Evidence that Plasmid-Borne Botulinum Neurotoxin Type B Genes Are Widespread among Clostridium botulinum Serotype B Strains

Background Plasmids that encode certain subtypes of the botulinum neurotoxin type B have recently been detected in some Clostridium botulinum strains. The objective of the present study was to investigate the frequency with which plasmid carriage of the botulinum neurotoxin type B gene ( bont /B) occurs in strains of C. botulinum type B, Ab, and A(B), and whether plasmid carriage is bont /B subtype-related. Methodology/Principal Findings PCR-Restriction fragment length polymorphism was employed to identify subtypes of the bont /B gene. Pulsed-field gel electrophoresis and Southern blot hybridization with specific probes were performed to analyze the genomic location of the bont /B subtype genes. All five known bont /B subtype genes were detected among the strains; the most frequently detected subtype genes were bont /B1 and /B2. Surprisingly, the bont /B subtype gene was shown to be plasmid-borne in >50% of the total strains. The same bont /B subtype gene was associated with the chromosome in some strains, whereas it was associated with a plasmid in others. All five known bont /B subtype genes were in some cases found to reside on plasmids, though with varying frequency (e.g., most of the bont /B1 subtype genes were located on plasmids, whereas all but one of the bont /B2 subtypes were chromosomally-located). Three bivalent isolates carried both bont /A and /B genes on the same plasmid. The plasmids carrying the bont gene were five different sizes, ranging from ∼55 kb to ∼245 kb. Conclusions/Significance The unexpected finding of the widespread distribution of plasmids harboring the bont /B gene among C. botulinum serotype B strains provides a chance to examine their contribution to the dissemination of the bont genes among heterogeneous clostridia, with potential implications on issues related to pathogenesis and food safety.