Spore germination of the psychrotolerant, red meat spoiler, Clostridium frigidicarnis.

AIMS: To determine germination triggers of Clostridium frigidicarnis, an important spoilage bacterium of chilled vacuum-packed meat. METHODS AND RESULTS: Germination of Cl. frigidicarnis spores in the presence of a range of potential nutrient and non-nutrient germinants was tested by monitoring the fall in optical density and by phase-contrast microscopy. The amino acid L-valine induced strong germination when paired with L-lactate in sodium phosphate under anaerobic conditions. Several other amino acids promoted germination when paired with L-lactate in sodium phosphate and the co-germinants NaHCO3 and L-cysteine. Heat activation, while not necessary for germination, increased the rate of germination. Spore germination was not observed when spores were incubated aerobically. CONCLUSIONS: Spores of psychrotolerant Cl. frigidicarnis germinated in the presence of L-valine in combination with L-lactate in sodium phosphate buffer under anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Anaerobic conditions, L-valine and L-lactate, have been identified as triggering germination in Cl. frigidicarnis, and are all present in packs of fresh, vacuum-packaged, red meat. This new information adds to what is known about red meat spoilage by cold tolerant clostridia and can be used to develop intervention strategies to prevent meat spoilage.

Lag time variability in individual spores of Clostridium botulinum.

Quantifying lag times from individual spores and the associated variability is an important part of understanding the hazard associated with spore-forming pathogens such as Clostridium botulinum. Knowledge of the underlying distribution would allow greater refinement of risk assessments. To date most studies have either examined lag time indirectly by measuring time to growth or have only examined the first stage of lag, germination. Recent studies have attempted to quantify the variability of spores during the different stages of lag phase and to examine the relationships between these stages. The effect of incubation temperature (22 °C, 15 °C, 10 °C or 8 °C), heat treatment (unheated or 80 °C for 20 s) and sodium chloride concentration in both the sporulation medium (0 or 3% w/v) or growth medium (0 or 2% w/v) on growth from individual spores has been examined. These studies found spores within a single population are very heterogeneous with large variability in all stages of lag. The duration and variability of times for germination, outgrowth and first doubling depended on both the historic treatment of the spores and the prevailing growth conditions, and the stage of lag most affected was treatment dependant.

Quantitative risk assessment for hazards that arise from non-proteolytic Clostridium botulinum in minimally processed chilled dairy-based foods.

A modular process risk model has been constructed that describes the manufacture of dairy dessert products and hazards that arise from non-proteolytic Clostridium botulinum. The model describes batch manufacture and consumer storage of a family size generic dairy dessert but includes a realistic quantification that could apply to a specific food product. The dairy dessert sector is an expanding part of the UK market. The model includes modules that describe spore loads in raw materials, spore inactivation during thermal processing, volume partition and the population kinetics for non-proteolytic C. botulinum during sequential isothermal storage regimes. Where possible elements of uncertainty and variability are identified explicitly. The model is constructed as a belief network from published data and expert opinions. The model provides marginal probabilities, and associated sensitivities, for a range of endpoint measures centred on the toxicity of a single retail unit after an extended period of storage. The decimal reduction time for non-proteolytic C. botulinum spore populations at the highest (hold) temperature of the primary thermal process and the highest temperature experienced during poorly controlled (consumer) storage are dominant factors determining risks. Priorities for additional information to support risk assessments have been identified.

Modelling the growth of Clostridium perfringens during the cooling of bulk meat.

A dynamic predictive model was developed to describe the effects of temperature, pH and NaCl concentration on the growth of Clostridium perfringens type A. The model for the specific growth rate was based on 81 growth curves generated in our laboratory or obtained from the publicly available ComBase database. Growth curves obtained during cooling were fitted with the dynamic model of Baranyi and Roberts. This made it possible to determine the parameter value reflecting the physiological state of C. perfringens after heating profiles typically applied to bulk meat. The model with the obtained parameters provided a good description of growth of C. perfringens in 24 heating/cooling curves generated specifically for this work (various non-isothermal treatments with a range of combinations of pH and NaCl concentration), and also for existing literature data. The dynamic model was implemented in Perfringens Predictor, a web-based application that can be accessed free of charge via www.combase.cc. It is anticipated that the use of this model and Perfringens Predictor will contribute to a reduction in the food poisoning incidence associated with C. perfringens.

The microbiological quality of hot water-washed broccoli florets and cut green beans.

