JCM Accepts, published online ahead of print on 1 July 2009

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J. Clin. Microbiol. doi:10.1128/JCM.00077-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Genetic characterization of Clostridium botulinum associated with type B infant botulism in Japan

Kaoru Umeda, Yoshiyuki Seto, Tomoko Kohda, Masafumi Mukamoto, and Shunji Kozaki^*

Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennoji-ku, Osaka 543-0026, Japan; Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan

^* To whom correspondence should be addressed. Email: kozaki{at}center.osakafu-u.ac.jp.

The 15 proteolytic C. botulinum type B strains, including three isolates associated with infant botulism in Japan, were genetically characterized by phylogenetic analysis of BoNT/B gene sequences, genotyping and BoNT/B gene location using PFGE for molecular epidemiological analysis of infant botulism in Japan. Strain Osaka05, isolated from a case in 2005, showed a unique BoNT/B gene sequence and was considered to be a new BoNT/B subtype by phylogenetic analysis. Strain Osaka06, isolated from a case in 2006, was classified as the B2 subtype, the same as strain 111, isolated from the case in 1995. The five isolates associated with infant botulism in the United States were classified into the B1 subtype. Isolates from food samples in Japan were divided into B1 and B2 subtypes, although no relation was shown with infant botulism by PFGE genotyping. The results of undigested DNA PFGE and Southern blot hybridization suggested that the BoNT/B gene was located on large plasmids (approximately 150 kbp, 260 kbp, 275 kbp or 280 kbp) in five strains belonging to three BoNT/B subtypes from various sources. BoNT/Osaka05 was suggested to have different antigenicity from BoNT/B1 and/B2 by sandwich ELISA using the recombinant BoNT/H_C domain. We established the multiplex PCR assay for BoNT/B subtyping, which will be useful for epidemiological studies of type B strains and their infectious diseases.