Perfringens Populations Research Articles

CONTROL OF CLOSTRIDIUM PERFRINGENS IN A MODEL ROAST BEEF BY SALTS OF ORGANIC ACIDS DURING CHILLING1

ABSTRACTControl of Clostridium perfringens germination and outgrowth by the following salts of organic acids, sodium lactate [Purasal™S/SP (Purasal); 1.50, 3.00 and 4.80%], sodium lactate supplemented with sodium diacetate [Purasal™ Opti.form™ (Optiform), 1.50, 3.00 and 4.80%], buffered sodium citrate [Ional™ (Ional), 0.75, 1.00 and 1.30]) and buffered sodium citrate supplemented with sodium diacetate [Ional Plus™ (Ional Plus), 0.75, 1.00 and 1.30%] was evaluated during continuous chilling of a model roast beef product. Beef rounds were ground through an 1/8′’ plate and NaCl, potato starch and potassium tetra pyrophosphate were added to final concentrations of 0.85, 0.25 and 0.20%, respectively, and mixed. Portions (250 g) of the meat were mixed with either Purasal (1.5, 3.0 or 4.8%), Optiform (1.5, 3.0 or 4.8%), Ional (0.75, 1.0 or 1.3%) or Ional Plus (0.75, 1.0 or 1.3%) along with a control that did not have any added antimicrobials. Each product (10 g) inoculated with C. perfringens spores (ca. 2.2 log10 spores/g) was packaged into vacuum bags (2 in. × 3 in.), vacuum sealed, heated to 60C within 1 h, and subsequently chilled from 54.4C to 7.2C in 18 or 21 h following exponential chilling rates. Products were sampled immediately after cooking to enumerate the C. perfringens populations (spores surviving heat treatment) and subsequent to chilling (total C. perfringens populations, including spores and vegetative cells resulting from germination and outgrowth of the spores). Chilling of cooked, model ground roast beef resulted in germination and outgrowth of C. perfringens spores; the population densities increased by 4.13 and 4.40 log10 CFU/g, following 18 and 21 h chill rates, respectively. Incorporation of Purasal (1.5–4.8%), Optiform (1.5–4.8%), Ional and Ional Plus (0.75–1.3%) substantially (P ± 0.05) inhibited germination and outgrowth of C. perfringens spores. Incorporation of antimicrobial ingredients resulted in ± 1.0 log10 CFU/g increase of the pathogen, except for model roast beef with Ional Plus at 0.75% concentration, following 18 h chilling rate. Similar results were obtained when 21 h chilling rate was followed, with roast beef containing ingredients (at all the concentrations) resulting in either reductions or ± 1.0 log10 CFU/g growth in total C. perfringens populations, except for Purasal and Ional Plus at 1.5 and 0.75% concentrations, respectively. Use of sodium salts of organic acids in formulation of model roast beef can reduce the risk of C. perfringens spore germination and outgrowth during extended chilling rates.

Influence of NaCl Content and Cooling Rate on Outgrowth of Clostridium perfringens Spores in Cooked Ham and Beef

The effect of NaCl concentration and cooling rate on the ability of Clostridium perfringens to grow from spore inocula was studied with the use of a process that simulates the industrial cooking and cooling of smoked boneless ham and beef roasts. NaCl was added to ground cooked hams A and B (which were commercially obtained) to obtain levels of 2.4, 3.1, 3.6, and 4.1% (wt/wt) and 2.8, 3.3, 3.8, and 4.3% (wt/wt), respectively, and to raw ground beef to obtain levels of 0, 1, 2, 3, and 4% (wt/wt). Ham C, a specially formulated, commercially prepared product, was supplemented with NaCl to obtain levels of 2.0, 2.5, 3.0, and 3.5%. The samples were inoculated with a three-strain mixture of C. perfringens spores to obtain concentrations of ca. 3 log10 CFU/g. Portions of meat (5 g each) were spread into thin layers (1 to 2 mm) in plastic bags, vacuum packaged, and stored at −40°C. Thawed samples were heated at 75°C for 20 min and subsequently cooled in a programmed water bath from 54.4 to ≤8.5°C in 15, 18, or 21 h. For the enumeration of C. perfringens, samples were plated on tryptose-sulfite-cycloserine agar and incubated in an anaerobic chamber at 37°C for 48 h. Population densities for cooked ham and beef increased as cooling time increased, and NaCl exerted a strong inhibitory effect on the germination and outgrowth of C. perfringens. For beef, while 3% NaCl completely arrested growth, pathogen numbers increased by ≥3, 5, and 5 log10 CFU/g in 15, 18, and 21 h, respectively, when the NaCl level was <2%. C. perfringens did not grow during cooling for 15, 18, or 21 h in ham samples containing ≥3.1% NaCl. Results obtained in this study suggest that a 15-h cooling time for cooked ham, which is normally formulated to contain >2% NaCl, would yield an acceptable product (with an increase of <1 log10 CFU/g in the C. perfringens count); however, for beef containing <2% NaCl, C. perfringens populations may reach levels high enough to cause illness.

