Synergistic interaction between pH and NaCl in the limits of germination and outgrowth of Clostridium sporogenes and Group I Clostridium botulinum vegetative cells and spores after heat treatment.

Group I Clostridium botulinum and Clostridium sporogenes are physiologically and genetically closely related. Both are widely distributed in the environment and can cause foodborne botulism. In this work, a physiological study was conducted with 37 isolates from spoiled canned food and five referenced strains of C. sporogenes (three isolates) and Group I C. botulinum (two isolates). Growth limits of vegetative cells were established as a function of pH and NaCl concentration in PYG modified medium (PYGm) at 30 °C for 48 days. The heat resistance of the spores was studied for 2 min and 10 min at 102 °C and 110 °C. This physiological study (pH, NaCl growth limits and heat resistance) allowed the selection of 14 isolates of C. sporogenes (twelve isolates) and Group I C. botulinum (two isolates) representative of the diversity found. This panel of 14 selected isolates (11 isolated from spoiled canned food and three reference strains), were whole genome sequenced, but no association of physiological and genetic characteristics could be detected. Finally, we studied the ability of spores to germinate and grow from 5 isolates (four C. sporogenes and one Group I C. botulinum), under stress conditions generated by pH and NaCl following a low intensity heat treatment. The accumulation of these 3 stresses creates synergies that will strongly reduce the probability of spore growth in pH and salt conditions where they usually proliferate. The effect is progressive as the conditions become drastic: the number of decimal reduction observed increases translating a probability of growth which decreases. This study provides a better understanding of the behaviour of C. sporogenes and Group I C. botulinum isolates and shows how the combination of pH, NaCl and heat treatment can help prevent or minimise foodborne botulism outbreaks.

Diversity of the Genomes and Neurotoxins of Strains of Clostridium botulinum Group I and Clostridium sporogenes Associated with Foodborne, Infant and Wound Botulism.

Clostridium botulinum Group I and Clostridium sporogenes are closely related bacteria responsible for foodborne, infant and wound botulism. A comparative genomic study with 556 highly diverse strains of C. botulinum Group I and C. sporogenes (including 417 newly sequenced strains) has been carried out to characterise the genetic diversity and spread of these bacteria and their neurotoxin genes. Core genome single-nucleotide polymorphism (SNP) analysis revealed two major lineages; C. botulinum Group I (most strains possessed botulinum neurotoxin gene(s) of types A, B and/or F) and C. sporogenes (some strains possessed a type B botulinum neurotoxin gene). Both lineages contained strains responsible for foodborne, infant and wound botulism. A new C. sporogenes cluster was identified that included five strains with a gene encoding botulinum neurotoxin sub-type B1. There was significant evidence of horizontal transfer of botulinum neurotoxin genes between distantly related bacteria. Population structure/diversity have been characterised, and novel associations discovered between whole genome lineage, botulinum neurotoxin sub-type variant, epidemiological links to foodborne, infant and wound botulism, and geographic origin. The impact of genomic and physiological variability on the botulism risk has been assessed. The genome sequences are a valuable resource for future research (e.g., pathogen biology, evolution of C. botulinum and its neurotoxin genes, improved pathogen detection and discrimination), and support enhanced risk assessments and the prevention of botulism.

Assessment of the risk of botulism from chilled, vacuum/modified atmosphere packed fresh beef, lamb and pork held at 3 °C-8 °C.

The safety of current UK industry practice (including shelf-life) for chilled, vacuum/modified atmosphere-packed fresh red meat (beef, lamb and pork) held at 3°C-8°C has been evaluated with respect to non-proteolytic Clostridium botulinum. UK industry typically applies a retail pack shelf-life at 3°C-8°C to 13 days for fresh red meat, with a maximum of 23 days for beef, 27 days for lamb, and 18 days for pork. An exposure assessment established that current commercial practice for fresh red meat provided strong protection with more than 1010 person servings marketed in the UK without association with foodborne botulism. A challenge test demonstrated that spores of non-proteolytic C. botulinum inoculated on chilled vacuum-packed fresh red meat did not lead to detectable neurotoxin at day 50 for beef, day 35 for lamb, or day 25 for pork (i.e. <40 pg type B toxin and type E toxin g-1 of meat). The products were visually spoiled many days before these end points. The exposure assessment and challenge test demonstrated the safety of current UK industry practices for the shelf-life of fresh, vacuum-packed beef, lamb and pork held at 3°C-8°C with respect to C. botulinum, and that botulinum neurotoxin was not detected within their organoleptic shelf-life.

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

The neurotoxin formed by Clostridium botulinum Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of C. botulinum Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse C. botulinum Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages-one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of C. botulinum and its neurotoxin genes, dissecting key aspects of C. botulinum Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.

