At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.

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.

Botulinum neurotoxins (BoNTs) produced by the bacteria Clostridium botulinum are the causative agent of human and animal botulism, a rare but serious and potentially deadly intoxication. Foodborne botulism is caused by the consumption of foods containing BoNTs, which results from contamination of foods with C. botulinum spores and toxin production by the bacteria during growth within the food. Validation of the safety of food products is essential in preventing foodborne botulism, however, limited guidance and standards exist for the selection of strains used in C. botulinum food challenge studies. Sequencing and genomics studies have revealed that C. botulinum is a large, diverse, and polyphyletic species, with physiologic and growth characteristics studied only in a few representatives. Little is known about potential growth competition or effects on toxin production between C. botulinum strains. In this study, we investigated an applied cocktail of ten C. botulinum strains, seven Group I and three Group II. Whole genome SNP alignments revealed that this strain cocktail encompasses the major clades of the Group I and II C. botulinum species. While growth competition appears to exist between several of the strains, the cocktail as a whole resulted in high levels of BoNT production.

Botulinum neurotoxins are a varied group of protein toxins that share similar structures and modes of activity. They include at least seven serotypes and over forty subtypes that are produced by seven different clostridial species. These bacterial species are not limited strictly to BoNT-producers as neuro-toxigenic and non-neuro-toxigenic members have been identified within each species. The nomenclature surrounding these toxins and associated bacteria has been evolving as new isolations and discoveries have arisen, resulting in challenges in diagnostic reporting, epidemiology and food safety studies, and in the application of therapeutic products. An understanding of the intricacies regarding the nomenclature of BoNTs and BoNT-producing clostridia is crucial for communication that allows for accurate reporting of information that is pertinent to each situation.

Cooked, uncured meat products packaged under reduced oxygen packaging conditions require the control of anaerobic and facultative anaerobic pathogens if they are held at temperatures greater than 3°C at retail or consumer level. The objective of this study was to determine the inhibition of Listeria monocytogenes and Clostridium botulinum in cooked, uncured shredded turkey and pork formulated with synthetic or clean-label antimicrobials. Treatments of shredded meat products were prepared with or without antimicrobials using turkey thigh or breast that were cooked to 85°C, shredded, and chilled before inoculation with the target pathogen. L. monocytogenes inoculated samples were stored at 7.2°C, whereas C. botulinum samples were stored at 12.8°C; triplicate samples were assayed every 2 weeks. In the first set of experiments, L. monocytogenes populations increased 2 to 3 logs within 2 weeks of storage at 7.2°C in both meat control treatments without antimicrobials and in pork with 4% lactate-diacetate blend (LD). A 1-log increase was observed in turkey with 4% LD and Pork with 2% cultured dextrose-vinegar-rosemary (CDVR) under the same storage conditions; a 1-log increase was observed in turkey with CDVR at 4 weeks. The second set of experiments tested the effect of pH reduction (to less than 5.5 by the addition of 0.5% citric acid) in combination with 2% CDVR when added to the brine precook or postcook during shredding. Populations of L. monocytogenes increased 4-log within 2 and 4 weeks at 7.2°C for the control turkey and pork formulations, respectively. No growth was observed in 12 weeks for any antimicrobial CDVR-CA treatments regardless of how antimicrobial was added. Similarly, botulinum toxin was detected in both control treatments at week 2 at 12.8°C, but no toxicity was observed in either antimicrobial treatment through 12 weeks. These data suggest that a combination of 2% cultured dextrose-vinegar-rosemary extract plus 0.5% citric acid to reduce pH inhibits the growth of L. monocytogenes and toxin production of C. botulinum in uncured shredded turkey and pork products stored under mild temperature abuse conditions for up to 12 weeks in reduced oxygen packaging.

The 2021 FSIS Stabilization Guidelines for Meat and Poultry Products (Appendix B) Option 1.2 limits Phase 1 cooling from 48.8 to 26.7 degrees C in uncured meats to 1 h. However, this time restriction is impractical to achieve in large-diameter whole-muscle products. The objective of this study was to compare the inhibitory effect of commercial dry vinegars (DVs) and cultured sugar-vinegar blends (CSVs) on Clostridium perfringens and Bacillus cereus in uncured beef and poultry products during extended cooling. Treatments (beef: 72-73% moisture, pH 6.2-6.3, 0.85-0.95% NaCl; turkey: 76-77% moisture, pH 6.5-6.7, 1.3-1.6% NaCl) included Controls without antimicrobials, and four DV and four CSV, each tested at 0.75 and 1.25%. Batches were inoculated with 2.5-log C. perfringens or B. cereus spores, vacuum-packaged, and cooked to 73 degrees C. Packages were cooled from 48.8 to 27 degrees C (Phase 1) in 3, 4, or 5 h; Phase 2 (27-12.8 degrees C) and Phase 3 (12.8-4 degrees C) were standardized for 5-h cooling each. Pathogens were enumerated on selective agar in triplicate samples assayed at precook, postcook, and at the end of Phase 1, 2, and 3 cooling. Experiments were conducted twice. B. cereus did not grow (4.5 log) in Control treatments when Phase 1 cooling was extended to >= 3 h. All 1.25% DV ingredients limited C. perfringens growth to <= 1-log when Phase 1 cooling was extended to 3 h but supported a >1-log increase when Phase 1 cooling was extended to 5 h. All 1.25% CSV inhibited growth under 3-h Phase 1 cooling; 1.25% CSV-A and >= 0.75% CSV-D inhibited growth in turkey during 5-h Phase 1 cooling, but inhibition with 1.25% CSV-C was inconsistent in beef. This study revealed that formulating uncured meats with 1.25% DV or certain CSV can extend Phase 1 cooling to 3 h. Although all ingredients inhibited growth when used at 0.75% or greater compared to a control, greater variability of inhibition was observed among CSV than for DV.