Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay

Abstract

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.

Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment

“…In reality, conventional PCR would feasibly detect strain IFR 18/139 as in silico PCR is more stringent and requires a complete sequence match. The findings therefore support the continued use of this in silico multiplex PCR [47].…”

“…Williamson and colleagues [47] have developed an in silico multiplex PCR assay to differentiate two major distinct lineages of C. botulinum Group II (i.e., type E toxin gene lineage, and type B/E/F toxin gene lineage; Figure 5). The sequencing work in the present study has greatly increased the number of genomes now publicly available, and we have evaluated whether these new additional genomes were correctly assigned using an established in silico multiplex PCR [47]. Of the 208 strains tested in the present study, the in silico PCR assigned 207 strains into the correct major lineage of C. botulinum Group II.…”

“…Genomic analysis of C. botulinum Group II indicates that it can be separated into two (or three) major lineages depending on the strains tested and criterion applied [3,5,[42][43][44][45][46][47][48][49]. The majority of strains forming type E neurotoxin are located in one lineage that is distantly separated from the other lineage that is dominated by strains forming subtype B4 neurotoxin and contains strains forming subtype F6 neurotoxin.…”

Variations in the Botulinum Neurotoxin Binding Domain and the Potential for Novel Therapeutics

“…It is not a trivial task to determine how BoNTs bind to their receptors on neuronal cell membranes, especially when trying to replicate the conditions in vivo. With the recent discovery of new BoNTs and BoNT-like molecules in other bacterial species, this raises questions regarding the evolution of the bont gene cluster, their ability to be transferred between species, the potential implications for biosafety, and the need for an agreed-upon consistent naming convention to avoid confusion and ambiguity [ 132 , 133 ]. Fast characterisation and the generation of neutralising antibodies against these novel toxins is required.…”

Proteomic analysis of four Clostridium botulinum strains identifies proteins that link biological responses to proteomic signatures

“…The genome was assembled with the SPAdes assembler v3.11.1 [ 15 ] and was annotated with Prokka v1.13 [ 16 ]. The 5 strains analyzed in this study were put into a broader phylogenetic context using core genome single nucleotide polymorphism (SNP) analysis similar to [ 17 ]. Genome assemblies were aligned to a reference assembly (GCF_000022765.1— C .…”