Genetic Diversity of the Flagellin Genes of Clostridium botulinum Groups I and II

Abstract

ABSTRACTBotulinum neurotoxins (BoNTs) are produced by phenotypically and genetically differentClostridiumspecies, includingClostridium botulinumand some strains ofClostridium baratii(serotype F) andClostridium butyricum(serotype E). BoNT-producing clostridia responsible for human botulism encompass strains of group I (secreting proteases, producing toxin serotype A, B, or F, and growing optimally at 37°C) and group II (nonproteolytic, producing toxin serotype E, B, or F, and growing optimally at 30°C). Here we report the development of real-time PCR assays for genotypingC. botulinumstrains of groups I and II based on flaVR (variable region sequence offlaA) sequences and theflaBgene. Real-time PCR typing of regions flaVR1 to flaVR10 andflaBwas optimized and validated with 62 historical and CanadianC. botulinumstrains that had been previously typed. Analysis of 210 isolates of European origin allowed the identification of four newC. botulinumflaVR types (flaVR11 to flaVR14) and one new flaVR type specific toC. butyricumtype E (flaVR15). The genetic diversity of the flaVR amongC. botulinumstrains investigated in the present study reveals the clustering of flaVR types into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolyticClostridium botulinum, subgroup 2 is made up of nonproteolyticC. botulinumonly, and subgroup 5 is specific toC. butyricumtype E. The genetic variability of the flagellin genes carried byC. botulinumand the possible association of flaVR types with certain geographical areas make gene profiling of flaVR andflaBpromising in molecular surveillance and epidemiology ofC. botulinum.