Genome-based discrimination between Group I Clostridium botulinum and Clostridium sporogenes strains: implications for bacterial taxonomy.

Weigand MR1, Pena-Gonzalez A2, Shirey TB3, Broeker RG3, Ishaq MK3, Konstantinidis KT4, Raphael BH5.

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Abstract

Taxonomic classification of Clostridium botulinum is based on the production of botulinum neurotoxin (BoNT) while closely-related, non-toxic organisms are classified as Clostridium 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 of C. botulinum Group I and C. sporogenes clearly 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 143 C. sporogenes clade-specific genetic signatures, a subset of which was further evaluated for their ability to correctly classify a panel of presumptive C. sporogenes strains by PCR. Genome sequencing of several C. sporogenes strains lacking these signatures confirmed that they clustered with C. botulinum strains in a core genome phylogenetic tree. Our analysis also identified C. botulinum strains that contained C. sporogenes clade-specific signatures and phylogenetically clustered with C. sporogenes strains. The genome sequences of two bontB2-containing strains belonging to the C. sporogenes clade contained regions with similarity to a bont-encoding plasmid (pCLD) while two different strains belonging to the C. botulinum clade encoded bontB2 on the chromosome. These results indicate that bont/B2 was likely acquired by C. sporogenes strains 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.