Risk presented to minimally processed chilled foods by psychrotrophic Bacillus cereus.

BACKGROUND: Spores of psychrotrophic Bacillus cereus may survive the mild heat treatments given to minimally processed chilled foods. Subsequent germination and cell multiplication during refrigerated storage may lead to bacterial concentrations that are hazardous to health. SCOPE AND APPROACH: This review is concerned with the characterisation of factors that prevent psychrotrophic B. cereus reaching hazardous concentrations in minimally processed chilled foods and associated foodborne illness. A risk assessment framework is used to quantify the risk associated with B. cereus and minimally processed chilled foods. KEY FINDINGS AND CONCLUSIONS: Bacillus cereus is responsible for two types of food poisoning, diarrhoeal (an infection) and emetic (an intoxication); however, no reported outbreaks of food poisoning have been associated with B. cereus and correctly stored commercially-produced minimally processed chilled foods. In the UK alone, more than 1010 packs of these foods have been sold in recent years without reported illness, thus the risk presented is very low. Further quantification of the risk is merited, and this requires additional data. The lack of association between diarrhoeal food poisoning and correctly stored commercially-produced minimally processed chilled foods indicates that an infectious dose has not been reached. This may reflect low pathogenicity of psychrotrophic strains. The lack of reported association of psychrotrophic B. cereus with emetic illness and correctly stored commercially-produced minimally processed chilled foods indicates that a toxic dose of the emetic toxin has not been formed. Laboratory studies show that strains form very small quantities of emetic toxin at chilled temperatures.

The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II.

Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca2+-DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II.

Identification of a novel botulinum neurotoxin gene cluster in Enterococcus.

The deadly neurotoxins of Clostridium botulinum (BoNTs) comprise eight serotypes (A-G; X). The neurotoxin gene cluster encoding BoNT and its accessory proteins includes an operon containing an ntnh gene upstream of the boNT gene. Another operon contains either ha (haemagglutinin) or orfX genes (of unknown function). Here we describe a novel boNT gene cluster from Enterococcus sp. 3G1_DIV0629, with a typical ntnh gene and an uncommon orfX arrangement. The neurotoxin (designated putative eBoNT/J) contains a metallopeptidase zinc-binding site, a translocation domain and a target cell attachment domain. Structural properties of the latter suggest a novel targeting mechanism with consequent implications for application by the pharmaceutical industry. This is the first complete boNT gene cluster identified in a non-clostridial genome.

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes.

The Gram-positive spore-forming anaerobe Clostridium sporogenes is a significant cause of food spoilage, and it is also used as a surrogate for C. botulinum spores for testing the efficacy of commercial sterilization. C. sporogenes spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of C. sporogenes spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual C. sporogenes wild-type and germination mutant spores. Most individual C. sporogenes spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at T1, all transitioned to rapid CaDPA release at Tlag1, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlys. T1, Tlag1, Trelease, and Tlys times for individual spores were heterogeneous, but ΔTrelease (Trelease - Tlag1) periods were relatively constant. However, variability in T1 (or Tlag1) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After Trelease, some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25-30% at Tlag2 prior to Tlys. This has not been seen with spores of other species. Almost all C. sporogenes spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a Tlag2 in L-alanine germination. Sublethal heat treatment potentiated C. sporogenes spore germination with L-alanine, primarily by shortening T1 times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, C. sporogenes spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in Bacillus spore germination.

Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature.

Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.

An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production.

Clostridium botulinum produces botulinum neurotoxins (BoNTs), highly potent substances responsible for botulism. Currently, mathematical models of C. botulinum growth and toxigenesis are largely aimed at risk assessment and do not include explicit genetic information beyond group level but integrate many component processes, such as signalling, membrane permeability and metabolic activity. In this paper we present a scheme for modelling neurotoxin production in C. botulinum Group I type A1, based on the integration of diverse information coming from experimental results available in the literature. Experiments show that production of BoNTs depends on the growth-phase and is under the control of positive and negative regulatory elements at the intracellular level. Toxins are released as large protein complexes and are associated with non-toxic components. Here, we systematically review and integrate those regulatory elements previously described in the literature for C. botulinum Group I type A1 into a population dynamics model, to build the very first computational model of toxin production at the molecular level. We conduct a validation of our model against several items of published experimental data for different wild type and mutant strains of C. botulinum Group I type A1. The result of this process underscores the potential of mathematical modelling at the cellular level, as a means of creating opportunities in developing new strategies that could be used to prevent botulism; and potentially contribute to improved methods for the production of toxin that is used for therapeutics.

Diversity of the Germination Apparatus in Clostridium botulinum Groups I, II, III, and IV.

