Strategy to inactivate Clostridium perfringens spores in meat products

The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100–200 MPa, 7 min) and elevated temperature (80 °C, 10 min); spore germination at high temperatures (55, 60 or 65 °C); and inactivation of germinated spores with elevated temperatures (80 and 90 °C, 10 and 20 min) and high pressure (586 MPa, at 23 and 73 °C, 10 min). Low pressures (100–200 MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80 °C, 10 min), and germinated at temperatures lethal for vegetative cells (≥55 °C) when incubated for 60 min with a mixture of l-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (∼4 decimal reduction) in meat by elevated temperatures (80–90 °C for 20 min) required a long germination period (55 °C for 60 min). However, similar inactivation level was reached with shorter germination period (55 °C for 15 min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568 MPa, 73 °C, 10 min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50 mM AK consisted: (i) a primary heat treatment (80 °C, 10 min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55 °C in about 20 min and further incubation at 55 °C for about 15 min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586 MPa at 73 °C for 10 min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C. perfringens.     

Inhibitory effects of nisin against Clostridium perfringens food poisoning and nonfood-borne isolates

The enterotoxigenic Clostridium perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and nonfood-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly resistant endospores, it has become a great concern to the meat industry to produce meat free of C. perfringens. In this study, we evaluated the antimicrobial effect of nisin against C. perfringens FP and NFB isolates. No inhibitory effect of nisin was observed against germination of spores of both FP and NFB isolates in laboratory medium. However, nisin effectively arrested outgrowth of germinated spores of C. perfringens in rich medium. Interestingly, germinated spores of NFB isolates possessed higher resistance to nisin than that of FP isolates. Furthermore, nisin exhibited inhibitory effect against vegetative growth of both FP and NFB isolates in laboratory medium, with vegetative cells of NFB isolates showing higher resistance than that of FP isolates. However, the antimicrobial activity of nisin against C. perfringens was significantly decreased in a meat model system. In conclusion, although nisin showed inhibitory effect against spore outgrowth and vegetative cells of C. perfringens FP and NFB isolates in laboratory conditions, no such effect was observed against C. perfringens spores inoculated into a meat model system.     

High pressure thermal processing for the inactivation of Clostridium perfringens spores in beef slurry

The spores of Clostridium perfringens can survive and grow in cooked/pasteurized meat, especially during the cooling of large portions. In this study, 600 MPa high pressure thermal processing (HPTP) at 75 °C for the inactivation of C. perfringens spores was compared with 75 °C thermal processing alone. The HPTP enhanced the inactivation of C. perfringens spores in beef slurry, resulting in 2.2 log reductions for HPTP vs. no reductions for thermal processing after 20 min. Then, the HPTP resistance of two C. perfringens spore strains in beef slurry at 600 MPa was compared and modeled, and the effect of temperature investigated. The NZRM 898 and NZRM 2621 exhibited similar resistance, and Weibull modeled well the log spore survivor curves. The spore inactivation increased when HPTP temperature was raised from 38 to 75 °C. The results confirm the advantage of high pressure technology to increase the thermal inactivation of C. perfringens spores in beef slurry.Industrial relevanceC. perfringens spores may cause food/meat poisoning as a result of improperly handled and prepared foods in industrial kitchens. Thermal processes at 100 °C or higher are generally carried out to ensure the elimination of these pathogenic spores. High pressure processing (HPP) is a food pasteurization technique which would help to maintain the sensorial and nutritional properties of food. Preservation of foods with HPP in conjunction with mild heat (HPTP) would enhance the spore inactivation compared to thermal processing alone at the same temperature, due to a known germination–inactivation mechanism. This technology, together with the application of Good Manufacturing Practices, including rapid cooling, is a good alternative to the traditional methods for producing safe processed meat and poultry products with enhanced sensory and nutritional quality.     

Inhibitory effects of polyphosphates on Clostridium perfringens growth, sporulation and spore outgrowth

This study evaluated the effects of various polyphosphates (SPP, STPP, SAPP and TSPP) on growth, sporulation and spore germination of Clostridium perfringens, and germination and outgrowth of C. perfrinegns spores in poultry meat. We have found that the requirements of polyP (0.8–1.0%) to inhibit C. perfringens bacterial growth were higher than those reported for other bacteria. Sub-lethal concentrations of polyP significantly (p<0.01) inhibited sporulation of C. perfringens by reducing sporulating cells (heat-resistant cells) 5–6 log₁₀. While C. perfringens spores were able to germinate in the presence of 1% STPP, their outgrowth was significantly (p<0.01) inhibited. Finally, a significant (p<0.01) reduction of survival of C. perfringens was observed when meat samples contaminated with a cocktail of spores of C. perfringens isolates carrying enterotoxin gene on the chromosome were treated with 1% STPP. Collectively, this study demonstrated the inhibitory effects of polyP on growth, sporulation and spore outgrowth of C. perfringens, and suggests that polyP can be used not only as an enhancer of the functional properties of meat products, but also as a promising C. perfringens antimicrobial agent.     

