Abstract
Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA gene sequencing and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades (clade I containing ATCC 7955 NCA3679 isolates 1961-2, 1990, and 2007 and clade II containing PA 3679 isolates NFL, UW, FDA, and Campbell and ATCC 7955 NCA3679 isolate 1961-4). The 16S maximum likelihood (ML) tree clustered both clades within proteolytic C. botulinum strains, with clade I forming a distinct cluster with other C. sporogenes non-PA 3679 strains. SNP analysis revealed that clade I isolates were more similar to the genomic reference PA 3679 (NCTC8594) genome (GenBank accession number AGAH00000000.1) than clade II isolates were. The genomic reference C. sporogenes PA 3679 (NCTC8594) genome and clade I C. sporogenes isolates were genetically distinct from those obtained from other sources (University of Wisconsin, National Food Laboratory, U.S. Food and Drug Administration, and Campbell's Soup Company). Thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates were. Considering the widespread use of C. sporogenes PA 3679 and its genetic information in numerous studies, the accurate identification and genetic characterization of C. sporogenes PA 3679 are of critical importance.
Highlights
Food-borne botulism is a neuroparalytic disease that results from the ingestion of botulinum neurotoxin (BoNT) produced by clostridia, including Clostridium botulinum and rare strains of Clostridium baratii and Clostridium butyricum
In a study by Weigand et al [24], whole-genome sequencing (WGS) was employed to show that strains of C. sporogenes and group I C. botulinum were distinctly grouped into two clades based on 2,016 core orthologues shared between these organisms
Pulsed-field gel electrophoresis (PFGE) was performed on the American Type Culture Collection (ATCC) 7955 NCA3679 1961 spore crop prior to streaking for isolation to ascertain the purity of the culture
Summary
Food-borne botulism is a neuroparalytic disease that results from the ingestion of botulinum neurotoxin (BoNT) produced by clostridia, including Clostridium botulinum and rare strains of Clostridium baratii and Clostridium butyricum. Group I (proteolytic) C. botulinum strains can be uniquely differentiated from the other groups based on their ability to form highly heat-resistant endospores. Spores of this organism are ubiquitously found in the environment and are generally presumed to be a natural contaminant of foods that come in contact with the soil [2]. That study showed that PA 3679 was serologically identical to two other putrefactive anaerobes, S2 and 9x, isolated from spoiled canned meats; all three strains were culturally and serologically distinct from C. sporogenes strain Spray [11]. The PA 3679 strain included in that study was C. sporogenes PA 3679 (NCTC8594) that was sequenced by Bradbury et al [25]
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