Michael W. Peck

Biology and Genomic Analysis of Clostridium botulinum

The ability to form botulinum neurotoxin is restricted to six phylogenetically and physiologically distinct bacteria (Clostridium botulinum Groups I-IV and some strains of C. baratii and C. butyricum). The botulinum neurotoxin is the most potent toxin known, with as little as 30-100 ng potentially fatal, and is responsible for botulism, a severe neuroparalytic disease that affects humans, animals, and birds. In order to minimize the hazards presented by the botulinum neurotoxin-forming clostridia, it is necessary to extend understanding of the biology of these bacteria. Analyses of recently available genome sequences in conjunction with studies of bacterial physiology are beginning to reveal new and exciting information on the biology of these dangerous bacteria. At the whole organism level, substantial differences between the six botulinum neurotoxin-forming clostridia have been reported. For example, the genomes of proteolytic C. botulinum (C. botulinum Group I) and non-proteolytic C. botulinum (C. botulinum Group II) are highly diverged and show neither synteny nor homology. It has also emerged that the botulinum neurotoxin-forming clostridia are not overtly pathogenic (unlike C. difficile), but saprophytic bacteria that use the neurotoxin to kill a host and create a source of nutrients. One important feature that has contributed to the success of botulinum neurotoxin-forming clostridia is their ability to form highly resistant endospores. The spores, however, also present an opportunity to control these bacteria if escape from lag phase (and hence growth) can be prevented. This is dependent on extending understanding of the biology of these processes. Differences in the genetics and physiology of spore germination in proteolytic C. botulinum and non-proteolytic C. botulinum have been identified. The biological variability in lag phase and its stages has been described for individual spores, and it has been shown that various adverse treatments extend different stages of lag phase. For example, heat treatment primarily extended germination, while incubation at a chilled temperature primarily extended outgrowth. The neurotoxin gene is present within a cluster of associated genes, and can be located on the chromosome, a plasmid or a bacteriophage. Two basic types of neurotoxin cluster have been identified. Evolution of the neurotoxin gene and cluster has occurred independently of the organism, and involved a series of recombination events but is still poorly understood. Factors affecting the regulation of neurotoxin formation also remain poorly understood, and will be the focus of much future research.

Modelling the growth, survival and death of microorganisms in foods: the UK Food Micromodel approach

Techniques for the development of mathematical models in the area of predictive microbiology have greatly improved recently, allowing better and more accurate descriptions of microbial responses to particular environmental conditions, thus enabling predictions of those responses to be made with greater confidence. Recognising the potential value of applying these techniques in the food industry, the Ministry of Agriculture, Fisheries and Food (MAFF) initiated a nationally coordinated five-year programme of research into the growth and survival of microorganisms in foods, with the aim of developing a computerised Predictive Microbiology Database in the UK. This initiative has resulted in the systematic generation of data, through protocols which ensure consistency of methodology, so that data in the database are truly comparable and compatible, and lead to reliable predictive models. The approaches taken by scientists involved in this programme are described and the various stages in the development of mathematical models summarized. It is hoped that this initiative and others being developed in the USA, Australia, Canada and other countries, will encourage a more integrated approach to food safety which will influence all stages of food production and, eventually, result in the development of an International Predictive Microbiology Database.

Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

BackgroundProteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray.ResultsWhole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI.ConclusionProteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks.

Clostridium botulinum and the safety of minimally heated, chilled foods: an emerging issue?

Foodborne botulism is caused by consumption of preformed botulinum neurotoxin, with as little as 30 ng of neurotoxin being potentially lethal. Consumption of minute quantities of neurotoxin‐containing food can result in botulism. In view of the severity of foodborne botulism, it is essential that new foods be developed safely without an increase in incidence of this disease. Minimally heated, chilled foods are a relatively new type of food, sales of which are currently increasing by about 10% per annum. These products meet consumer demand for high‐quality foods that require little preparation time. Their safety and quality depends on mild heat treatment, chilled storage, restricted shelf life and sometimes on intrinsic properties of the foods. The principal microbiological hazard is nonproteolytic Clostridium botulinum, and there is a concern that this may become an emerging issue. A considerable amount of research and development over the last 15 years has underpinned the safe production of commercial, minimally heated, chilled foods with respect to foodborne botulism, and it is essential that safe food continues to be developed. In particular, the desire to use lighter heat processes and a longer shelf life presents a challenge that will only be met by significant developments in quantitative microbiological food safety.

Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature.

