Characterization of Clostridium botulinum strains producing BoNT/F4 or BoNT/F5.

Raphael BH1, Bradshaw M, Kalb SR, Joseph LA, Lúquez C, Barr JR, Johnson EA, Maslanka SE.

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Abstract

Botulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as types A or B. Type F neurotoxin gene sequences have been further classified into 7 toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/F5, and bont/A2). In this study, a total of 8 strains containing bont/F4 and 7 strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5-. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-encoding plasmid among strains of diverse genomic backgrounds.