New Insights into the Genetic Diversity of Clostridium botulinum Group III through Extensive Genome Exploration

Abstract

Animal botulism is caused by group III Clostridium botulinum strains producing type C and D toxins, or their chimeric forms C/D and D/C. Animal botulism is considered an emerging disease in Europe, notably in poultry production. Before our study, 14 genomes from different countries were available in the public database, but none were from France. In order to investigate the genetic relationship of French strains with different geographical areas and find new potential typing targets, 17 strains of C. botulinum group III were sequenced (16 from France and one from New Caledonia). Fourteen were type C/D strains isolated from chickens, ducks, guinea fowl and turkeys and three were type D/C strains isolated from cattle. The New Caledonian strain was a type D/C strain. Whole genome sequence analysis showed the French strains to be closely related to European strains from C. botulinum group III lineages Ia and Ib. The investigation of CRISPR sequences as genetic targets for differentiating strains in group III proved to be irrelevant for type C/D due to a deficient CRISPR/Cas mechanism, but not for type D/C. Conversely, the extrachromosomal elements of type C/D strains could be used to generate a genetic ID card. The highest level of discrimination was achieved with SNP core phylogeny, which allowed differentiation up to strain level and provide the most relevant information for genetic epidemiology studies and discrimination.

Exploration of the Diversity of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Systems in Clostridium novyi sensu lato

“…Shared identical spacers were investigated first ( Supplementary Table S2 ). Interestingly, the distribution of strains based on shared spacers was very similar to the distribution previously obtained using Gegenees ( Skarin and Segerman, 2014 ) or based on SNP phylogeny profiling ( Woudstra et al, 2016 ). For example, 17 C. botulinum BoNT C/D strains from lineage Ia shared more than 90% of their spacers ( Supplementary Table S2 ).…”

Substrate cleavage and duration of action of botulinum neurotoxin type FA (“H, HA”)

“…Sequences from over 40 subtypes have been published (Hill and Smith, 2013; Hill et al, 2015; Montecucco and Rasotto, 2015; Peck et al, 2017). In addition, members of BoNT/C and /D serotypes, which cause large scale avian and bovine botulism outbreaks (Friend and Milton, 1999; Souillard et al, 2014; Woudstra et al, 2016), include several chimeric BoNTs denoted D/C or C/D and consisting of various LCH N -H C combinations (Hedeland et al, 2011; Nakamura et al, 2013; Takeda et al, 2005; Woudstra et al, 2016). Intra-serotype chimeric toxins combining HC and LC domains of subtypes within one serotype have also been reported including BoNT/A2 and BoNT/F5 (Hill et al, 2007; Raphael et al, 2010).…”

Botulinum Neurotoxin Detection Methods for Public Health Response and Surveillance

“…Whole genome sequencing (WGS) has demonstrated the high genomic diversity of toxigenic clostridia, leading to the identification of novel genotypes (Gonzalez-Escalona et al, 2014a , b ; Weedmark et al, 2015 ; Williamson et al, 2016 ). SNP-based analysis of whole genomic data and core genome MLST approaches coupled with phylogenetic analysis generally provide sufficient resolution to differentiate between toxigenic clostridial strains, even those with absolute nucleotide identity at the bont gene sequence and botulinum toxin gene cluster (Gonzalez-Escalona et al, 2014b ; Weedmark et al, 2015 ; Woudstra et al, 2016 ). However, nucleic acid methods including WGS depend on culture enrichment techniques which frequently require 5 days for successful isolation (Haim and Timothy, 1998 ; Cheng et al, 2016 ).…”