Evaluation of a Clostridium perfringens Predictive Model, Developed under Isothermal Conditions in Broth, To Predict Growth in Ground Beef during Cooling

Proper temperature control is essential in minimizing Clostridium perfringens germination, growth, and toxin production. The U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) offers two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log₁₀ CFU/g increase of C. perfringens and no growth of Clostridium botulinum. A mathematical model developed by Juneja et al. (Food Microbiol. 16:335-349, 1999) may be helpful in determining if the C. perfringens performance standard has been achieved, but this model has not been extensively validated. The objective of this study was to validate the Juneja 1999 model in ground beef under a variety of changing temperature and temperature abuse situations. The Juneja 1999 model consistently underpredicted growth of C. perfringens during exponential cooling of ground beef. The model also underpredicted growth of C. perfringens in ground beef cooled at two different rates. The results presented here show generally good agreement with published data on the growth of C. perfringens in similar products. The model error may be due to faster-than-expected exponential growth rates in ground beef during cooling or an error in the mathematical formulation of the model.     

DETECTION of ENTEROTOXIGENIC CLOSTRIDIUM PERFRINGENS IN GROUND BEEF

Clostridium perfringens is widely distributed in foods. This experiment was performed to assess occurrence of C. perfringens cultures and toxigenic strains isolated from ground beef Samples (118) were collected from 20 locations in Northeast Kansas and the number of C. perfringens was enumerated in these samples by Fung's Double-tube method with tryptose sulfite cycloserine agar medium. Out of 118 samples, 54 (46%) were found positive for C. perfringens. Pure isolates of C. perfringens were further grown in cooked meat medium for 24 h at 42C then heat shocked at 75C for 20 min and inoculated into modified Duncan and Strong medium for production of C. perfringens enterotoxin. Presence of enterotoxin was tested by the reverse passive latex agglutination test (Oxoid), which can detect enterotoxin up to a minimum level of 2 ng/mL. the data indicate that 46% of the beef samples harbored C. perfringens , but only 32 (6%) of 525 isolates were found to produce enterotoxin. This study emphasized the importance of continued surveillance of C. perfringens in meats and meat products and assessment of the toxigenesis of isolates.     

Growth of Clostridium perfringens in cooked chili during cooling

U.S. Department of Agriculture regulations require that brick chili be cooled from 48.9 degrees C to 4.4 degrees C within 2 h of cooking, but processors may not always be able to comply. Studies were conducted to evaluate the extent of bacterial multiplication resulting from outgrowth of germinated Clostridium perfringens spores experimentally inoculated into chili and incubated at various temperatures. Inoculated samples were heated (75 degrees C for 20 min) to activate spores, quickly equilibrated, and held at one of five desired temperatures for 6 h. No growth was observed for C. perfringens in samples held at 26.7 degrees C and below for 6 h, but growth was observed by 6 h in samples held at 32.2 degrees C and after 2 h in samples held at temperatures between 37.8 degrees C and 48.9 degrees C. Using isothermal growth data, we developed a simple model for predicting the growth of bacteria with time under exponential cooling conditions. The model predicts both the lag phase and the numbers of bacteria at specific times during the growth phase. It was developed by using isothermal growth data and tested by using temperature-varying growth data from experiments with spores of C. perfringens in chili. Actual data agreed closely with predicted results. The results should be useful for evaluating the hazard potential for growth of C. perfringens in chili.     

Effect of sample transport systems on survival of bacteria in ground beef.

The effects of two transport systems and cryoprotective agents on the survival of bacteria in ground beef samples were evaluated. Survival of Clostridium perfringens in ground beef samples after simulated transport (72 h) was higher (about 99%) in Dry Ice than in Trans Temp shipping units (-3 degrees C). There were no significant differences between the two transport systems in survival of coliforms, Escherichia coli, Staphylococcus aureus, or aerobic bacteria. Mixing ground beef samples at a ratio of 1:1 (wt/vol) with 10, 20, or 30% buffered solutions of dimethyl sulfoxide or glycerol before freezing improved the survival of C. perfringens and coliforms in both transport systems. Recovery of E. coli was significantly higher with the addition of 10% dimethyl sulfoxide before Dry Ice transport. Addition of 10% dimethyl sulfoxide resulted in a 100% recovery of both S. aureus and aerobic bacteria from ground beef after simulated transport in Trans Temp shipping units. The use of cryoprotective agents can improve the survival of bacteria during transport of ground beef samples.     

Growth of Clostridium perfringens in cooked chili during cooling.

