Perfringens Vegetative Cells Research Articles

Combined Effect of Nigella sativa and Kefir on the Live Performance and Health of Broiler Chickens Affected by Necrotic Enteritis.

Coccidiosis and necrotic enteritis (NE) are prevalent poultry ailments worldwide, leading to decreased live performance and elevated mortality rates without antibiotic usage. This study evaluated Nigella sativa (black cumin) seeds (BCS) and kefir as alternatives to antibiotics for broilers. An in vivo study over a 28-day period, using 384 Cobb 500 male broilers organized into six treatment groups as part of a completely randomized block experimental design was conducted. Each treatment group included eight replicates, with each replicate containing eight birds. The treatments included positive control, negative control, antibiotic control, 5% BCS in feed, 20% kefir in drinking water, and a combination of 5% BCS and 20% kefir. NE was induced in broilers by administering ~5000 oocysts of Eimeria maxima orally on day 14, followed by inoculation with about 108 CFU/mL of Clostridium perfringens (Cp) (strain Cp#4) on days 19, 20, and 21. Live performance metrics including feed intake, body weight gain, and feed conversion were assessed in broilers. Additionally, NE disease outcomes such as lesion scores, mortality rates, and Cp populations in cecum were determined during the study. The BCS, kefir, and the combination had no detrimental effect on broiler live performance. BCS-treated and combination groups had lower NE scores (p > 0.05) in comparison to the positive control and exhibited no significant difference (p > 0.05) from antibiotic control. Additionally, treatment groups and antibiotic control were not significantly different (p > 0.05) in mortality, whereas the BCS and kefir combination significantly reduced (p < 0.05) mortality to 14.1% compared to 31.3% for the positive control. C. perfringens vegetative cells significantly decreased (p < 0.05) in treatments with BCS, kefir, and their combination on days 22 and 28 compared to the positive control. On day 22, Cp sores were significantly lower (p < 0.05) for the kefir and combination treatments compared to the positive control. In conclusion, BCS and kefir successfully reduced C. perfringens infection and mortality without any detrimental impact on broiler live performance with the combined treatment being the most effective. These results suggest that BCS and kefir could serve as potential alternatives to antibiotics in managing NE.

Occurrence of Clostridium perfringens vegetative cells and spores throughout an industrial production process of black soldier fly larvae (Hermetia illucens)

The main use of black soldier fly larvae (Hermetia illucens) is currently as an animal feed ingredient. While the bacterial community of the larvae has been characterised repeatedly via sequencing, microbiological safety assessment based on culture-dependent techniques is still scarce. This study focused on the occurrence of the spore-forming foodborne pathogen Clostridium perfringens during rearing and consecutive processing of the larvae, based on observations in a single rearing facility. C. perfringens vegetative cells and spores were determined, in addition to total viable counts, total aerobic spore counts and intrinsic parameters including pH, water activity and moisture content. All samples were obtained from an industrial production plant. In a preliminary experiment, substrate ingredients and dried larvae were analysed, but the larvae were produced with a previous batch of the substrate mixture. A second, more detailed, experiment was performed where all samples were collected sequentially from the same production run (substrate ingredients, substrate mixture, starting larvae, harvested larvae, residue, dried larvae and stored dried larvae). In the two experiments, (presumptive) C. perfringens, as determined on tryptose sulphite cycloserine agar, was found at low numbers in the ingredients and in the second experiment it was also found in the substrate mixture. Over the two experiments, total C. perfringens counts (i.e. vegetative cells plus spores) ranged between 3.0±0.1 and &lt;1.2±0.5 log cfu/g and C. perfringens spores ranged between 2.5±0.1 and &lt;1.0±0.0 log cfu/g. Interestingly, vegetative cells and spores of C. perfringens were below the detection limit in all larvae samples. Therefore, it appears that at this production site and based on the samples investigated, the pathogen did not colonise the larvae. However, these results indicate that insect producers should monitor this pathogen among others, and install good hygiene practices to avoid contamination.