Control of Clostridium perfringens Germination and Outgrowth by Buffered Sodium Citrate during Chilling of Roast Beef and Injected Pork

Inhibition of the germination and outgrowth of Clostridium perfringens by buffered sodium citrate (Ional) and buffered sodium citrate supplemented with sodium diacetate (Ional Plus) during the abusive chilling of roast beef and injected pork was evaluated. Beef top rounds or pork loins were injected with a brine containing NaCl, potato starch, and potassium tetrapyrophosphate to yield final in-product concentrations of 0.85, 0.25, and 0.20%, respectively. Products were ground and mixed with Ional or Ional Plus at 0, 0.5, 1.0, and 2.0%. Each product was mixed with a three-strain C. perfringens spore cocktail to obtain final spore concentrations of ca. 2.5 log10 spores per g. Chilling of roast beef from 54.4 to 7.2°C resulted in C. perfringens population increases of 1.51 and 5.27 log10 CFU/g for 18- and 21-h exponential chill rates, respectively, while chilling of injected pork resulted in increases of 3.70 and 4.41 log10 CFU/g. The incorporation of Ional into the roast beef formulation resulted in C. perfringens population reductions of 0.98, 1.87, and 2.47 log10 CFU/g with 0.5, 1.0, and 2.0% Ional, respectively, over 18 h of chilling, while ≥1.0% Ional Plus was required to achieve similar reductions (reductions of 0.91 and 2.07 log10 CFU/g were obtained with 1.0 and 2.0% Ional Plus, respectively). An Ional or Ional Plus concentration of ≥1.0% was required to reduce C. perfringens populations in roast beef or injected pork chilled from 54.4 to 7.2°C in 21 h. Cooling times for roast beef or injected pork products after heat processing can be extended to 21 h through the incorporation of ≥1.0% Ional or Ional Plus into the formulation to reduce the potential risk of C. perfringens germination and outgrowth.

Clostridium perfringens and foodborne infections

Clostridium perfringens type A food poisoning is one of the more common in the industrialised world. This bacterium is also responsible for the rare but severe food borne necrotic enteritis. C. perfringens enterotoxin (CPE) has been shown to be the virulence factor responsible for causing the symptoms of C. perfringens type A food poisoning. CPE is a single polypeptide chain with a molecular weight of 3.5 kDa that binds to receptors on the target epithelial cells. Through a unique four-step membrane action it finally causes a breakdown in normal plasma membrane permeability properties. Genetic studies of cpe have shown that cpe can be either chromosomal or plasmid-borne and that only a small minority of the global C. perfringens population is cpe positive. CPE expression appears to be transcriptionally regulated during sporulation, at least in part, by regulatory factors that are common to all C. perfringens isolates.

Predictive model for growth of Clostridium perfringens at temperatures applicable to cooling of cooked meat

The objective of this study was to develop a model to predict the growth of Clostridium perfringens from spores at temperatures applicable to the cooling of cooked meat products. The growth medium used was trypticase-peptone-glucose-yeast extract broth. C. perfringens population counts were determined at appropriate intervals by plating onto tryptose-sulfite-cycloserine agar. C. perfringens growth from spores was not observed at a temperature of 51°C for up to 3 weeks. It was found that, generally, for relative growth of more than 12 log10, the use of the logistic function provided a better prediction than the use of the Gompertz function. The two parameters: germination, outgrowth and lag (GOL) time, and exponential growth rate, EGR, were determined using the logistic function. The exponential growth rates and the reciprocal of the GOL times were fitted to the square root Ratkowsky model, using temperature as the independent variable. Applying multi-variate statistical procedures, confidence intervals were computed on the prediction of the amount of relative growth for a given temperature. Closed form equations are developed that allow for predicting growth for a general cooling scenario and the standard error of the prediction. These equations depend upon microbiological assumptions of the effect of history of the GOL times for gradual changes in temperature. For example, for cooling from 50°C to 10°C in 8 h, the equations predict a relative growth of 3·37 with an upper 97·5% confidence limit of 6·73.

