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Journal of Clinical Microbiology, September 2009, p. 2720-2728, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00077-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Genetic Characterization of Clostridium botulinum Associated with Type B Infant Botulism in Japan{triangledown}

Kaoru Umeda,1,2 Yoshiyuki Seto,2 Tomoko Kohda,2 Masafumi Mukamoto,2 and Shunji Kozaki2*

Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennoji-ku, Osaka 543-0026, Japan,1 Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano-shi, Osaka 598-0048, Japan2

Received 14 January 2009/ Returned for modification 2 March 2009/ Accepted 24 June 2009

The 15 proteolytic Clostridium botulinum type B strains, including 3 isolates associated with infant botulism in Japan, were genetically characterized by phylogenetic analysis of boNT/B gene sequences, genotyping, and determination of the boNT/B gene location by using pulsed-field gel electrophoresis (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 a 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 the B1 and the B2 subtypes, although no relation with infant botulism was shown by PFGE genotyping. The results of PFGE and Southern blot hybridization with undigested DNA suggested that the boNT/B gene is 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. The botulinum neurotoxin (BoNT) of Osaka05 was suggested to have an antigenicity different from the antigenicities of BoNT/B1 and BoNT/B2 by a sandwich enzyme-linked immunosorbent assay with the recombinant BoNT/B-C-terminal domain. We established a multiplex PCR assay for BoNT/B subtyping which will be useful for epidemiological studies of type B strains and the infectious diseases that they cause.

* Corresponding author. Present address: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano-shi, Osaka 598-0048, Japan. Phone: 81-72-463-5690. Fax: 81-72-463-5691. E-mail: kozaki{at}center.osakafu-u.ac.jp

{triangledown} Published ahead of print on 1 July 2009.

Journal of Clinical Microbiology, September 2009, p. 2720-2728, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00077-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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