AIMS: To determine the effect of hot water washing on the microbiological quality of cut broccoli florets and trimmed green beans. METHODS AND RESULTS: Broccoli florets and trimmed beans were washed for 90 s in tap water at either 20 degrees C or 52 degrees C and stored at 7 and 10 degrees C. The numbers of naturally occurring aerobic mesophilic organisms, Pseudomonas spp., Enterobacteriaceae, yeast and moulds and lactobacilli or lactic acid bacteria were enumerated at intervals for up to 2 weeks. The ability of Listeria monocytogenes, Bacillus cereus and Escherichia coli O157:H7 inoculated onto the tissue post heat treatment to survive or grow was also measured to mimic the effect of postprocess contamination. Using a hot wash treatment improved the initial appearance of the vegetables and resulted in a small, but significant, reduction in populations of all groups of endogenous flora measured. The number of yeast and moulds on the vegetables washed at 52 degrees C remained below the levels observed on the 20 degrees C washed vegetables throughout the observation period, but Pseudomonas spp., lactobacilli and Enterobacteriaceae were better able to grow on the hot-washed vegetables such that the counts at the end of storage were greater on hot-washed than ambient-washed vegetables. All three of the pathogens tested were better able to grow on hot-washed broccoli and beans than on equivalent product washed at 20 degrees C. CONCLUSIONS: Hot water washing can be used to control enzymic browning or yeast and moulds growth but it can also allow more rapid and extensive growth by pathogens and spoilage organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Reduced protection against growth by pathogens means that the hot wash treatment of vegetables should be used with caution and requires careful assessment of risk.

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.

Germination and growth from spores: variability and uncertainty in the assessment of food borne hazards.

We have developed a model for the variability of spore lag times and shown that variability has an important role in the quantitative assessment of risks associated with spore forming bacteria in food. The model includes two sequential independent delay times that contribute to the lag time for a single spore. We have shown that a population of variable spores also has a variable lag time, and we have emphasised the significance of this variability in quantitative representations of population dynamics for small populations. We have made a Bayesian estimate for the extent of the variability in spore lag times and made a comparison with direct microscopic observations of individual spores of nonproteolytic Clostridium botulinum. We conclude that Bayesian inference is a practical method for quantifying variability and hence a significant element in the development of quantitative risk assessments for hazards associated with spore forming bacteria.

Probabilistic representation of the exposure of consumers to Clostridium botulinum neurotoxin in a minimally processed potato product.

We have examined the potential of a well-specified, minimally processed potato product as a vehicle for the exposure of consumers to Clostridium botulinum neurotoxin. The product is a relatively simple combination of raw potato flakes, flour, starch and other minor ingredients and has an extended lifetime under refrigeration conditions. A combination of information and data, from a variety of sources that includes the manufacturer, has shown that the product is particularly safe with respect to non-proteolytic C. botulinum hazards. The model concentrates on a simple end point, the toxicity of an individual retail unit of the product at the point of consumer preparation, which is related to an individual risk. The probabilistic analysis was built using Bayesian Belief Network (BBN) techniques.

Prevalence of Clostridium species and behaviour of Clostridium botulinum in gnocchi, a REPFED of italian origin.

Sales and consumption of refrigerated processed foods of extended durability (REPFEDs) have increased many-fold in Europe over the last 10 years. The safety and quality of these convenient ready-to-eat foods relies on a combination of mild heat treatment and refrigerated storage, sometimes in combination with other hurdles such as mild preservative factors. The major hazard to the microbiological safety of these foods is Clostridium botulinum. This paper reports on the prevalence and behaviour of proteolytic C. botulinum and non-proteolytic C. botulinum in gnocchi, a potato-based REPFED of Italian origin. Attempts to isolate proteolytic C. botulinum and non-proteolytic C. botulinum from gnocchi and its ingredients were unsuccessful. Based on assessment of the adequacy of the methods used, it was estimated that for proteolytic C. botulinum there was < 25 spores/kg of gnocchi and < 70 spores/kg of ingredients. The total anaerobic microbial load of gnocchi and its ingredients was low, with an estimated 1 MPN/g in processed gnocchi. Most of the anaerobic flora was facultatively anaerobic. A few obligately anaerobic bacteria were isolated from gnocchi and its ingredients and belonged to different Clostridium species. The protection factor, number of decimal reductions in the probability of toxigenesis from a single spore, was determined for eight different gnocchi formulations by challenge test studies. For all gnocchi stored at 8 degrees C (as recommended by the manufacturer) or 12 degrees C (mild temperature abuse), growth and toxin production were not detected in 75 days. The protection factor was >4.2 for proteolytic C. botulinum, and >6.2 for non-proteolytic C. botulinum. When inoculated packs were stored at 20 degrees C (severe temperature abuse), toxin production in 75 days was prevented by the inclusion of 0.09% (w/w) sorbic acid (protection factors as above), however in the absence of sorbic acid the packs became toxic before the end of the intended shelf-life and the protection factors were lower. Providing sorbic acid (0.09% w/w) is included in the gnocchi, the safety margin would seem to be very large with respect to the foodborne botulism hazard.