An overview of Clostridium perfringens enterotoxin

Clostridium perfringens enterotoxin (CPE) is considered to be the virulence factor responsible for causing the symptoms of C. perfringens type A food poisoning and may also be involved in other human and veterinary illnesses. CPE has a unique four-step membrane action that apparently involves: (1) CPE binding to a 50,000 mol. wt mammalian protein receptor, forming a small complex of 90,000 mol. wt; (2) the development of a post-binding physical change to this small complex; this physical change could represent either the insertion of CPE into the membrane or a conformational change to small complex; (3) an interaction between this physically changed small complex and a 70,000 mol. wt mammalian protein, forming a large, 160,000 mol. wt complex in membranes; and (4) a breakdown in normal plasma membrane permeability properties for small (< 200,000 mol. wt) molecules. Structure-function analyses have identified a receptor binding region at the C-terminus of CPE and indicate that residues in the N-terminal half of CPE are required for the second step in CPE action to occur. Finally, cpe genetic studies are in their infancy but already indicate that cpe can be either chromosomal or plasmid-borne and that only a tiny minority of the global C. perfringens population is cpe positive. CPE expression appears to be transcriptionally regulated during sporulation, at least in part, by regulatory factors that are common to all C. perfringens isolates.

Growth of Clostridium perfringens from spore inocula in sous-vide turkey products.

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag ground turkey (pH 6) that included 0.3% (w/w) sodium pyrophosphate, and sodium chloride at 0, 1, 2, or 3% (w/w). The packages were processed to an internal temperature of 71.1 degrees C, ice chilled and stored at various temperatures. The total C. perfringens population was determined by plating diluted samples on tryptose-sulfite-cycloserine agar followed by anaerobic incubation at 37 degrees C for 48 h. At 28 degrees C, the addition of 3% salt in turkey was effective in delaying growth for 12 h. At 15 degrees C, growth occurred at a relatively slow rate in the presence of 1-2% salt. Vegetative cells were not observed even after 28 days of storage in the presence of 3% salt. C. perfringens growth was not observed at 4 degrees C regardless of salt levels. The D-values ranged from 23.2 min (no salt) to 17.7 min (3% salt). Cyclic and static temperature abuse of refrigerated products for 8 h did not lead to growth by C. perfringens from a spore inoculum.

Growth of Clostridium perfringens in Fish Fillets Packaged with a Controlled Carbon Dioxide Atmosphere at Abuse Temperatures

The growth of Clostridium perfringens inoculated in fish fillets of jack mackerel subsequently packaged under a controlled carbon dioxide gas atmosphere (40% CO2, 60% N2) was investigated at marginal growth (15°C) and stimulative ambient (30°C) handling temperatures. The fish fillets were inoculated with C. perfringens, packaged either with air or the modified controlled carbon dioxide atmosphere and stored at 15°C and 30°C. No increase in the C. perfringens population in the fish was noted regardless of the type of packaging at 15°C in 3 days storage time, when all samples were spoiled. C. perfringens rapidly increased in the abuse temperature (30°C) after a 2- to 4-h lag phase regardless of the package type, but growth was significantly more stimulated under the controlled carbon dioxide gas atmosphere within 6 h of storage time. When fish fillets inoculated with C. perfringens were stored at 5°C for 24 h before being held at 30°C for 6 h, C. perfringens did not grow during the abuse-temperature storage. This suggests a reduction of a health hazard risk by the organism when the distribution temperature of the fish fillets is strictly controlled below 5°C. However, the combination of two temperature-abuse events during distribution and consumer handling may lead to a higher food-poisoning risk by the organism in controlled CO2 modified atmosphere-packaged fish compared to air-packaged fish. Product control of the initial contamination of organisms at low levels during raw fish processing will prevent food poisoning

