Abstract
Clostridium botulinum is a Gram-positive, spore-forming anaerobic bacterium that produces botulinum neurotoxin (BoNT). Closing their genomes provides information about their neurotoxin clusters’ arrangement(s) and their location (e.g., chromosome or plasmid) which cannot be assessed using draft genomes. Therefore, we tested the use of long-read sequencing (nanopore sequencing) in combination with short-read sequencing to close two toxin-producing strains. These genomes could be used by the Public Health Emergency Preparedness and Response staff during botulism outbreaks. The genomes of two toxin-producing C. botulinum strains, one from an environmental sample (83F_CFSAN034202) and the other from a clinical sample (CDC51232_CFSAN034200) were sequenced using MinION and MiSeq devices. The genomes, including the chromosomes and the plasmids, were closed by a combination of long-read and short-read sequencing. They belonged to different C. botulinum sequence types (STs), with 83F belonging to ST4 and CDC51232 to ST7. A whole genome single nucleotide polymorphism (SNP) analysis clustered these two strains with strains in lineage 2 (e.g., 6CDC297) and 4 (e.g., NCTC2916) from Group I, respectively. These two strains were also bivalent strains with the BoNTB and BoNTA4 clusters located in the larger plasmid for CDC51232, and the BoNTB and BoNTA1 clusters located both in the chromosome for 83F. Overall, this study showed the advantage of combining these two sequencing methods to obtain high quality closed C. botulinum genomes that could be used for SNP phylogenies (source tracking) as well as for fast identification of BoNT clusters and their gene arrangements.
Highlights
Clostridium botulinum is a Gram-positive, spore-forming anaerobic bacterium that produces botulinum neurotoxin (BoNT) (Gill, 1982)
The MinION run for strain 83F produced a total of 239,626 reads with a read length N50 of 5 kb, while the run for CDC15232 produced a total of 963,433 reads with a read length N50 of 3 kb
Ninety-seven percent of the reads for 83F strain (232,387) and CDC15232 strain (932,465) were classified as belonging to C. botulinum by the What’s in my pot” (WIMP) workflow included in the Epi2me cloud service (Oxford Nanopore Technologies, Oxford, United Kingdom), confirming the identity and purity of these two
Summary
Clostridium botulinum is a Gram-positive, spore-forming anaerobic bacterium that produces botulinum neurotoxin (BoNT) (Gill, 1982). The C. botulinum serotypes are defined by the neurotoxins produced by these organisms which are serologically different and seven serotypes have been described, designated by the letters A through G (Shapiro et al, 1998), with type H still. The toxin types can be subdivided into subserotypes based on their genetic diversity if they encoded a BoNT that differs in their amino acid sequence by at least 2.6% (Peck et al, 2017).Four of the seven serotypes (A, B, E, and F) have been linked with human botulism, with the majority of the cases due to serotypes A and B (Hill et al, 2007; Johnson and Montecucco, 2008). BoNTs have therapeutic applications, regardless of being dangerous biohazard agents, such as in the treatment of various muscle spasm disorders and for cosmetic purposes (Segura-Aguilar and Kostrzewa, 2006; Dressler, 2008; Pickett and Perrow, 2011)
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