Genetic Diversity Among Botulinum Neurotoxin Producing Clostridial Strains

Bioscience, Computing, Computational and Statistical Sciences and Theoretical Divisions, Los Alamos National Laboratory, Los Alamos, NM 87545; Integrated Toxicology Division, United States Army Medical Institute of Infectious Diseases (USAMRIID), Fort Detrick, MD 21702; Department of Food Microbiology and Toxicology, Food Research Institute (FRI), University of Wisconsin, Madison, WI 53706; Defense Biology Division, Lawrence Livermore National Laboratory, Livermore, CA 94551; Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, Rm 3C-38, San Francisco General Hospital, 1001 Potrero Ave, San Francisco, CA 94110

^* To whom correspondence should be addressed. Email: marksj{at}anesthesia.ucsf.edu.

	Abstract

Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria which have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT A-G). Analysis of the16S rRNA sequences confirmed earlier identification of at least four distinct genomic backgrounds (Groups I-IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into sub-groups. Sequencing of the BoNT genes from multiple strains of serotypes A, B and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed varying degrees of inter-relatedness and recombination, as had been previously noted for the NTNH gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for treatment of botulism.