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

Abstract

ABSTRACTWe report the development of real-time PCR assays for genotypingClostridium botulinumgroup III targeting the newly definedC. novyi sensu latogroup; the nontoxic nonhemagglutinin (NTNH)-encoding genentnh; the botulinum neurotoxin (BoNT)-encoding genesbont/C,bont/C/D,bont/D, andbont/D/C; and the flagellin (fliC) gene. The genetic diversity offliCamongC. botulinumgroup III strains resulted in the definition of five major subgroups namedfliC-I tofliC-V. Investigation offliCsubtypes in 560 samples, with various European origins, showed thatfliC-I was predominant and found exclusively in samples contaminated byC. botulinumtype C/D,fliC-II was rarely detected, no sample was recorded asfliC-III orfliC-V, and onlyC. botulinumtype D/C samples tested positive forfliC-IV. The lack of genetic diversity of the flagellin gene ofC. botulinumtype C/D would support a clonal spread of type C/D strains in different geographical areas.fliC-I tofliC-III are genetically related (87% to 92% sequence identity), whereasfliC-IV fromC. botulinumtype D/C is more genetically distant from the otherfliCtypes (with only 50% sequence identity). These findings suggestfliC-I tofliC-III have evolved in a common environment and support a different genetic evolution forfliC-IV. A combination of theC. novyi sensu lato,ntnh,bont, andfliCPCR assays developed in this study allowed better characterization ofC. botulinumgroup III and showed the group to be less genetically diverse thanC. botulinumgroups I and II, supporting a slow genetic evolution of the strains belonging toC. botulinumgroup III.