Clostridium botulinum is a highly dangerous pathogen that forms very resistant endospores that are ubiquitous in the environment, and which, under favorable conditions germinate to produce vegetative cells that multiply and form the exceptionally potent botulinum neurotoxin. To improve the control of botulinum neurotoxin-forming clostridia, it is important to understand the mechanisms involved in spore germination. Here we present models for spore germination in C. botulinum based on comparative genomics analyses, with C. botulinum Groups I and III sharing similar pathways, which differ from those proposed for C. botulinum Groups II and IV. All spores germinate in response to amino acids interacting with a germinant receptor, with four types of germinant receptor identified [encoded by various combinations of gerA, gerB, and gerC genes (gerX)]. There are three gene clusters with an ABC-like configuration; ABC [gerX1], ABABCB [gerX2] and ACxBBB [gerX4], and a single CA-B [gerX3] gene cluster. Subtypes have been identified for most germinant receptor types, and the individual GerX subunits of each cluster show similar grouping in phylogenetic trees. C. botulinum Group I contained the largest variety of gerX subtypes, with three gerX1, three gerX2, and one gerX3 subtypes, while C. botulinum Group III contained two gerX1 types and one gerX4. C. botulinum Groups II and IV contained a single germinant receptor, gerX3 and gerX1, respectively. It is likely that all four C. botulinum Groups include a SpoVA channel involved in dipicolinic acid release. The cortex-lytic enzymes present in C. botulinum Groups I and III appear to be CwlJ and SleB, while in C. botulinum Groups II and IV, SleC appears to be important.

Systematic Assessment of Nonproteolytic Clostridium botulinum Spores for Heat Resistance.

UNLABELLED: Heat treatment is an important controlling factor that, in combination with other hurdles (e.g., pH, aw), is used to reduce numbers and prevent the growth of and associated neurotoxin formation by nonproteolytic C. botulinum in chilled foods. It is generally agreed that a heating process that reduces the spore concentration by a factor of 10(6) is an acceptable barrier in relation to this hazard. The purposes of the present study were to review the available data relating to heat resistance properties of nonproteolytic C. botulinum spores and to obtain an appropriate representation of parameter values suitable for use in quantitative microbial risk assessment. In total, 753 D values and 436 z values were extracted from the literature and reveal significant differences in spore heat resistance properties, particularly those corresponding to recovery in the presence or absence of lysozyme. A total of 503 D and 338 z values collected for heating temperatures at or below 83°C were used to obtain a probability distribution representing variability in spore heat resistance for strains recovered in media that did not contain lysozyme. IMPORTANCE: In total, 753 D values and 436 z values extracted from literature sources reveal significant differences in spore heat resistance properties. On the basis of collected data, two z values have been identified, z = 7°C and z = 9°C, for spores recovered without and with lysozyme, respectively. The findings support the use of heat treatment at 90°C for 10 min to reduce the spore concentration by a factor of 10(6), providing that lysozyme is not present during recovery. This study indicates that greater heat treatment is required for food products containing lysozyme, and this might require consideration of alternative recommendation/guidance. In addition, the data set has been used to test hypotheses regarding the dependence of spore heat resistance on the toxin type and strain, on the heating technique used, and on the method of D value determination used.

Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts.

Analysis of more than 150 Clostridium botulinum Group II type E genomes identified a small fraction (6%) where neurotoxin-encoding genes were located on plasmids. Seven closely related (134-144 kb) neurotoxigenic plasmids of subtypes E1, E3, and E10 were characterized; all carried genes associated with plasmid mobility via conjugation. Each plasmid contained the same 24-kb neurotoxin cluster cassette (six neurotoxin cluster and six flanking genes) that had split a helicase gene, rather than the more common chromosomal rarA. The neurotoxin cluster cassettes had evolved as separate genetic units which had either exited their chromosomal rarA locus in a series of parallel events, inserting into the plasmid-borne helicase gene, or vice versa. A single intact version of the helicase gene was discovered on a nonneurotoxigenic form of this plasmid. The observed low frequency for the plasmid location may reflect one or more of the following: 1) Less efficient recombination mechanism for the helicase gene target, 2) lack of suitable target plasmids, and 3) loss of neurotoxigenic plasmids. Type E1 and E10 plasmids possessed a Clustered Regularly Interspaced Short Palindromic Repeats locus with spacers that recognized C. botulinum Group II plasmids, but not C. botulinum Group I plasmids, demonstrating their long-term separation. Clostridium botulinum Group II type E strains also carry nonneurotoxigenic plasmids closely related to C. botulinum Group II types B and F plasmids. Here, the absence of neurotoxin cassettes may be because recombination requires both a specific mechanism and specific target sequence, which are rarely found together.

Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials.

We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg(-1). Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments.

Impact of Clostridium botulinum genomic diversity on food safety.