Effect of meat ingredients (sodium nitrite and erythorbate) and processing (vacuum storage and packaging atmosphere) on germination and outgrowth of Clostridium perfringens spores in ham during abusive cooling

The effect of nitrite and erythorbate on Clostridium perfringens spore germination and outgrowth in ham during abusive cooling (15 h) was evaluated. Ham was formulated with ground pork, NaNO₂ (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 547 ppm). Ten grams of meat (stored at 5 °C for 3 or 24 h after preparation) were transferred to a vacuum bag and inoculated with a three-strain C. perfringens spore cocktail to obtain an inoculum of ca. 2.5 log spores/g. The bags were vacuum-sealed, and the meat was heat treated (75 °C, 20 min) and cooled within 15 h from 54.4 to 7.2 °C. Residual nitrite was determined before and after heat treatment using ion chromatography with colorimetric detection. Cooling of ham (control) stored for 3 and 24 h, resulted in C. perfringens population increases of 1.46 and 4.20 log CFU/g, respectively. For samples that contained low NaNO₂ concentrations and were stored for 3 h, C. perfringens populations of 5.22 and 2.83 log CFU/g were observed with or without sodium erythorbate, respectively. Residual nitrite was stable (p > 0.05) for both storage times. Meat processing ingredients (sodium nitrite and sodium erythorbate) and their concentrations, and storage time subsequent to preparation of meat (oxygen content) affect C. perfringens spore germination and outgrowth during abusive cooling of ham.     

Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology

Spores of Bacillus sporothermodurans are known to be contaminant of dairy products and to be extremely heat-resistant. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk stability. In this study, the nutrient-induced germination of B. sporothermodurans LTIS27 spores was studied. Testing the effect of 23 nutrient elements to trigger an important germination rate of B. sporothermodurans spores, only d-glucose, l-alanine, and inosine were considered as strong independent germinants. Both inosine and l-alanine play major roles as co-germinants with several other amino acids. A central composite experimental design with three factors (l-alanine, d-glucose, and temperature) using response surface methodology was used to optimize the nutrient-induced germination. The optimal rate of nutrient-induced germination (100%) of B. sporothermodurans spores was obtained after incubation of spore for 60 min at 35 °C in presence of 9 and 60 mM of d-glucose and l-alanine, respectively. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination, which will be beneficial for screening of B. sporothermodurans in milk after induction their germination. Moreover, the chosen method of optimization of the nutrient-induced germination was efficient in finding the optimum values of three factors.     

Clostridial spore germination versus bacilli: genome mining and current insights

Bacilli and clostridia share the characteristic of forming metabolically inactive endospores. Spores are highly resistant to adverse environmental conditions including heat, and their ubiquitous presence in nature makes them inevitable contaminants of foods and food ingredients. Spores can germinate under favourable conditions, and the following outgrowth can lead to food spoilage and foodborne illness. Germination of spores has been best studied in Bacillus species, but the process of spore germination is less well understood in anaerobic clostridia. This paper describes a genome mining approach focusing on the genes related to spore germination of clostridia. To this end, 12 representative sequenced Bacillus genomes and 24 Clostridium genomes were analyzed for the distribution of known and putative germination-related genes and their homologues. Overall, the number of ger operons encoding germinant receptors is lower in clostridia than in bacilli, and some Clostridium species are predicted to produce cortex-lytic enzymes that are different from the ones encountered in bacilli. The in silico germination model constructed for clostridia was linked to recently obtained experimental data for selected germination determinants, mainly in Clostridium perfringens. Similarities and differences between germination mechanisms of bacilli and clostridia will be discussed.     