Biofilm and Spore Formation of Clostridium perfringens and Its Resistance to Disinfectant and Oxidative Stress.

Clostridium perfringens is a major human pathogen that causes gastroenteritis via enterotoxin production and has the ability to form spores and biofilms for environmental persistence and disease transmission. This study aimed to compare the disinfectant and environmental resistance properties of C. perfringens vegetative cells and spores in planktonic and sessile conditions, and to examine the nucleotide polymorphisms and transcription under sessile conditions in C. perfringens strains isolated from meat. The sporulation rate of sessile C. perfringens TYJAM-D-66 (cpe+) was approximately 19% at day 5, while those of CMM-C-80 (cpe−) and SDE-B-202 (cpe+) were only 0.26% and 0.67%, respectively, at day 7. When exposed to aerobic conditions for 36 h, TYJAM-D-66, CMM-C-80, and SDE-B-202 vegetative cells showed 1.70 log, 5.36 log, and 5.67 log reductions, respectively. After treatment with sodium hypochlorite, the survival rates of TYJAM-D-66 vegetative cells (53.6%) and spores (82.3%) in biofilms were higher than those of planktonic cells (9.23%). Biofilm- and spore-related genes showed different expression within TYJAM-D-66 (–4.66~113.5), CMM-C-80 (–3.02~2.49), and SDE-B-202 (–5.07~2.73). Our results indicate the resistance of sessile cells and spores of C. perfringens upon exposure to stress conditions after biofilm formation.

Modeling for Survival of Clostridium perfringens in Saeng-sik, a Powdered Ready-to-Eat Food with Low Water Activity

We developed a mathematical model to predict the survival of C. perfringens in food. C. perfringens vegetative cells and spores were inoculated into dried powder food. The aw of saeng-sik was below 0.1. Weibull and Davey models can successfully describe the survival of C. perfringens. The developed model can be applied to samples with different microbial communities.

Combined effect of high pressure and vinegar addition on the control of Clostridium perfringens and quality in nitrite-free emulsion-type sausage

Abstract We evaluated the combined effect of high pressure (HP) and vinegar addition on control of Clostridium perfringens and quality in emulsion-type pork sausages. Sausages were manufactured with different levels of commercial white distilled vinegar made with jasmine tea extract (0, 1, and 2%) and sodium nitrite (0.02%, control). The sausages except for sodium nitrite treatment were subjected to HP at 500 MPa (four cycles and each for 3 min). After storage, the combined treatment of HP and vinegar showed the lowest number of C. perfringens vegetative cells (4.8-log CFU/g reduction) among the all treatments. The combined treatment also inhibited the growth of C. perfringens spores for five weeks (stored at 4 °C for two weeks followed by at 20 °C for three weeks). Among all treatment combinations, the best pH, water-holding capacity and chewiness, were found with the HP-vinegar sausage. In addition, vinegar treatment inhibited the lipid oxidation of sausage throughout the storage. Use of HP and vinegar addition inhibited growth of C. perfringens without observed adverse effects on quality of the emulsion-type sausage. Industrial relevance Currently, the use of natural additives and novel processing technologies for replacing sodium nitrite in meat products is gaining attention owing to increase in consumer demand for nitrite-free and safe meat products. The study showed that the addition of 1% (w/v) of vinegar and pressure treatment at 500 MPa (four cycles and each for 3 min) can replace sodium nitrite to inhibit growth of C. perfringens in emulsion-type sausages.

