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Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.

ABSTRACT: BACKGROUND:Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. RESULTS:Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. CONCLUSIONS:This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found.

SUBMITTER: Williamson CH

PROVIDER: S-EPMC4778365 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.

Williamson Charles H D CH Sahl Jason W JW Smith Theresa J TJ Xie Gary G Foley Brian T BT Smith Leonard A LA Fernández Rafael A RA Lindström Miia M Korkeala Hannu H Keim Paul P Foster Jeffrey J Hill Karen K

BMC genomics 20160303

<h4>Background</h4>Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neuro ...[more]

PMID: 26939550

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Project description:Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha+] or orfX+). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters.IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha+ or orfX+) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

Project description:Shiga toxin-producing Escherichia coli (STEC) O121 is among the top six non-O157 serogroups that are most frequently associated with severe disease in humans. While O121:H19 is predominant, other O121 serotypes have been frequently isolated from environmental samples, but their virulence repertoire is poorly characterized. Here, we sequenced the complete genomes of two animal isolates belonging to O121:H7 and O121:H10 and performed comparative genomic analysis with O121:H19 to assess their virulence potential. Both O121:H7 and O121:H10 strains carry a genome comparable in size with the O121:H19 genomes and belong to phylogroup B1. However, both strains appear to have evolved from a different lineage than the O121:H19 strains according to the core genes-based phylogeny and Multi Locus Sequence Typing. A systematic search of over 300 E. coli virulence genes listed in the Virulence Factor DataBase revealed a total of 73 and 71 in O121:H7 and O121:H10 strains, respectively, in comparison with an average of 135 in the O121:H19 strains. This variation in the virulence genes repertoire was mainly attributed to the reduction in the number of genes related to the Type III Secretion System in the O121:H7 and O121:H10 strains. Compared to the O121:H19 strains, the O121:H7 strain carries more adherence and toxin genes while the O121:H10 strain carries more genes related to the Type VI Secretion System. Although both O121:H7 and O121:H10 strains carry the large virulence plasmid pEHEC, they do not harbor all pEHEC virulence genes in O121:H19. Furthermore, unlike the O121:H19 strains, neither the O121:H7 nor O121:H10 strain carried the Locus of Enterocyte Effacement, OI-122, nor the tellurite resistance island. Although an incomplete Locus of Adhesion and Autoaggregation (LAA) was identified in the O121:H7 and O121:H10 strains, a limited number of virulence genes were present. Consistently, both O121:H7 and O121:H10 strains displayed significant reduced cytotoxicity than either the O157:H7 strain EDL933 or the O121:H19 strain RM8352. In fact, the O121:H7 strain RM8082 appeared to cause minimal cytotoxicity to Vero cells. Our study demonstrated distinct evolutionary lineages among the strains of serotypes O121:H19, O121:H10, and O121:H7 and suggested reduced virulence potentials in STEC strains of O121:H10 and O121:H7.

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Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.

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Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.

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