Characterisation of non-toxigenic Clostridium spp. strains, to use as surrogates for non-proteolytic Clostridium botulinum in chilled food challenge testing

Parker, Moira D.; Barrett, Paul; Shepherd, Jill; Price, Laura; Bull, S.D.

doi:10.1016/j.mimet.2014.11.011

Cited by 15 publications

(12 citation statements)

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“…The diversity within BoNT-producing Clostridia should be considered when studying these microbes. For example, appropriate strains must be chosen for conducting microbiological challenge tests used for evaluating the risk of BoNT-producing Clostridia or botulinum neurotoxins contaminating food [ 101 – 103 ].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2016

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BackgroundClostridium 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.ResultsComparative 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2502-z) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2016

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“…The developed qPCR method is interesting for studies of both naturally non-toxigenic C. botulinum isolates and mutants where botulinum neurotoxin genes have been removed. This is relevant, as an increasing number of such isolates has been described within the last few years (Brunt et al, 2020;Clauwers et al, 2016;Gonzalez-Angulo et al, 2020;Parker et al, 2015).…”

Section: Qpcr Enumeration Of Cb-mixmentioning

confidence: 99%

Extensive growth and growth boundary model for non-proteolytic Clostridium botulinum – Evaluation and validation with MAP and smoked foods

2022

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“…However, a suitable validated surrogate for gIICb has been lacking to date. Recently, Parker et al (19) studied previously isolated natural nontoxigenic Clostridium species that could be used as surrogates for gIICb. Two out of the three strains analyzed showed equal or faster growth than toxigenic gIICb under most (but not all) stress conditions (low temperature, reduced pH, and water activity [a w ]), making them potentially useful for challenge studies with refrigerated foods, at least under some conditions.…”

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Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

Clauwers

Vanoirbeek

Delbrassinne

et al. 2016

Appl Environ Microbiol

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Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia. IMPORTANCEThe nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia. Botulism, caused by the botulinum neurotoxin (BoNT) produced by Clostridium botulinum, is a rare but severe paralytic illness in humans and animals. Botulinum toxins are 150-kDa proteins with zinc endopeptidase activity, consisting of two subunits, a 100-kDa heavy chain and a 50-kDa light chain. The heavy chain is responsible for binding and translocation of the light chain into the cytosol of neuronal cells, whereas the light chain cleaves SNARE proteins that are involved in the docking of acetylcholine-containing vesicles and fusion to the presynaptic membrane. When the SNARE proteins are cleaved, neurotransmitter release is inhibited, leading to the paralysis of the corresponding muscle (1, 2).C. botulinum is a strictly anaerobic bacterium that thrives in decaying organic matter in soils and sediments of ponds, lakes, and oceans. It also forms dormant endospores that are highly resilient to hostile conditions and therefore can be found widespread in the environment. These spores may contaminate foods via raw materials or other sources, possibly leading to food-borne botuli...

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Characterisation of non-toxigenic Clostridium spp. strains, to use as surrogates for non-proteolytic Clostridium botulinum in chilled food challenge testing

Cited by 15 publications

References 50 publications

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

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

Extensive growth and growth boundary model for non-proteolytic Clostridium botulinum – Evaluation and validation with MAP and smoked foods

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

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