +81-3-5791-6127. E-mail: kawahara-k@kitasato.or.jp 433 tered an outbreak of ETEC infection in a cafeteria of a research institute in Tokyo. The bacterium isolated in high frequency from the stool specimen of the patients was E. coli 025. The patients suffered from diarrhea with slight fever, which continued for 2 or 3 days (T. Suzuki, personal communications). The strains isolated independently from 10 patients were examined by PCR primer sets for enterotoxin (LT, STIa and STIb) genes, shiga toxin (STX) (STXl and STX2) genes, and other virulence genes (invE and ipaH), and were proved to have STIb gene (Y. Kagami, unpublished data). These reports suggested that E. coli 025 strains which produce STIb have been frequently isolated recently in the Kanto district of Japan. Therefore, investigations of the characteristic...