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

Abstract

BackgroundPlasmids 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 FindingsPCR-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/SignificanceThe 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.

Genetic Characterization and Comparison of Clostridium botulinum Isolates from Botulism Cases in Japan between 2006 and 2011

“…Subtype B5 is known as a bivalent B subtype (bvB or Bbv) and is often found in bivalent strains like 657 and Bf (Table 4). However, there are also reports of strains that carry bont/B5 as the single toxin gene in the genome (47). Ehime2011 is thought to be genetically related to bivalent strains, as demonstrated by wholegenome SNP phylogenetic analysis (Fig.…”

“…3A and 7A). C. botulinum strains reportedly carry plasmids of a variety of sizes (41,47,51,52); however, small and medium plasmids without bont genes have not been extensively characterized. The analysis of small plasmids in C. botulinum may be useful for strain characterization and discrimination.…”

Characterization of Toxin Plasmids in Clostridium perfringens Type C Isolates

“…Similar plasmid incompatibility issues may also affect the virulence of other pathogenic clostridia. For example, although plasmids exist that encode only botulinum neurotoxin serotype A or B (13,23), all surveyed Clostridium botulinum stains producing both neurotoxin serotypes B and A carry these two neurotoxin genes on the same plasmid (13,22). This observation might open the possibility that plasmids encoding only serotype B or serotype A cannot be stably maintained together in the same C. botulinum cell.…”

Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

“…The botG cluster is carried by a plasmid (10). The botA, botB, and botF clusters were initially found in the bacterial chromosome; however, plasmidborne toxin genes have recently been shown for subtypes A3 (11), B1, B2, the nonproteolytic type B (12)(13)(14), and some bivalent subtypes, such as Ab, Ba, and Bf (15). The two available C. botulinum type E genome sequences (NCBI) show chromosomal botE clusters, as is the case with neurotoxigenic C. butyricum (16).…”