Principles of some novel rapid dipstick methods for detection and characterization of verotoxigenic Escherichia coli.

AIMS: The verotoxigenic Escherichia coli (VTEC) serotype most commonly associated with verotoxin (VT) production is O157:H7, but other serotypes have also been implicated in food-borne illness. These serotypes exhibit much greater genetic and biochemical diversity than E. coli O157:H7, making screening for all VTEC difficult. Here we describe development and testing of novel multi-analyte antibody-based dipstick methods for presumptive detection of VTEC cells and VTs, including non-O157 serotypes. METHODS AND RESULTS: The dipsticks are formatted as paddle-style and lateral flow devices. Test materials included raw milk, minced beef, apple juice and salami, spiked with VTEC. Prototype paddle dipsticks gave 47 of 48 E. coli O157-positive samples correct, and, simultaneously, 27 of 31 O26-positive samples correct, across the four food types. Prototype lateral flow dipsticks gave 12 of 12 E. coli O157-positive milk samples correct and, simultaneously, 28 of 28 positive VT samples correct. CONCLUSIONS: This work demonstrates that simple and rapid detection of more than one VTEC characteristic (toxin production and type, serogroup) is possible in a single dipstick test device, directly from a food enrichment culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of simple easy-to-use rapid methods for simultaneous detection and preliminary characterization of VTEC will enable the risk presented by all VTEC to be more thoroughly assessed (e.g. in surveillance studies, outbreak investigations).

Variability in spore germination response by strains of proteolytic Clostridium botulinum types A, B and F.

AIMS: The objective of the study was to evaluate the variability of germination response of 10 strains of proteolytic Clostridium botulinum. METHODS AND RESULTS: An automated turbidometric method was used to follow the fall in optical density. Spores of proteolytic Cl. botulinum germinated in response to l-alanine alone, with rate and extent of germination increased by addition of l-lactate or bicarbonate ions. Other hydrophobic amino acids also triggered germination of spores of proteolytic Cl. botulinum but not AGFK and inosine, germinants for Bacillus subtilis or B. cereus. CONCLUSIONS: Unlike spores of nonproteolytic Cl. botulinum, all proteolytic Cl. botulinum germinate in hydrophobic l-amino acids without l-lactate. However, a great variability of response to germinant is evidenced between the species. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of a model strain to study germination of Cl. botulinum spores should consider the variability in sensitivity to germinants shown in this work. In particular, the sequenced strain ATCC 3502 may not be the most appropriate model for germination studies.

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions.

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.

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.

Thermal inactivation of Escherichia coli O157:H7.

Verotoxin-producing Escherichia coli O157:H7 is a cause of serious foodborne illness. It has a very small infectious dose and so it is vital to eliminate this pathogen from food. As heat treatment is the method of bacterial destruction most frequently used in food processing, accurate prediction of thermal death rates is necessary to achieve desired safety margins whilst minimizing processing. In most studies thermal inactivation has been described using first-order reaction kinetics and D-values. Whilst this approach does not seem justified on a theoretical basis, and may increase inaccuracy, there is no doubt that it is convenient and in many cases provides an adequate description of thermal death. A review of published data on the measured thermal inactivation of E. coli O157:H7 shows no strong evidence that a heat treatment of 70 degrees C for 2 min (or equivalent) fails to deliver a 6-decimal reduction in cell numbers.

A predictive model that describes the effect of prolonged heating at 70 to 90 degrees C and subsequent incubation at refrigeration temperatures on growth from spores and toxigenesis by nonproteolytic Clostridium botulinum in the presence of lysozyme.

Refrigerated processed foods of extended durability such as cook-chill and sous-vide foods rely on a minimal heat treatment at 70 to 95 degrees C and then storage at a refrigeration temperature for safety and preservation. These foods are not sterile and are intended to have an extended shelf life, often up to 42 days. The principal microbiological hazard in foods of this type is growth of and toxin production by nonproteolytic Clostridium botulinum. Lysozyme has been shown to increase the measured heat resistance of nonproteolytic C. botulinum spores. However, the heat treatment guidelines for prevention of risk of botulism in these products have not taken into consideration the effect of lysozyme, which can be present in many foods. In order to assess the botulism hazard, the effect of heat treatments at 70, 75, 80, 85, and 90 degrees C combined with refrigerated storage for up to 90 days on growth from 10(6) spores of nonproteolytic C. botulinum (types B, E, and F) in an anaerobic meat medium containing 2,400 U of lysozyme per ml (50 microg per ml) was studied. Provided that the storage temperature was no higher than 8 degrees C, the following heat treatments each prevented growth and toxin production during 90 days; 70 degrees C for >/=2,545 min, 75 degrees C for >/=463 min, 80 degrees C for >/=230 min, 85 degrees C for >/=84 min, and 90 degrees C for >/=33.5 min. A factorial experimental design allowed development of a predictive model that described the incubation time required before the first sample showed growth, as a function of heating temperature (70 to 90 degrees C), period of heat treatment (up to 2,545 min), and incubation temperature (5 to 25 degrees C). Predictions from the model provided a valid description of the data used to generate the model and agreed with observations made previously.