Effect of grape seed extract on heat resistance of Clostridium perfringens vegetative cells in sous vide processed ground beef

The heat resistance (57.5–65 °C) of a three-strain cocktail of Clostridium perfringens vegetative cells in sous vide processed ground beef supplemented with 0–3% grape seed extract (GSE) was quantified. The surviving cell population was enumerated on tryptose–sulfite–cycloserine agar. The decimal reduction (D)-values in beef that included no GSE were 67.11, 17.15, 4.02, and 1.62 min at 57.5, 60, 62.5, and 65 °C, respectively. Addition of 1.0% GSE resulted in concomitant decrease in heat resistance as evidenced by reduced bacterial D-values. The D-values in beef with added 1.0% GSE were 62.89, 13.70, 3.47 and 1.46 min at 57.5, 60, 62.5, and 65 °C, respectively. The heat resistance was further decreased when the GSE concentration in beef was increased to 2 or 3%. The z-values in beef with or without GSE were similar, ranging from 4.41 to 4.56 °C. The results of this study would be beneficial to the retail and institutional food service establishments in estimating re-heating time and temperature for sous vide processed ground beef to ensure safety against C. perfringens.

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

Abstract 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 1.9 log CFU/mL (or g) compared to those of the control (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 relevance Clostridium 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). The results reveal a potential technology for bio-protecting chicken meat from C. perfringens growth.

Inactivation Strategies for Clostridium perfringens Spores and Vegetative Cells.

Clostridium perfringens is an important pathogen to human and animals and causes a wide array of diseases, including histotoxic and gastrointestinal illnesses. C. perfringens spores are crucial in terms of the pathogenicity of this bacterium because they can survive in a dormant state in the environment and return to being live bacteria when they come in contact with nutrients in food or the human body. Although the strategies to inactivate C. perfringens vegetative cells are effective, the inactivation of C. perfringens spores is still a great challenge. A number of studies have been conducted in the past decade or so toward developing efficient inactivation strategies for C. perfringens spores and vegetative cells, which include physical approaches and the use of chemical preservatives and naturally derived antimicrobial agents. In this review, different inactivation strategies applied to control C. perfringens cells and spores are summarized, and the potential limitations and challenges of these strategies are discussed.

Unique Regulatory Mechanism of Sporulation and Enterotoxin Production in Clostridium perfringens

Clostridium perfringens causes gas gangrene and gastrointestinal (GI) diseases in humans. The most common cause of C. perfringens-associated food poisoning is the consumption of C. perfringens vegetative cells followed by sporulation and production of enterotoxin in the gut. Despite the importance of spore formation in C. perfringens pathogenesis, the details of the regulation of sporulation have not yet been defined fully. In this study, microarray and bioinformatic analyses identified a candidate gene (the RNA regulator virX) for the repression of genes encoding positive regulators (Spo0A and sigma factors) of C. perfringens sporulation. A virX mutant constructed in the food poisoning strain SM101 had a much higher sporulation efficiency than that of the wild type. The transcription of sigE, sigF, and sigK was strongly induced at 2.5 h of culture of the virX mutant. Moreover, the transcription of the enterotoxin gene was also strongly induced in the virX mutant. Western blotting confirmed that the levels of enterotoxin production were higher in the virX mutant than in the wild type. These observations indicated that the higher levels of sporulation and enterotoxin production in the virX mutant were specifically due to inactivation of the virX gene. Since virX homologues were not found in any Bacillus species but were present in other clostridial species, our findings identify further differences in the regulation of sporulation between Bacillus and certain Clostridium species. The virX RNA regulator plays a key role in the drastic shift in lifestyle of the anaerobic flesh eater C. perfringens between the vegetative state (for gas gangrene) and the sporulating state (for food poisoning).

Efficiency of a reheating step to inactivate Clostridium perfringens vegetative cells: How to measure it?