Persistence strategies of Bacillus cereus spores isolated from dairy silo tanks

Survival of Bacillus cereus spores of dairy silo tank origin was investigated under conditions simulating those in operational dairy silos. Twenty-three strains were selected to represent all B. cereus isolates (n = 457) with genotypes (RAPD-PCR) that frequently colonised the silo tanks of at least two of the sampled eight dairies. The spores were studied for survival when immersed in liquids used for cleaning-in-place (1.0% sodium hydroxide at pH 13.1, 75 °C; 0.9% nitric acid at pH 0.8, 65 °C), for adhesion onto nonliving surfaces at 4 °C and for germination and biofilm formation in milk. Four groups with different strategies for survival were identified. First, high survival (log 15 min kill ≤1.5) in the hot-alkaline wash liquid. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterised by slow germination in rich medium and well preserved viability when exposed to heating at 90 °C. Fourth, spores capable of germinating at 8 °C and possessing the cspA gene. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot-alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus.     

Inactivation of Bacillus cereus spores using a combined treatment of UV-TiO2 photocatalysis and high hydrostatic pressure

Bacillus cereus spores are resistant to high hydrostatic pressure (HHP) processing treatment. A combination of UV-TiO₂ photocatalysis (UVTP for 10 min) and two cycles of 600 MPa HHP treatment for 10 min for the first cycle and 1 min for the second cycle (UVTP-2HHP) at ambient temperature was applied to inactivate B. cereus spores inoculated on a solidified agar matrix (SAM) used as a model matrix. Two cycles of HHP treatment were used as a strategy for induction of spore germination, followed by inactivation. UVTP and 2 cycles of HHP resulted in a 5.0-log CFU/cm² spore reduction (initial spore count was 6.6 log CFU/cm²), including an approximate 0.8-log CFU/cm² reduction due to a synergistic effect. The inactivation mechanism of UVTP pretreatment was related to lipid peroxidation of the spore membrane based on the level of malondialdehyde (MDA) making spores susceptible to the HHP treatment. Flow cytometry and transmission electron microscopic (TEM) analyses showed severe physiological alteration and structural damage to spores after the combined treatment. UVTP and 2 cycles of HHP showed potential for effective inactivation of B. cereus to ensure food safety from B. cereus spores on food products.Practical applicationsInactivation of bacterial spores remains a technical challenge for HHP and other interventions because spores are highly resistant to high pressure. Pretreatment with UVTP followed by two cycles of HHP resulted in reduction in B. cereus spores due to a synergistic effect. This hurdle technology of UVTP and HHP can help food industry in ensuring food safety against the Bacillus spores.     

Inactivation of Clostridium sporogenes spores on stainless-steel using heat and an organic acidic chemical agent

The efficacy of a food grade acidic chemical agent for the reduction of Clostridium sporogenes spores on a stainless steel surface was investigated. The chemical agent was a combination of selected fatty acids and lactate esters. Distilled deionized water and 35% hydrogen peroxide were used as negative and positive controls, respectively. Approximately 3 log cfu reductions in viable spore numbers were detected on the steel surfaces for all treatments at room temperature, except the controls. Reductions in the viable spore numbers significantly increased with increasing exposure times and concentrations of the acidic agent. Five log reduction of viable spore number was achieved after 10 min treatment with the 10% agent solution at 68 °C. No viable spores were observed on the 10% agent treated sample after a 60 min exposure time at 75 °C. This research showed that the acidic sanitizer tested in this study could be used to reduce the number of C. sporogenes spores on stainless steel surfaces.     

Effect of food residues on efficiency of surfactant disinfectants against food related pathogens adhered on polystyrene and ceramic surfaces

The adhesion of pathogenic bacteria on food contact surfaces increases the risk of cross-contamination in the food industry. However, food-borne disease introduced by the production process can be mitigated by surfactant use. This study investigates the effect of food residues (milk, beef gravy and tuna gravy) on the bactericidal efficiency of benzalkonium chloride (BAC) and alkyldiaminoethylglycine hydrochloride (AGH). The test was conducted on pathogenic bacteria (Escherichia coli O26, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and B. cereus spores) dried and adhered to the surfaces of polystyrene and ceramic dishes at room temperature for 1.5 h. The protein and lipid rich food residues protected the bacterial cells from dehydration and from the adverse effects of disinfectants, although bacterial numbers were decreased after drying and the surfaces were clearly sterilized after disinfectant treatment at typical concentrations (0.5 mg/ml–2.0 mg/ml) for 10 min. Following general and proper washing processes, the bactericidal effect of the disinfectants became clearly visible. These results indicate that applying a proper washing process prior to disinfectant treatment can prevent cross-contamination.     

