Differentiation of Clostridium botulinum Serotype A Strains Using Multiple-Locus Variable Number Tandem Repeat Analysis

Bioscience and Computing, Computational and Statistical Sciences Divisions, Los Alamos National Laboratory, Los Alamos, NM 87545; Defense Biology Division, Lawrence Livermore National Laboratory, Livermore, CA 94551; Integrated Toxicology Division, United States Army Medical Institute of Infectious Diseases (USAMRIID), Fort Detrick, MD 21702

^* To whom correspondence should be addressed. Email: khill{at}lanl.gov.

	Abstract

Ten variable number tandem repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of BoNT/A1-A4 subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B-G, including five bivalent strains, and two strains of the closely related species, Clostridium sporogenes were also tested. Amplified fragment length polymorphism (AFLP) analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The ten VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus variable number tandem repeat analysis (MLVA) of the 59 BoNT/A1-A4 strains and three bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1-A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.