Molecular Gene Profiling of Clostridium botulinum Group III and Its Detection in Naturally Contaminated Samples Originating from Various European Countries

We report the development of real-time PCR assays for genotyping Clostridium botulinum group III targeting the newly defined C. novyi sensu lato group; the nontoxic nonhemagglutinin (NTNH)-encoding gene ntnh ; the botulinum neurotoxin (BoNT)-encoding genes bont/C , bont/C/D , bont/D , and bont/D/C ; and the flagellin ( fliC ) gene. The genetic diversity of fliC among C. botulinum group III strains resulted in the definition of five major subgroups named fliC -I to fliC -V. Investigation of fliC subtypes in 560 samples, with various European origins, showed that fliC -I was predominant and found exclusively in samples contaminated by C. botulinum type C/D, fliC -II was rarely detected, no sample was recorded as fliC -III or fliC -V, and only C. botulinum type D/C samples tested positive for fliC -IV. The lack of genetic diversity of the flagellin gene of C. botulinum type C/D would support a clonal spread of type C/D strains in different geographical areas. fliC -I to fliC -III are genetically related (87% to 92% sequence identity), whereas fliC -IV from C. botulinum type D/C is more genetically distant from the other fliC types (with only 50% sequence identity). These findings suggest fliC -I to fliC -III have evolved in a common environment and support a different genetic evolution for fliC -IV. A combination of the C. novyi sensu lato , ntnh , bont , and fliC PCR assays developed in this study allowed better characterization of C. botulinum group III and showed the group to be less genetically diverse than C. botulinum groups I and II, supporting a slow genetic evolution of the strains belonging to C. botulinum group III.