Growth from spores of nonproteolytic Clostridium botulinum in heat-treated vegetable juice.

Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80 degrees C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80 degrees C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation.

Oxygen sensitivity of heated cells of Escherichia coli O157:H7.

Following defined heat treatments (55 degrees C for 100 min, 50 degrees C for 5 min, 61 degrees C for l min), a 6 decimal (6-D) reduction was obtained when cells of Escherichia coli O157:H7 were enumerated in aerobic growth medium. Part of this reduction (3-D) was due to thermal inactivation (as determined when cells were enumerated in anaerobic growth medium), and part (3-D) was due to the inability of sub-lethally heat-injured cells of E. coli O157:H7 to grow in the presence of oxygen. When held anaerobically, the injured cells regained their ability to grow in the presence of oxygen. Following heating at 59 degrees C for 5 min, repair took 4 h at 30 degrees C, 48 h at 20 degrees C, 95 h at 10 degrees C, but did not occur in 816 at 5 degrees C. Recovery from sub-lethal heat injury was not influenced by heat shock. These findings are relevant to the safety of minimally-heated foods.

Investigation of the ability of proteolytic Clostridium botulinum to multiply and produce toxin in fresh Italian pasta.

The ability of proteolytic Clostridium botulinum (types A, B, and F) to produce toxin in filled fresh Italian pasta (tortelli) packed under a modified atmosphere was investigated. Four types of tortelli (filled with artichoke, meat, ricotta-spinach, or salmon) were inoculated with a suspension of heat-shocked spores to give an initial concentration of approximately 10(3) spores per piece. Samples were incubated at both 12 and 20 degrees C for up to 50 days and examined at selected time intervals for the presence of toxin by an ELISA and the mouse test. Toxin was not detected in any tortelli stored at 12 degrees C. When storage was at 20 degrees C, toxin was detected in the salmon-filled tortelli at day 30, in the meat and ricotta-spinach tortelli at day 50, but not in the artichoke-filled tortelli at day 50.

Effect of oxygen concentration and redox potential on recovery of sublethally heat-damaged cells of Escherichia coli O157:H7, Salmonella enteritidis and Listeria monocytogenes.

The measured heat resistance of cells of Escherichia coli O157:H7, Salmonella enteritidis and Listeria monocytogenes was up to eightfold greater when they were grown, heated and recovered anaerobically rather than aerobically. Measured heat resistance was highest when anaerobic gas mixtures were used (time at 59 degrees C for a 6-decimal (6-D) reduction of E. coli O157:H7, 19-24 min); moderate when low concentrations of oxygen (0.5-1%) were included (time for a 6-D reduction, 5-17 min); and lowest when higher concentrations of oxygen (2-40%) were used (time for a 6-D reduction, 3 min). This effect was principally attributed to the recovery conditions, and a greater effect was noted at lower heating temperatures. The use of reduced oxygen concentration (< 2% O2), e.g. packing under an anaerobic gas mixture or a vacuum, might therefore increase the risk of these pathogens surviving heat treatments applied to food. It is also possible that foods that are packed in air but with a low redox potential might allow the survival of heated cells, and thus the anticipated level of safety might not be achieved.

Redox potential affects the measured heat resistance of Escherichia coli O157:H7 independently of oxygen concentration.

Cells of Escherichia coli O157:H7 were heat-treated at 59 degrees C and enumerated in (i) anaerobic medium with a low redox potential, (ii) anaerobic media with the oxidizing agents potassium ferricyanide or 2,6-dichloroindophenol (DPIP) added to raise the redox potential, (iii) aerobic medium with a high redox potential and (iv) aerobic medium with the reducing agent dithiothreitol added to lower the redox potential. The measured heat-resistance was greatest when the enumeration medium was highly anaerobic due to the absence of oxygen and the presence of hydrogen and cysteine HCl. Measured heat resistance was influenced by the redox potential of the enumeration medium independently of the chemical used to adjust it and therefore, independently of the presence of oxygen. Sub-lethally heat-damaged cells regained their ability to grow in media of high redox potential at a similar rate whether the redox potential was increased by the addition of potassium ferricyanide, DPIP or oxygen.