The deadly botulinum neurotoxin formed by Clostridium botulinum is the causative agent of foodborne botulism. The increasing availability of C. botulinum genome sequences is starting to allow the genomic diversity of C. botulinum Groups I and II and their neurotoxins to be characterised. This information will impact on microbiological food safety through improved surveillance and tracing/tracking during outbreaks, and a better characterisation of C. botulinum Groups I and II, including the risk presented, and new insights into their biology, food chain transmission, and evolution.

New Elements To Consider When Modeling the Hazards Associated with Botulinum Neurotoxin in Food.

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most potent biological substances known to mankind. BoNTs are the agents responsible for botulism, a rare condition affecting the neuromuscular junction and causing a spectrum of diseases ranging from mild cranial nerve palsies to acute respiratory failure and death. BoNTs are a potential biowarfare threat and a public health hazard, since outbreaks of foodborne botulism are caused by the ingestion of preformed BoNTs in food. Currently, mathematical models relating to the hazards associated with C. botulinum, which are largely empirical, make major contributions to botulinum risk assessment. Evaluated using statistical techniques, these models simulate the response of the bacterium to environmental conditions. Though empirical models have been successfully incorporated into risk assessments to support food safety decision making, this process includes significant uncertainties so that relevant decision making is frequently conservative and inflexible. Progression involves encoding into the models cellular processes at a molecular level, especially the details of the genetic and molecular machinery. This addition drives the connection between biological mechanisms and botulism risk assessment and hazard management strategies. This review brings together elements currently described in the literature that will be useful in building quantitative models of C. botulinum neurotoxin production. Subsequently, it outlines how the established form of modeling could be extended to include these new elements. Ultimately, this can offer further contributions to risk assessments to support food safety decision making.

The pattern of growth observed for Clostridium botulinum type A1 strain ATCC 19397 is influenced by nutritional status and quorum sensing: a modelling perspective.

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most poisonous substances known to mankind. However, toxin regulation and signals triggering synthesis as well as the regulatory network and actors controlling toxin production are unknown. Experiments show that the neurotoxin gene is growth phase dependent for C. botulinum type A1 strain ATCC 19397, and toxin production is influenced both by culture conditions and nutritional status of the medium. Building mathematical models to describe the genetic and molecular machinery that drives the synthesis and release of BoNT requires a simultaneous description of the growth of the bacterium in culture. Here, we show four plausible modelling options which could be considered when constructing models describing the pattern of growth observed in a botulinum growth medium. Commonly used bacterial growth models are unsuitable to fit the pattern of growth observed, since they only include monotonic growth behaviour. We find that a model that includes both the nutritional status and the ability of the cells to sense their surroundings in a quorum-sensing manner is most successful at explaining the pattern of growth obtained for C. botulinum type A1 strain ATCC 19397.

Apertures in the Clostridium sporogenes spore coat and exosporium align to facilitate emergence of the vegetative cell.

Clostridium sporogenes forms highly heat resistant endospores, enabling this bacterium to survive adverse conditions. Subsequently, spores may germinate, giving rise to vegetative cells that multiply and lead to food spoilage. Electron microscopy was used to visualise changes in spore structures during germination, emergence and outgrowth. C. sporogenes spores were surrounded by an exosporium that was oval in shape and typically 3 µm in length. An aperture of 0.3-0.4 µm was observed at one end of the exosporium. The rupture of the spore coats occurs adjacent to the opening in the exosporium. The germinated cell emerges through this hole in the spore coat and then through the pre-existing aperture in the exosporium, before eventually being released, leaving behind a largely intact exosporium with an enlarged aperture (0.7-1.0 µm) and coat shell. The formation of this aperture, its function and its alignment with the spore coat is discussed.

A possible route for foodborne transmission of Clostridium difficile?

Spores of toxigenic Clostridium difficile and spores of food-poisoning strains of Clostridium perfringens show a similar prevalence in meats. Spores of both species are heat resistant and can survive cooking of foods. C. perfringens is a major cause of foodborne illness; studies are needed to determine whether C. difficile transmission by a similar route is a cause of infection.

Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II.

Recent developments in whole genome sequencing have made a substantial contribution to understanding the genomes, neurotoxins and biology of Clostridium botulinum Group I (proteolytic C. botulinum) and C. botulinum Group II (non-proteolytic C. botulinum). Two different approaches are used to study genomics in these bacteria; comparative whole genome microarrays and direct comparison of complete genome DNA sequences. The properties of the different types of neurotoxin formed, and different neurotoxin gene clusters found in C. botulinum Groups I and II are explored. Specific examples of botulinum neurotoxin genes are chosen for an in-depth discussion of neurotoxin gene evolution. The most recent cases of foodborne botulism are summarised.