Clostridium perfringens is responsible for foodborne diseases often associated with processed meats in institutions. This study investigated the behavior of C. perfringens in beef-in-sauce products in a French hospital. In the studied process, the final reheating step makes the inactivation of C. perfringens vegetative cells possible. The aim of this study was thus to combine microbial, thermal and dose response modeling to propose, for this reheating step, three new control measures which are as efficient as current French regulation while also being easier to apply. These measures were based on thermal process duration (DA53), final temperature in food (FTF) and sum of temperatures-minutes above 53 °C (ST53) required to achieve food safety. A criterion was defined to describe the ability of the proposed control measures to lower the risk of foodborne illness related to the consumption of products with high populations of C. perfringens vegetative cells before reheating.To estimate acceptable values of DA53, FTF and ST53, variability and uncertainty were taken into account separately. A two dimensional Monte Carlo simulation enabled the definition of 14 measure thresholds which made sure with a 97.5% confidence that less than 20%, 10% or 5% of exposed consumers in a specified population would experience diarrhea. The three proposed control measures, single and combined, were then compared by estimating the duration required to reach control measure thresholds. This required duration differs significantly from a control measure to the other, with the lowest duration for the combination of FTF and DA53.

Detection of Enterotoxigenic Clostridium perfringens in Meat Samples by Using Molecular Methods

To prevent food-borne bacterial diseases and to trace bacterial contamination events to foods, microbial source tracking (MST) methods provide important epidemiological information. To apply molecular methods to MST, it is necessary not only to amplify bacterial cells to detection limit levels but also to prepare DNA with reduced inhibitory compounds and contamination. Isolates carrying the Clostridium perfringens enterotoxin gene (cpe) on the chromosome or a plasmid rank among the most important food-borne pathogens. Previous surveys indicated that cpe-positive C. perfringens isolates are present in only ∼5% of nonoutbreak food samples and then only at low numbers, usually less than 3 cells/g. In this study, four molecular assays for the detection of cpe-positive C. perfringens isolates, i.e., ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP), were developed and evaluated for their reliability using purified DNA. For use in the artificial contamination of meat samples, DNA templates were prepared by three different commercial DNA preparation kits. The four molecular assays always detected cpe when >10³ cells/g of cpe-positive C. perfringens were present, using any kit. Of three tested commercial DNA preparation kits, the InstaGene matrix kit appeared to be most suitable for the testing of a large number of samples. By using the InstaGene matrix kit, the four molecular assays efficiently detected cpe using DNA prepared from enrichment culture specimens of meat samples contaminated with low numbers of cpe-positive C. perfringens vegetative cells or spores. Overall, the current study developed molecular assay protocols for MST to detect the contamination of foods with low numbers of cells, and at a low frequency, of cpe-positive C. perfringens isolates.

The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells.

GROWTH INHIBITION OF CLOSTRIDIUM PERFRINGENS VEGETATIVE CELLS AND SPORES USING CHICKEN IMMUNOGLOBULIN Y

ABSTRACT Egg yolk antibody (IgY) was isolated by the water dilution method from the egg yolk of chickens immunized with Clostridium perfringens vegetative cells and spores. Specific binding activity of IgY against C. perfringens vegetative cells and spores remained relatively high during the immunization period (up to 9 weeks). The titer of specific IgY against C. perfringens spores was 1.4‐fold less than that of specific IgY against C. perfringens vegetable cells. The specific IgY powder (10 µg/mL) was found to inhibit the growth of C. perfringens vegetative cells or C. perfringens spores in a liquid medium. The difference of C. perfringens vegetative cell growth between the treatment and control groups was 8.9 × 106 colony forming units (cfu)/mL at 8 h of incubation and 9.95 × 107 cfu/mL at 24 h of incubation. Significant cfu reductions in C. perfringens spores were also observed with specific IgY powder at 24 h of incubation.PRACTICAL APPLICATIONSIgY antibody exerts an antimicrobial activity against pathogens by binding, immobilizing and consequently reducing or inhibiting the growth, replication or colony forming ability of pathogenic bacteria; thus, it is proved to be a viable alternative for antibiotics and preservatives. In this study, IgY against Clostridium perfringens can be used to replace chemical preservatives in food industries. Because IgY functions well at low temperature, it can be used to inhibit the growth of Clostridia which germinate in refrigerated storage conditions, thus preventing foodborne enterotoxicity caused by such bacteria. In practical applications, natural antimicrobial IgY antibody can be applied to meat products for the improvement of food safety.

