A new BoNT/B subtype detected from infant botulism in Japan

Abstract

Clostridium botulinum type B has recently been isolated from food-borne and infant botulism cases. The BoNT/B in these cases was divided into two main subtypes, B1 and B2, based on analysis of the amino acid sequences. The genetic properties of the C. botulinum type B strains in relation to infant botulism in Japan (strains 111, Osaka05 and Osaka06) were analyzed by sequencing of the bont/B and nontoxic component genes (ha70, ha17, ha33, p21 and ntnh), PFGE genotyping and southern blot hybridization with bont/B probe. Strain Osaka05 produced particular BoNT/B, which were grouped to a novel variant on phylogenetic analysis. The substitutions of amino acid residues were concentrated in the heavy chain, especially in the HCC subdomain. Strains 111 and Osaka06 were classified as subtype B2. The nontoxic component gene arrangements within these subtypes were identical to each other. Multiplex PCR method for BoNT/B subtyping (B1, B2 and Osaka05) was newly established based on bont/B nucleotide sequences variability. Three isolates from infant botulism in Japan were identified as different genotypes based on Sma I-digested PFGE. The BoNT/B encoding gene was shown to be located on large plasmids (approximately 150 or 260–280 kbp) for various subtype strains including isolates from infant botulism in Japan using Southern blot hybridization after nondigested PFGE with bont/B probe.