OUTGROWTH OF CLOSTRIDIUM PERFRINGENS SPORES IN COOK‐IN‐BAG BEEF PRODUCTS1

ABSTRACTClostridium perfringens growth from a spore inoculum was investigated in vacuum‐packaged, cook‐in‐bag ground beef that included 0.3% (w/w) sodium pyrophosphate, pH 5.5 or 7.0, and salt (sodium chloride) at 0 or 3% (w/w). The packages were processed to an internal temperature of 71.1C, ice chilled and stored at various temperatures. The total C. perfringens population was determined by plating diluted samples on tryptose‐sulfite‐cycloserine agar followed by anaerobic incubation for 48 h at 37C. At 28C, a combination of 3% salt and pH 5.5 was effective in delaying growth for 24 h. At 15C, growth occurred within 6 days in samples with pH 7.0, but was delayed until day 8 in the presence of 3% salt at pH 7.0. Vegetative cells were not observed even after 21 days 21 of storage at 15C in the presence 3% salt at pH 5.5. C. perfringens growth was not observed at 4C regardless of pH or salt levels. The D‐values ranged from 23.33 min (3% salt, pH 7.0) to 13.99 min (3% salt, pH 5.5). Cyclic and static temperature abuse of refrigerated products for 12–15 h did not permit C. perfringens growth. However, temperature abuse of cook‐in‐bag beef products for periods longer than 15 h in the absence of salt led to growth of C. perfringens from a spore inoculum.

The effect of temperature abuse on Clostridium perfringens in cooked turkey stored under air and vacuum

The potential for growth of Clostridium perfringens in aerobic and anaerobic (vacuum) packaged cooked ground turkey was investigated. Samples of autoclaved ground turkey were inoculated with ∼3·0 log10 cfu g-1 of C. perfringens strain NCTC 8238 or NCTC 8239, packaged and stored at various temperatures. Vegetative growth and heat-resistant spores were enumerated by plating unheated and heated (75°C for 20 min) samples, respectively, on tryptose-sulfite-cycloserine agar. The type of atmosphere influenced the growth of C. perfringens at 15 and 28°C. Both strains grew to about 7 logs within 9 h anaerobically and by 24 h aerobically at 28°C. While aerobic growth was slow at 15°C, mean log10 cfu g-1 increased anaerobically by 4-4·5 logs by day 8 for both strains. Spores were not found at 4 and 15°C, but were detected as early as 24 h at 2°C under anaerobic conditions in both strains. C. perfringens population stabilized or slowly decreased at 4°C. Cyclic and static temperature abuse of refrigerated products for 5 h will not permit C. perfringens growth. However, temperature abuse of such products for relatively long periods may lead to high and dangerous numbers of organisms. Reheating such products to an internal temperature of 65°C before consumption would prevent food-poisoning since the vegetative cells were killed.

Effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens introduced into chickens

The effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens (KGW 851) newly introduced into chickens was studied. Four groups of chickens consisted of: (1) Uninoculated controls; (2) inoculated with C perfringens (KGW 851); (3) inoculated with E tenella; and (4) inoculated with C perfringens (KGW 851) followed by E tenella. Five chickens in each group were necropsied on each of days 0, 3, 5, 7, 10 and 14 after the E tenella inoculation. The mean total C perfringens counts in the caecal contents increased at five days after E tenella inoculation and reached maximum counts at seven days after the inoculation. Also lincomycin-resistant C perfringens readily established itself at approximately one 10th of the total C perfringens population in the presence or absence of E tenella infection.

BEHAVIOR OF CLOSTRIDIUM PERFRINGENS IN PRECOOKED CHILLED GROUND BEEF GRAVY DURING COOLING, HOLDING, AND REHEATING

The suitability of handling practices used in school kitchens was evaluated using ground beef gravy that was contaminated with Clostridium perfringens. Cooked gravy was cooled to 110 F (43.5 C) and inoculated with a mixture of vegetative cells and spores of C. perfringens NCTC 8239 to provide approximately 10,000 organisms/g. After inoculation, gravy was packed in bags, refrigerated for 16 h, held for 5 h at 82 F (28 C) or 42 F (5.5 C), and then heated in a compartment steamer for 35 min or until the temperature of the gravy at the center of the pan reached 165 F (74 C). C. perfringens was enumerated at intervals during cooling, holding, and heating of the gravy. The number of viable cells after 16 h of refrigeration at 42 F (5.5 C) was influenced by the first 6 h of cooling when the temperature of the gravy was in the range that permitted growth of C. perfringens (65–122 F; 18.5–50 C). Plate counts of gravy held for 5 h at 82 F (28 C) or 42 F (5.5 C) indicated stablization of the C. perfringens population. When 165 F (74 C) was the final temperature to which the gravy was heated, no viable cells of C. perfringens were found.