U.S. Department of Agriculture regulations require that brick chili be cooled from 48.9 degrees C to 4.4 degrees C within 2 h of cooking, but processors may not always be able to comply. Studies were conducted to evaluate the extent of bacterial multiplication resulting from outgrowth of germinated Clostridium perfringens spores experimentally inoculated into chili and incubated at various temperatures. Inoculated samples were heated (75 degrees C for 20 min) to activate spores, quickly equilibrated, and held at one of five desired temperatures for 6 h. No growth was observed for C. perfringens in samples held at 26.7 degrees C and below for 6 h, but growth was observed by 6 h in samples held at 32.2 degrees C and after 2 h in samples held at temperatures between 37.8 degrees C and 48.9 degrees C. Using isothermal growth data, we developed a simple model for predicting the growth of bacteria with time under exponential cooling conditions. The model predicts both the lag phase and the numbers of bacteria at specific times during the growth phase. It was developed by using isothermal growth data and tested by using temperature-varying growth data from experiments with spores of C. perfringens in chili. Actual data agreed closely with predicted results. The results should be useful for evaluating the hazard potential for growth of C. perfringens in chili.     

Effect of sample transport systems on survival of bacteria in ground beef.

The effects of two transport systems and cryoprotective agents on the survival of bacteria in ground beef samples were evaluated. Survival of Clostridium perfringens in ground beef samples after simulated transport (72 h) was higher (about 99%) in Dry Ice than in Trans Temp shipping units (-3 degrees C). There were no significant differences between the two transport systems in survival of coliforms, Escherichia coli, Staphylococcus aureus, or aerobic bacteria. Mixing ground beef samples at a ratio of 1:1 (wt/vol) with 10, 20, or 30% buffered solutions of dimethyl sulfoxide or glycerol before freezing improved the survival of C. perfringens and coliforms in both transport systems. Recovery of E. coli was significantly higher with the addition of 10% dimethyl sulfoxide before Dry Ice transport. Addition of 10% dimethyl sulfoxide resulted in a 100% recovery of both S. aureus and aerobic bacteria from ground beef after simulated transport in Trans Temp shipping units. The use of cryoprotective agents can improve the survival of bacteria during transport of ground beef samples.     

Bacteriological quality and shelf life of ground beef.

The bacteriological quality of unfrozen raw ground beef was evaluated after 0, 3, 6, 9, 12, 15, and 18 days of storage at 29 +/- 1 F (-1.7 +/- 0.6 C). At the time of fabrication, all of the ground beef samples contained 10(6) or fewer total aerobic and psychrotrophic bacteria/g; 81% contained 100 or fewer coliforms/g; 94% contained 100 or fewer Escherichia coli/g; and all of the samples contained 100 or fewer coagulase-positive Staphylococcus aureus and Clostridium perfringens/g. Total aerobic and psychrotrophic bacteria increased by 1 log between 3 and 18 days of storage. Coliform and E. coli counts decreased during storage, whereas coagulase-positive S. aureus and C. perfringens counts did not change significantly. These data indicate that meat processors, wholesalers, and retailers could improve the bacteriological quality and prolong the shelf life of ground beef packaged in oxygen-impermeable film if the temperature of product never exceeded 29 +/- 1 F (-1.7 +/- 0.6 C).     

Bacteriological quality and shelf life of ground beef.

The bacteriological quality of unfrozen raw ground beef was evaluated after 0, 3, 6, 9, 12, 15, and 18 days of storage at 29 +/- 1 F (-1.7 +/- 0.6 C). At the time of fabrication, all of the ground beef samples contained 10(6) or fewer total aerobic and psychrotrophic bacteria/g; 81% contained 100 or fewer coliforms/g; 94% contained 100 or fewer Escherichia coli/g; and all of the samples contained 100 or fewer coagulase-positive Staphylococcus aureus and Clostridium perfringens/g. Total aerobic and psychrotrophic bacteria increased by 1 log between 3 and 18 days of storage. Coliform and E. coli counts decreased during storage, whereas coagulase-positive S. aureus and C. perfringens counts did not change significantly. These data indicate that meat processors, wholesalers, and retailers could improve the bacteriological quality and prolong the shelf life of ground beef packaged in oxygen-impermeable film if the temperature of product never exceeded 29 +/- 1 F (-1.7 +/- 0.6 C).     

