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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.

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum . BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum . The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 10 1 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).

Here, we report the genome sequence of a rare bivalent strain of Clostridium botulinum , A2B3 87. The strain was isolated from a foodborne botulism case that occurred in Italy in 1995. The case was characterized by rapid evolution of the illness and failure of conventional treatments.