Radio frequency heating of comminuted meats - considerations in relation to microbial challenge studies

Bacillus cereus and Clostridium perfringens vegetative cell and spore cocktails in maximum recovery diluent (MRD) were inoculated into pork luncheon meat to challenge a previously developed radio frequency (RF) cooking protocol. After RF cooking and cooling microbial enumeration results showed a reduction in B. cereus vegetative cell and spores of 5.4 and 1.8 log 10 cfu g −1, respectively while the corresponding reduction for C. perfringens vegetative cells and spores were 6.8 and 4.1 log 10 cfu g −1, respectively. However, post cooking temperatures within the product were lower than anticipated. Subsequent analysis of product thermal and dielectric properties indicated that MRD addition and compositional variations within meat ingredients altered thermal and dielectric properties which in turn contributed to reduced and less uniform temperatures. The study shows that for RF microbial challenge studies, adjustment of product formulation prior to MRD addition is critical to ensure a similar composition to the normal product and a true picture of microbial inactivation.

An evaluation of Clostridium perfringens media

The objective of this experiment was to compare a range of agars in terms of their suitability as cultural media for three different strains of Clostridium perfringens vegetative cells and recovery media for thermally treated C. perfringens spores. The eight media tested were; tryptose sulphite cycloserine + egg yolk emulsion (EYE) (TSC+), sulphite cycloserine − EYE (TSC−), Shahidi–Ferguson perfringens agar + EYE (SFP+), Shahidi–Ferguson perfringens agar − EYE (SFP−), oleandomycin polymyxin sulfadiazine perfringens (OPSP) (pour plate (OPSP PP) and spread plate (OPSP SP)), reinforced clostridial agar (RCA) and brain heart infusion agar (BHI). The three strains used were C. perfringens DSM 11784, C. perfringens NCTC 08237 and C. perfringens NCTC 10614. The optimum growth of the three C. perfringens vegetative cell strains was obtained on RCA and TSC, also, the addition of EYE did not improve growth. For thermally treated and untreated spores, RCA and BHI were the only media of those tested that supported the germination and growth of the spores. The results indicate that TSC− and RCA best cultured the three C. perfringens strains, while RCA was a significantly ( P ⩾ 0.05) better medium for recovering thermally treated and untreated C. perfringens spores than any other media examined in this study. This research offers definitive data with respect to recovery and growth rate of C. perfringens on a number of commercial media and could be used as a reference by researchers when choosing an appropriate medium for use in isolating and recovering C. perfringens.

Experimental Effect of Ozone upon Some Indicator Bacteria for Preservation of an Ecologically Protected Watery System

In the frame of a project concerning the evaluation of the pollution level and the ozone experimental effect upon some bacterial indicators of a protected ecosystem, water samples were estimated microbiologically before and after saturation with ozone gas for 5, 10, 15, 20 and 30 min. We found that the ozone treatment decreased the concentration in all different bacterial indicators by reducing their number for more than six times after 15 min of application. Total inactivation was observed for C. perfringens vegetative cells. Since there are complex relations between the microbial loading the water characteristics and the action of ozone, further studies are needed to determine the optimum conditions of ozone effect, as its use consists of a promising technology.

Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll

The aim of this study was to design a thermal treatment(s) for pork luncheon roll, which would destroy Bacillus cereus and Clostridium perfringens vegetative cells and spores. B. cereus and C. perfringens vegetative and spore cocktails were used to inoculate luncheon meat. Samples were subjected to different temperatures and removal times. The decimal-reduction times ( D-values) were calculated by linear regression analysis ( D=−1/slope of a plot of log surviving cells versus time). The log 10 of the resulting D-values were plotted against their corresponding temperatures to calculate (−1/slope of the curve) the thermal resistance ( z-values) of each cocktail. The D-values for vegetative cells ranged from 1 min (60 °C) to 33.2 min (50 °C) for B. cereus and from 0.9 min (65 °C) to 16.3 min (55 °C) for C. perfringens. The D-values for B. cereus spores ranged from 2.0 min (95 °C) to 32.1 min (85 °C) and from 2.2 min (100 °C) to 34.2 min (90 °C) for C. perfringens. The z-values were calculated to be 6.6 and 8.5 °C for B. cereus vegetative and spores, respectively, and 7.8 and 8.4 °C for C. perfringens vegetative cells and spores, respectively. The D-values of B. cereus and C. perfringens suggest that a mild cook of 70 °C for 12 s and 1.3 min would achieve a 6 log reduction of B. cereus and C. perfringens vegetative cells, respectively. The equivalent reduction of B. cereus and C. perfringens spores would require the pork luncheon meat to be heated for 36 s at 105 and 110 °C, respectively. The results of this study provide the thermal inactivation data necessary to design a cooking protocol for pork luncheon roll that would inactivate B. cereus and C. perfringens vegetative cells and spores. The data may also be used in future risk assessment studies.

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.

Increased Inactivation of Ozone-Treated Clostridium perfringens Vegetative Cells and Spores on Fabricated Beef Surfaces Using Mild Heat

Ozone treatment of beef surfaces enhanced the effectiveness of cooking temperatures ranging from 45 to 75°C against enterotoxin-producing strains of Clostridium perfringens. Vegetative cells on beef surfaces at an initial concentration of 5.59±0.17 log CFU/g were reduced significantly (P < 0.05) to 4.09±0.72 log CFU/g and 3.50±0.90 log CFU/g after combined treatments with aqueous ozone (5 ppm) and subsequent heating at 45 and 55°C, respectively. Spores on the beef surface were likewise significantly reduced from an initial concentration of 2.94±0.37 log spores per g to 2.07±0.38 log spores per g and 1.70±0.37 log spores per g after the combined treatment with aqueous ozone (5 ppm) and subsequent heating at 55 and 75°C, respectively. Fluorescent nucleic acid stains were used with confocal fluorescence microscopy to show that spores remaining attached to the meat were protected from treatment-specific injury. This study provides evidence for the decreased resistance of both vegetative cells and spores of C. perfringens with ozone treatment that is followed by heat treatment at temperatures that would not otherwise be as effective, thus lowering the requirements for cooking beef while maintaining a margin of safety.

Heat Treatment Adaptations in Clostridium perfringens Vegetative Cells

Vegetative cells of Clostridium perfringens enterotoxigenic strains NCTC 8679, NCTC 8238, and H6 were grown at 37°C followed by a 60-min exposure to 28°C or 46°C. D10-values, as a measure of thermal resistance at 60°C, were significantly lower for 28°C exposures as compared with cultures given 37 and 46°C exposures. Following refrigeration at 4°C for 24 h, D10-values for the 37 and 46°C samples could not be differentiated from 28°C samples. Western immunoblot analyses of lysates from heat-adapted cells also detected the increased expression of proteins reacting with antiserum directed against the molecular chaperonins from Escherichia coli; GroEL, DnaJ, and the small acid soluble protein from Bacillus subtilis, SspC. Differential scanning calorimetry (DSC) identified thermal transitions corresponding to ribosomal protein denaturations at 72.1 ± 0.5°C. Any cellular heat adaptations in the DSC profiles were lost following refrigeration for several days to simulate minimally processed food storage conditions. Further analyses of high-speed pellets from crude cell extract fractions using two-dimensional gel electrophoresis detected the differential gene expression of at least four major proteins in heat-adapted vegetative cells of C. perfringens. N-terminal amino acid analyses identified two of the proteins as glyceraldehyde3-phosphate dehydrogenase and rubrerythrin. Both appear to have roles in this anaerobe under stressful conditions.