Clostridium Perfringens Vegetative Cells Research Articles

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 <1.2±0.5 log cfu/g and C. perfringens spores ranged between 2.5±0.1 and <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.

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.

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.

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.

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.

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.

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.

Interactive Effects of Temperature, Initial pH, Sodium Chloride, and Sodium Pyrophosphate on the Growth Kinetics of Clostridium perfringens

The interactive effects of temperature (12 to 42°C), initial pH (5.5 to 7), sodium chloride (0 to 3%) and sodium pyrophosphate concentrations (0 to 0.3%) on the growth in Trypticase-peptone-glucose-yeast extract broth of a three-strain mixture of Clostridium perfringens vegetative cells were determined. The number of viable C. perfringens cells was determined at appropriate intervals by plating on tryptose-sulfite-cycloserine agar. Growth data were analyzed by the Gompertz equation; the gompertz B and M parameters were then used to calculate lag-phase duration, exponential growth rate, generation time, and maximum population-density values. The data indicated that the growth kinetics of C. perfringens were dependent on the interaction of the four variables, particularly in regard to exponential growth rates and lag-phase durations. Cubic models based on the natural logarithm transformation of lag-phase duration and generation time were evaluated and appeared to adequately fit the data. The data suggest that sodium pyrophosphate can have significant bacteriostatic activity against C. perfringens and may provide processed meats with a degree of protection against this microorganism, particularly if employed in conjunction with a combination of acidic pH, high salt concentrations, and adequate refrigeration.

Post‐packaging Pasteurization Reduces CIostridiur perfringens and Other Bacteria in Precooked Vacuum‐packaged Beef Loin Chunks

ABSTRACTPrecooked beef loin chunks were vacuum packaged after inoculation with Clostridium perfringens vegetative cells or spores. The control group was uninoculated. Chunks were either nonpasteurized or pasteurized, and stored at 4°C for up to 85 days. Pasteurization reduced populations of vegetative cells (VC) and vegetative cells from spores (VCS) of C. perfringens on beef chunks throughout 85 days of refrigerated storage. Without pasteurization, constant populations of VC and VCS were maintained for 28 and 65 days, respectively. Indige‐nous microflora populations of nonpasteurized beef chunks increased for up to 28 days and remained high thereafter. Pasteurization prevented the microflora from exceeding populations of 200 log10/cm2 (surface) or mL (broth) for 85 days of storage. Pasteurization increased the shelf‐life of precooked beef loin chunks.

A 48 kilodalton enterotoxin-related protein from Clostridium perfringens vegetative and sporulating cells

Sporulating and vegetative cell extracts of enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens were examined by Western blotting using enterotoxin antiserum. A 48-kDa enterotoxin-related protein was found in vegetative and sporulating cell extracts of both toxin types. The results question previous reports about the ability of vegetative cells and presumptive enterotoxin-negative strains of this organism to produce enterotoxin, a protein with a molecular weight of 34 kDa.

Growth and Survival of Clostridium perfringens in Rare Beef Prepared in a Water Bath

The low-temperature, long-time cooking of beef, using either a water bath or a conventional oven, resulted in partial inactivation of Clostridium perfringens vegetative cells. Beef roasts were cooked in a water bath for process times calculated to inactivate low and high levels of C. perfringens vegetative cells. Cooking beef in a water bath to an internal temperature of 60 C and holding for at least 12 min, as required by the USDA, reduced a population of C. perfringens by approximately 3 log cycles. To decrease the risk of subsequent outgrowth of C. perfringens, roasts (⩽ 1.5 kg) may be subjected to a process calculated for a 12-log reduction in population, which would include holding times of 2.3 h or longer at an internal temperature of 60 C. Recommendations are given for cooking and cooling roasts to minimize microbiological problems.

Effect of Freezing on Incidence and Levels of Clostridium perfringens in Mechanically Deboned Chicken Meat

Paucity of information about the effect of plant practices on microbiological condition of comminuted chicken meat prompted this investigation. Commercially deboned chicken backs and necks were analyzed for levels of aerobic organisms, incidence and levels of Clostridium perfringens vegetative cells and spores before and after 4—6 weeks at —23° C. Initially vegetative cells were isolated more frequently than spores. Frozen storage significantly reduced incidence and levels of vegetative cells and spores but did not affect levels of aerobic organisms. After frozen storage (a common industrial practice), with good food handling practices, C. perfringens should pose no undue hazard when comminuted chicken meat is incorporated into other food products.

SURVIVAL OF Clostridium perfringens IN REFRIGERATED AND FROZEN MEAT AND POULTRY ITEMS

ABSTRACTRefrigerated and frozen meats and poultry products from a retail source were examined for incidence of Clostridium perfringens vegetative cells and spores. Foods included raw ground beef, beef and pork liver, and precooked frozen beef and poultry items. Inoculated samples were also frozen and stored for various periods up to 42 days at ‐29°C. Survival of C. perfringens spores in frozen food samples and in a bacteriological medium was high, but vegetative cells were virtually eliminated. Thawing at low temperature for 2 days caused further reduction of vegetative cells, but not of spores. Approximately 50% of 32 retail frozen precooked food samples were positive for vegetative cells, and about 15% demonstrated spores of C. perfringens.

Inhibition of Clostridium perfringens by heated combinations of nitrite, sulfur, and ferrous or ferric ions.

Heating mixtures of sodium nitrite, cysteine, and either ferrous sulfate or ferric chloride at 121 C for 20 min at pH 6.5 or 6.3 produced a potent inhibitor of Clostridium perfringens vegetative cells and spores when added to previously heat-sterilized fluid thioglycolate medium. When the mixtures containing FeSO4 at pH 5.2 or FeCl3 at pH 2.7 were heated, the inhibitory effect was not produced. These responses seem to eliminate the possibility that cysteine nitrosothiol is the agent responsible for the heated-nitrite inhibition known as the Perigo effect. The variable pH responses also cast doubt upon the role of the black Roussin salt as the agent of the Perigo effect.

RESPONSE OF CLOSTRIDIUM PERFRINGENS SPORES AND VEGETATIVE CELLS TO TEMPERATURE VARIATION.

The vegetative cells and spores of four strains of Clostridium perfringens were examined to determine the effect of lowered and elevated temperatures. Spores were produced by following the method of Ellner, and vegetative cells were obtained from thioglycolate cultures. After exposure to freezing or refrigeration temperatures (-17.7 and 7.1 C, respectively), only small numbers of the vegetative cells were recovered. After similar treatment, 16 to 58% of the spores were recovered. Essentially no vegetative cells and few spores survived holding at 80 C for 10 min. Although all strains were isolated from food, only one strain of the four studied had its origin in a food-poisoning outbreak, and it had been carried on laboratory media for approximately 10 years.