The effect of combinations of temperature (2°, 3°, 4°, 5°, 8° and 10°C), pH (5·0–7·2) and NaCl (0·1–5·0% w/w) on growth from spores of non‐proteolytic Clostridium botulinum types B, E and F was determined using a strictly anaerobic medium. Inoculated media were observed weekly for turbidity, and tests were made for the presence of toxin in conditions that approached the limits of growth. Growth and toxin production were detected at 3°C in 5 weeks, at 4°C in 3/4 weeks and at 5°C in 2/3 weeks. The resulting data define growth/no growth boundaries with respect to low temperature, pH, NaCl and incubation time. This is important in assessment of the risk of growth and toxin production by non‐proteolytic Cl. botulinum in minimally processed chilled foods.

Predictive model of the effect of CO2, pH, temperature and NaCl on the growth of Listeria monocytogenes

The growth responses of L. monocytogenes as affected by CO2 concentration (0-100% v/v, balance nitrogen), NaCl concentration (0.5-8.0% w/v), pH (4.5-7.0) and temperature (4-20 degrees C) were studied in laboratory medium. Growth curves were fitted using the model of Baranyi and Roberts, and specific growth rates derived from the curve fit were modelled. Predictions for specific growth rate, doubling time and time to a 1000-fold increase could be made for any combination of conditions within the matrix. Predictions of growth from the model were compared with published data and this showed the model to be suitable for predicting growth of L. monocytogenes in a range of foods packaged under a modified atmosphere.

Clostridium botulinum and the safety of refrigerated processed foods of extended durability

Refrigerated processed foods of extended durability (e.g. sousvide foods) rely on a mild heat treatment followed by storage at chill temperature for safety and preservation. The principal microbiological hazard in such foods is growth of nonproteolytic Clostridium botulinum . Recent research has identified combinations of mild heat treatment and subsequent refrigerated storage that, when combined with a specified shelf life, provide a defined safety margin with respect to nonproteolytic C. botulinum . This article discusses microbiological concerns associated with such minimally processed foods, describes recent studies of factors affecting the survival and growth of non-proteolytic C. botulinum , and focuses on the use of combination processes to ensure product safety.

Clostridium botulinum in the post-genomic era

Foodborne botulism is a severe neuroparalytic disease caused by consumption of botulinum neurotoxin formed by strains of proteolytic Clostridium botulinum and non-proteolytic C. botulinum during their growth in food. The botulinum neurotoxin is the most potent substance known, with as little as 30-100 ng potentially fatal, and consumption of just a few milligrams of neurotoxin-containing food is likely to be sufficient to cause illness and potentially death. In order to minimise the foodborne botulism hazard, it is necessary to extend understanding of the biology of these bacteria. This process has been recently advanced by genome sequencing and subsequent analysis. In addition to neurotoxin formation, endospore formation is also critical to the success of proteolytic C. botulinum and non-proteolytic C. botulinum as foodborne pathogens. The endospores are highly resistant, and enable survival of adverse treatments such as heating. To better control the botulinum neurotoxin-forming clostridia, it is important to understand spore resistance mechanisms, and the physiological processes involved in germination and lag phase during recovery from this dormant state.

Research on factors allowing a risk assessment of spore-forming pathogenic bacteria in cooked chilled foods containing vegetables: a FAIR collaborative project

Vegetables are frequent ingredients of cooked chilled foods and are frequently contaminated with spore-forming bacteria (SFB). Therefore, risk assessment studies have been carried out, including the following: hazard identification and characterisation--from an extensive literature review and expertise of the participants, B. cereus and C. botulinum were identified as the main hazards; exposure assessment--consisting of determination of the prevalence of hazardous SFB in cooked chilled foods containing vegetables and in unprocessed vegetables, and identification of SFB representative of the bacterial community in cooked chilled foods containing vegetables, determination of heat-resistance parameters and factors affecting heat resistance of SFB, determination of the growth kinetics of SFB in vegetable substrate and of the influence of controlling factors, validation of previous work in complex food systems and by challenge testing and information about process and storage conditions of cooked chilled foods containing vegetables. The paper illustrates some original results obtained in the course of the project. The results and information collected from scientific literature or from the expertise of the participants are integrated into the microbial risk assessment, using both a Bayesian belief network approach and a process risk model approach, previously applied to other foodborne hazards.

Predictive model of the effect of temperature, pH and sodium chloride on growth from spores of non-proteolytic Clostridium botulinum

Non-proteolytic strains of Clostridium botulinum are capable of growth at chill temperatures and thus pose a potential hazard in minimally-processed chilled foods. The combined effect of pH (5.0-7.3), NaCl concentration (0.1-5.0%) and temperature (4-30 degrees C) on growth of non-proteolytic C. botulinum in laboratory media was studied. Growth curves at various combinations of pH, NaCl concentration and temperature were fitted by the Gompertz and Baranyi models, and parameters derived from the curve-fit were modelled. Predictions of growth from the models were compared with data in the literature and this showed them to be suitable for use with fish, meat and poultry products. This model should contribute to ensuring the safety of minimally-processed foods with respect to non-proteolytic C. botulinum.