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Applied and Environmental Microbiology, November 2000, p. 4992-4997, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Genetic Analysis of Type E Botulinum Toxin-Producing Clostridium butyricum Strains†

Xingmin Wang,1 Tsuneo Maegawa,1 Tadahiro Karasawa,1 Shunji Kozaki,2 Kentaro Tsukamoto,2 Yotaku Gyobu,3 Kiyotaka Yamakawa,3 Keiji Oguma,4 Yoshihiko Sakaguchi,4 and Shinichi Nakamura1,*

Department of Bacteriology, School of Medicine, Kanazawa University, Kanazawa 920-8640,1 Department of Veterinary Science, College of Agriculture, Osaka Prefecture University, Sakai, Osaka 599-8531,2 Department of Bacteriology, Toyama Institute of Health, Toyama 939-0363,3 and Department of Bacteriology, Okayama University Medical School, Okayama 700-8558,4 Japan

Received 19 April 2000/Accepted 28 August 2000

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.

* Corresponding author. Mailing address: Department of Bacteriology, School of Medicine, Kanazawa University, Kanazawa 920-8640, Japan. Phone: 81-76-265-2200. Fax: 81-76-234-4230. E-mail: nakamura{at}med.kanazawa-u.ac.jp.

dagger We dedicate this work to the memory of C. L. Hatheway.

Applied and Environmental Microbiology, November 2000, p. 4992-4997, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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