Protective role of Lactobacillus fermentum R6 against Clostridium perfringens in vitro and in chicken breast meat under temperature abuse conditions

Six bacterial species were evaluated to determine their inhibitory effects on Clostridium perfringens in vitro (brain heart infusion broth) and in situ (chicken breast meat) under temperature abuse conditions (4 ± 1 °C for 12 h, followed by 7 h at 28 ± 1 °C and then 4 ± 1 °C for 53 h). During abusive storage, rapid growth of C. perfringens from vegetative cell and spore inocula was observed, exhibiting a 2.68–3.37 log CFU/mL (or g) increase in bacterial counts. In the presence of Pediococcus pentosaceus P1 or Lactobacillus fermentum R6, the counts of C. perfringens remained unchanged in the samples containing vegetative cells at the end of storage (P < 0.05); for those containing spores, the germination and outgrowth were also effectively inhibited, decreasing in bacterial counts of > 1.9 log CFU/mL (or g) compared to those of the control (P < 0.05). The pH of chicken meat was slightly declined by 0.09 in the presence of L. fermentum (P > 0.05), and the inhibitory effect against C. perfringens was ascribed to non-acid antimicrobial substances. These results indicate a potential solution for bio-protecting chicken meat from C. perfringens growth.Industrial relevanceClostridium perfringens is a common pathogen that contaminates meat and meat products, but the organism cannot multiply under cold chain conditions at 4 °C. However, it was reported that temperature abuses commonly occurred during the transportation, storage or retail display of the food chill chain. During the abusive storage, C. perfringens could grow rapidly, which may lead to food poisoning. It is a serious problem for food safety.In this study, Lactobacillus fermentum R6 was found to show effective inhibition on both the growth of C. perfringens vegetative cells and the germination and outgrowth of its spores in chicken meat (P < 0.05) under temperature abuse conditions, and also it had a minimal effect on the pH of the meat (P > 0.05). The results reveal a potential technology for bio-protecting chicken meat from C. perfringens growth.     

Germination and outgrowth of spores of Bacillus cereus group members: diversity and role of germinant receptors

Bacillus cereus is a Gram-positive, facultative anaerobic, endospore-forming toxicogenic human pathogen. Endospores are highly specialized, metabolically dormant cell types that are resistant to extreme environmental conditions, including heat, dehydration and other physical stresses. B. cereus can enter a range of environments, and can in its spore form, survive harsh conditions. If these conditions become favorable, spores can germinate and grow out and reach considerable numbers in a range of environments including processed foods. Certainly the last decade, when consumer preferences have shifted to mildly processed food, new opportunities arose for spore-forming spoilage and pathogenic organisms. Only rigorous methods have been shown to be capable of destroying all spores present in food, thus a shift toward e.g., milder heat preservation strategies, may result in low but significant amounts of viable spores in food products. Hence, the need for a mild spore destruction strategy is eminent including control of spore outgrowth. Consequently, there is a large interest in triggering spore germination in foodstuffs, since germinated spores have lost the extreme resistance of dormant spores and are relatively easy to kill. Another option could be to prevent germination so that no dangerous levels can be reached. This contribution will focus on germination and outgrowth characteristics of B. cereus and other members of the B. cereus group, providing an overview of the niches these spore-formers can occupy, the signals that trigger germination, and how B. cereus copes with these wake-up calls in different environments including foods, during food processing and upon interaction with the human host.     

Effect of Rooibos (Aspalathus linearis) on Growth Control of Clostridium perfringens and Lipid Oxidation of Ready‐to‐Eat Jokbal (Pig's Trotters)

This study investigated the antimicrobial effects of rooibos (tea extract), potassium lactate (PL) and sodium diacetate (SDA) mixture alone or in combinations on the growth of Clostridium perfringens vegetative cell and spore in ready‐to‐eat (RTE) Jokbal (pig's trotters). Addition of a combination of 10% rooibos and 4% PL + SDA inhibit growth of C. perfringens vegetative cell in Jokbal at 24 °C and 36 °C. The significant inhibition on germination and growth of C. perfringens spores was also observed in Jokbal with a combination of 10% rooibos and 4% PL + SDA (PL: 2.24%, SDA: 0.16%) at 24 °C. The Jokbal treated with 10% rooibos and 4% PL + SDA mixture had significantly (P < 0.05) lower TBARS values than the control at 10 and 24 °C. The lipid oxidation inhibition effect was the highest (P < 0.05) in anaerobic packed Jokbal with 10% rooibos. The addition of a combination of 10% rooibos and 4% PL + SDA during the processing of Jokbal prevented the growth of C. perfringens and the germination and growth of C. perfringens spores at room temperature. This study shows rooibos tea as a valuable natural food preservative in meat products.     

