Dataset Information

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data.

ABSTRACT: Clostridium botulinum group II isolates (n = 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core SNP methods were consistent in terms of clade-level isolate classification; however, core SNP analysis showed higher resolution capability. Furthermore, core SNP analysis correctly distinguished isolates by outbreak and location. This study illustrated the utility of next-generation sequence-based typing approaches for isolate characterization and source attribution and identified discrete SNP loci and MLST alleles for isolate comparison.

SUBMITTER: Weedmark KA

PROVIDER: S-EPMC4551264 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data.

Weedmark K A KA Mabon P P Hayden K L KL Lambert D D Van Domselaar G G Austin J W JW Corbett C R CR

Applied and environmental microbiology 20150626 17

Clostridium botulinum group II isolates (n = 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of differe ...[more]

PMID: 26116673

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Project description:BackgroundClostridium botulinum is a group of four physiologically and phylogenetically distinct bacteria that produce botulinum neurotoxin. While studies have characterised variability between strains of Group I (proteolytic) C. botulinum, the genetic and physiological variability and relationships between strains within Group II (non-proteolytic) C. botulinum are not well understood. In this study the genome of Group II strain C. botulinum Eklund 17B (NRP) was sequenced and used to construct a whole genome DNA microarray. This was used in a comparative genomic indexing study to compare the relatedness of 43 strains of Group II C. botulinum (14 type B, 24 type E and 5 type F). These results were compared with characteristics determined from physiological tests.ResultsWhole genome indexing showed that strains of Group II C. botulinum isolated from a wide variety of environments over more than 75 years clustered together indicating the genetic background of Group II C. botulinum is stable. Further analysis showed that strains forming type B or type F toxin are closely related with only toxin cluster genes targets being unique to either type. Strains producing type E toxin formed a separate subset. Carbohydrate fermentation tests supported the observation that type B and F strains form a separate subset to type E strains. All the type F strains and most of type B strains produced acid from amylopectin, amylose and glycogen whereas type E strains did not. However, these two subsets did not differ strongly in minimum growth temperature or maximum NaCl concentration for growth. No relationship was found between tellurite resistance and toxin type despite all the tested type B and type F strains carrying tehB, while the sequence was absent or diverged in all type E strains.ConclusionsAlthough Group II C. botulinum form a tight genetic group, genomic and physiological analysis indicates there are two distinct subsets within this group. All type B strains and type F strains are in one subset and all type E strains in the other.

Project description:The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt "bookmark" sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and "bookmark"-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique "watermark" through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.

Dataset Information

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data.

Publications

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data.

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