Implications of Genome-Based Discrimination between Clostridium botulinum Group I and Clostridium sporogenes Strains for Bacterial Taxonomy

Abstract

ABSTRACTTaxonomic classification ofClostridium botulinumis based on the production of botulinum neurotoxin (BoNT), while closely related, nontoxic organisms are classified asClostridium sporogenes. However, this taxonomic organization does not accurately mirror phylogenetic relationships between these species. A phylogenetic reconstruction using 2,016 orthologous genes shared among strains ofC. botulinumgroup I andC. sporogenesclearly separated these two species into discrete clades which showed ∼93% average nucleotide identity (ANI) between them. Clustering of strains based on the presence of variable orthologs revealed 143C. sporogenesclade-specific genetic signatures, a subset of which were further evaluated for their ability to correctly classify a panel of presumptiveC. sporogenesstrains by PCR. Genome sequencing of severalC. sporogenesstrains lacking these signatures confirmed that they clustered withC. botulinumstrains in a core genome phylogenetic tree. Our analysis also identifiedC. botulinumstrains that containedC. sporogenesclade-specific signatures and phylogenetically clustered withC. sporogenesstrains. The genome sequences of twobont/B2-containing strains belonging to theC. sporogenesclade contained regions with similarity to abont-bearing plasmid (pCLD), while two different strains belonging to theC. botulinumclade carriedbont/B2on the chromosome. These results indicate thatbont/B2was likely acquired byC. sporogenesstrains through horizontal gene transfer. The genome-based classification of these species used to identify candidate genes for the development of rapid assays for molecular identification may be applicable to additional bacterial species that are challenging with respect to their classification.