Microbiota of cocoa powder with particular reference to aerobic thermoresistant spore-formers

The microbiological criteria of commercial cocoa powder are defined in guidelines instituted by the cocoa industry. Twenty-five commercial samples were collected with the aim of assessing the compliance with the microbiological quality guidelines and investigating the occurrence and properties of aerobic Thermoresistant Spores (ThrS). Seventeen samples complied with the guidelines, but one was positive for Salmonella, five for Enterobacteriaceae and two had mould levels just exceeding the maximum admissible level. The treatment of the cocoa powder suspensions from 100 °C to 170 °C for 10 min, revealed the presence of ThrS in 36% of the samples. In total 61 ThrS strains were isolated, of which the majority belonged to the Bacillus subtilis complex (65.6%).Strains resporulation and spore crops inactivation at 110 °C for 5 min showed a wide diversity of heat-resistance capacities. Amplified fragment length polymorphism analysis revealed not only a large intraspecies diversity, but also different clusters of heat-resistant spore-forming strains. The heat-resistance of spores of six B. subtilis complex strains was further examined by determination of their D and z-values.We concluded that B. subtilis complex spores, in particular those from strain M112, were the most heat-resistant and these may survive subsequent preservation treatments, being potentially problematic in food products, such as chocolate milk.     

Flow cytometric assessment of Bacillus spore response to high pressure and heat

The physiological response of Bacillus licheniformis spores to high pressure and thermal inactivation in sodium citrate buffer and nutrient broth was investigated using multiparameter flow cytometry. Spores were treated by heat-only at 121 °C, by high pressure at 150 MPa (37 °C), or by a combined high pressure and heat treatment at 600 MPa and 77 °C, and then dual stained with the fluorescent dyes SYTO 16 and propidium iodide (PI). For pressure treated spores, but not heat-only treated spores, four distinct sub-populations were detected by flow cytometry, and for these we suggest a three step model of inactivation involving a germination step following hydrolysis of the spore cortex, an unknown step, and finally an inactivation step with physical compromise of the spore's inner membrane.

Industrial relevance

This preliminary study offers a simple and fast flow cytometric method for the rapid assessment of the physiological state of bacterial spores following high pressure and thermal processing. An improved understanding of the mechanisms of spore inactivation will aid in the food safety assessment of pressure assisted thermal sterilisation in particular, and also assist in the commercialisation of these processes facilitating adoption by industry.     

Inhibition of Clostridium perfringens Spore Germination and Outgrowth by Lemon Juice and Vinegar Product in Reduced NaCl Roast Beef

Abstract: Inhibition of Clostridium perfringens spore germination and outgrowth in reduced sodium roast beef by a blend of buffered lemon juice concentrate and vinegar (MoStatin LV1) during abusive exponential cooling was evaluated. Roast beef containing salt (NaCl; 1%, 1.5%, or 2%, w/w), blend of sodium pyro‐ and poly‐phosphates (0.3%), and MoStatin LV1 (0%, 2%, or 2.5%) was inoculated with a 3‐strain C. perfringens spore cocktail to achieve final spore population of 2.5 to 3.0 log CFU/g. The inoculated products were heat treated and cooled exponentially from 54.4 to 4.4 °C within 6.5, 9, 12, 15, 18, or 21 h. Cooling of roast beef (2.0% NaCl) within 6.5 and 9 h resulted in <1.0 log CFU/g increase in C. perfringens spore germination and outgrowth, whereas reducing the salt concentration to 1.5% and 1.0% resulted in >1.0 log CFU/g increase for cooling times longer than 9 h (1.1 and 2.2 log CFU/g, respectively). Incorporation of MoStatin LV1 into the roast beef formulation minimized the C. perfringens spore germination and outgrowth to <1.0 log CFU/g, regardless of the salt concentration and the cooling time. Practical Application: Cooked, ready‐to‐eat meat products should be cooled rapidly to reduce the risk of Clostridium perfringens spore germination and outgrowth. Meat processors are reducing the sodium chloride content of the processed meats as a consequence of the dietary recommendations. Sodium chloride reduces the risk of C. perfringens spore germination and outgrowth in meat products. Antimicrobials that contribute minimally to the sodium content of the product should be incorporated into processed meats to assure food safety. Buffered lemon juice and vinegar can be incorporated into meat product formulations to reduce the risk of C. perfringens spore germination and outgrowth during abusive cooling.     

