Clostridium botulinum, from toxin and flagellin genotyping to Whole Genome Sequencing : an insight into genetic diversity of human and animal botulism associated clostridia’s

Clostridium botulinum is the etiologic agent of botulism, a deadly paralytic disease that can affects both human and animals. Different bacteria, producing neurotoxins type A to H, are responsible for the disease. They are separated into different groups (I to VI) on the basis of their phenotypical and biological characteristics. Human botulism is mainly due to Groups I and II producing neurotoxins A, B, E and F, with type H recently discovered. Also C. butyricum and C. baratii species (Groups V and VI), producing toxins type F and E respectively, are scarcely reported. C. argentinense Group IV, producing toxin type G, which has been suspected to be associated with infant botulism in Argentina. Animal botulism is mainly due to Group III, which is constituted by C. novyi sensu lato species. They produce toxin types C, D and their mosaic variants. Botulinum neurotoxins are the most powerful toxin known to date with as little as 70 µg enough to kill a person by food poisoning. Therefore, it received a great deal of attention. Botulinum neurotoxins have been deeply studied, especially human related toxins compared to animal. The toxins found to be useful for medical or cosmetic (Botox) treatments, but it was also used as a biological warfare agent, and for bioterrorism. Its extreme potency is equal to its dangerousness. Therefore, governments show concerns of its potential misuse as a bioterrorism weapon; research programs are funded to study and raise awareness about both the toxins and the producing organisms. My PhD work was structured by the different projects I was involved in, which were related to C. botulinum detection and typing, like BIOTRACER and AniBioThreat European projects, the French national CBRN program, or the NRL for avian botulism. The main transversal objective I followed lead me to develop new methods to trace back the origin of C. botulinum contamination, in case of a deliberate, accidental or naturally occurring botulism outbreak. I investigated flagellin genes as potential genetic targets for typing C. botulinum Group I-II and III, responsible for human and animal botulism respectively. Flagellin genes flaA and flaB showed the investigated C. botulinum Group I and II strains to cluster into 5 major groups and up to 15 subgroups, some being specific for certain geographical areas, and flaB being specific to C. botulinum type E. Flagellin fliC gene investigated in C. botulinum Group III showed to cluster into five groups, with fliC-I and fliC-IV associated to type C/D and D/C respectively, being not discriminative enough to differentiate highly genetically related strains. I also studied the prevalence of mosaic toxin genes in C. botulinum Group III in animal botulism, mainly in poultry and bovine. The results brought out the mosaic toxin types C/D and D/C to be predominant in the samples investigated throughout Europe. Finally, I explored the full genome sequences of 14 types C/D and 3 types D/C C. botulinum Group III strains, mainly originating from French avian and bovine botulism outbreaks. Analyses of their genome sequences showed them to be closely related to other European strains from Group III. While studying their genetic content, I was able to point out that the extrachromosomal elements of strains type C/D could be used to generate a genetic ID card. Investigation of Crispr typing method showed to be irrelevant for type C/D, due to a deficient Crispr-Cas mechanism, but deserve more investigation for type D/C. The highest level of discrimination was achieved while using SNP core phylogeny, which allowed distinguishing up to the strain level. Here are the results I’m going to develop in this manuscript