Does proximity to neighbours affect germination of spores of non-proteolytic Clostridium botulinum?

It is recognised that inoculum size affects the rate and extent of bacterial spore germination. It has been proposed that this is due to spores interacting: molecules released from germinated spores trigger germination of dormant neighbours. This study investigated whether changes to the total number of spores in a system or proximity to other spores (local spore density) had a more significant effect on interaction between spores of non-proteolytic Clostridium botulinum strain Eklund 17B attached to defined areas of microscope slides. Both the number of spores attached to the slides and local spore density (number of spores per mm²) were varied by a factor of nine. Germination was observed microscopically at 15 °C for 8 h and the probability of, and time to, germination calculated from image analysis measurements. Statistical analysis revealed that the effect of total spore number on the probability of germination within 8 h was more significant than that of proximity to neighbours (local spore density); its influence on germination probability was approximately four-times greater. Total spore number had an even more significant affect on time to germination; it had a nine-fold greater influence than proximity to neighbours. The applied models provide a means to characterise, quantitatively, the effect of the total spore number on spore germination relative to the effect of proximity to neighbouring spores.     

Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage

Due to changes in the design of industrial food processing and increasing international trade, highly thermoresistant spore-forming bacteria are an emerging problem in food production. Minimally processed foods and products with extended shelf life, such as milk products, are at special risk for contamination and subsequent product damages, but information about origin and food quality related properties of highly heat-resistant spore-formers is still limited. Therefore, the aim of this study was to determine the biodiversity, heat resistance, and food quality and safety affecting characteristics of aerobic spore-formers in the dairy sector. Thus, a comprehensive panel of strains (n = 467), which originated from dairy processing environments, raw materials and processed foods, was compiled. The set included isolates associated with recent food spoilage cases and product damages as well as isolates not linked to product spoilage. Identification of the isolates by means of Fourier-transform infrared spectroscopy and molecular methods revealed a large biodiversity of spore-formers, especially among the spoilage associated isolates. These could be assigned to 43 species, representing 11 genera, with Bacillus cereus s.l. and Bacillus licheniformis being predominant. A screening for isolates forming thermoresistant spores (TRS, surviving 100 °C, 20 min) showed that about one third of the tested spore-formers was heat-resistant, with Bacillus subtilis and Geobacillus stearothermophilus being the prevalent species. Strains producing highly thermoresistant spores (HTRS, surviving 125 °C, 30 min) were found among mesophilic as well as among thermophilic species. B. subtilis and Bacillus amyloliquefaciens were dominating the group of mesophilic HTRS, while Bacillus smithii and Geobacillus pallidus were dominating the group of thermophilic HTRS. Analysis of spoilage-related enzymes of the TRS isolates showed that mesophilic strains, belonging to the B. subtilis and B. cereus groups, were strongly proteolytic, whereas thermophilic strains displayed generally a low enzymatic activity and thus spoilage potential. Cytotoxicity was only detected in B. cereus, suggesting that the risk of food poisoning by aerobic, thermoresistant spore-formers outside of the B. cereus group is rather low.     

Novel insights into the epidemiology of Clostridium perfringens type A food poisoning

Clostridium perfringens food poisoning ranks among the most common gastrointestinal diseases in developed countries. The disease is caused by C. perfringens enterotoxin (CPE) encoded by cpe and produced by less than 5% of C. perfringens type A strains. Molecular epidemiological research in the past 15 years has focused on the reservoirs and routes of cpe-positive C. perfringens aiming to clarify the role and epidemiology of chromosomal and plasmid-borne cpe-carrying strains. This literature review highlights novel aspects in the epidemiology of CPE-mediated diseases. We suggest that (1) chromosomal and plasmid-borne cpe-carrying C. perfringens strains are genetically and epidemiologically distinct and have adapted to different environments; (2) not only chromosomal but also plasmid-borne cpe-carrying C. perfringens strains cause food poisonings; (3) other CPE-mediated diseases, such as antibiotic-associated and sporadic diarrhea, associated with plasmid-borne cpe-positive strains, may be food-related; (4) the role of animals as the main reservoir of cpe-positive C. perfringens needs to be reconsidered; (5) humans serve as an important reservoir of cpe-positive C. perfringens, introducing a contamination risk into foods through handling; and (6) the current standard procedures to diagnose C. perfringens food poisoning fail to detect and isolate many C. perfringens strains, distorting the epidemiological understanding of C. perfringens food poisoning.