Effect of Soy Proteins on the Growth of Clostridium perfringens

Proteins that are used to fabricate imitation foods such as synthetic meats were evaluated for stimulative or inhibitory effects on the growth of Clostridium perfringens. Growth rate and extent were measured in thioglycolate medium without dextrose. This liquid medium contains Trypticase (BBL) which served as the protein control. For comparison, various soy proteins, synthetic meats, beef, turkey, sodium caseinate, and combinations of each were substituted for Trypticase. Meat loaf systems were also employed to determine the effects of protein additives to meat under actual meat loaf conditions. Growth of C. perfringens type A, strain S40, was measured in the respective media at 45 C at a pH of 7.0 and an E(h) of below -300 mv. Viable populations were enumerated by agar plate techniques on Trypticase-sulfite-yeast-citrate-agar incubated anaerobically (90% N(2)-10% CO(2)) for 18 hr at 35 C. When compared to Trypticase, some soy proteins had stimulative effects on the growth of C. perfringens, whereas sodium caseinate and some soy proteins were inhibitory. In liquid medium in which meat or soy meat was the source of protein, there was a marked stimulation by beef, chicken, and soy beef. Soy chicken supported growth at a rate less than observed with Trypticase. Under actual meat loaf conditions, the addition of soy meat or protein additives to beef did not affect the growth of C. perfringens. The addition of protein additives to turkey meat loaves significantly enhanced the rate of growth of C. perfringens. The stimulative effects of some soy proteins are significant in relation to control of foodborne disease.     

Development and validation of experimental protocols for use of cardinal models for prediction of microorganism growth in food products

An experimental protocol to validate secondary-model application to foods was suggested. Escherichia coli, Listeria monocytogenes, Bacillus cereus, Clostridium perfringens, and Salmonella were observed in various food categories, such as meat, dairy, egg, or seafood products. The secondary model validated in this study was based on the gamma concept, in which the environmental factors temperature, pH, and water activity (aw) were introduced as individual terms with microbe-dependent parameters, and the effect of foodstuffs on the growth rates of these species was described with a food- and microbe-dependent parameter. This food-oriented approach was carried out by challenge testing, generally at 15 and 10 degrees C for L. monocytogenes, E. coli, B. cereus, and Salmonella and at 25 and 20 degrees C for C. perfringens. About 222 kinetics in foods were generated. The results were compared to simulations generated by existing software, such as PMP. The bias factor was also calculated. The methodology to obtain a food-dependent parameter (fitting step) and therefore to compare results given by models with new independent data (validation step) is discussed in regard to its food safety application. The proposed methods were used within the French national program of predictive microbiology, Sym'Previus, to include challenge test results in the database and to obtain predictive models designed for microbial growth in food products.     

Effect of Potassium Sorbate on Salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum in Cooked, Uncured Sausage

Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate.     

Nonradioactive colony hybridization assay for detection and enumeration of enterotoxigenic Clostridium perfringens in raw beef.

A DNA probe endolabeled with digoxigenin by PCR was developed to detect and enumerate enterotoxigenic Clostridium perfringens in raw beef. After 2 h of hybridization, membranes were developed by using an anti-digoxigenin-alkaline phosphatase conjugated antibody. The resulting chromogenic reaction allowed us to detect and enumerate < or = 10 CFU of C. perfringens per g.     

Method for Estimating the Presence of Clostridium perfringens in Food

The methods currently used for the enumeration of Clostridium perfringens in food are often inadequate because of the rapid loss of viability of this organism when the sample is frozen or refrigerated. A method for estimating the presence of C. perfringens in food which utilizes the hemolytic and lecithinase activities of alpha toxin was developed. The hemolytic activity was measured in hemolysin indicator plates. Lecithinase activity of the extract was determined by the lecithovitellin test. Of 34 strains of C. perfringens associated with foodborne disease outbreaks, 32 produced sufficient alpha toxin in roast beef with gravy and in chicken broth to permit a reliable estimate of growth in these foods. Alpha toxin was extracted from food with 0.4 m saline buffered (at pH 8.0) with 0.05 mN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and concentrated by dialysis against 30% polyethylene glycol. A detectable quantity of alpha toxin was produced by approximately 10(6)C. perfringens cells per g of substrate, and the amount increased in proportion to the cell population. Results obtained with food samples responsible for gastroenteritis in humans indicate that a correlation can be made between the amount of alpha toxin present and previous growth of C. perfringens in food regardless of whether the organisms are viable when the examination is performed.     

Assessment of the Sanitary Effectiveness of Holding Temperatures on Beef Cooked at Low Temperature

Beef cubes cooked at low temperature were surface inoculated and incubated at 43.3 through 53.3 C to establish temperature limits for growth of staphylococci, salmonellae, and Clostridium perfringens. A greater than 99% reduction in staphylococci was achieved after 6 hr at 48.8 C, of salmonellae at 51.1 C, and of C. perfringens at 53.3 C. There were no survivors of a mixed inoculum of Staphylococcus aureus, Salmonella enteritidis, and C. perfringens after 12 hr at 51.1 C.     

Beneficial effect of catalase treatment on growth of Clostridium perfringens.

Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media.     

Beneficial effect of catalase treatment on growth of Clostridium perfringens.

Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media.     

Design of species-specific primers to identify 13 species of Clostridium harbored in human intestinal tracts

The genus Clostridium is dominant in human intestinal tracts and plays an important role in human health. We designed species-specific primers to identify 13 species of Clostridium (C. perfringens, C. paraputrificum, C. bifermentans, C. difficile, C. clostridiiforme, C. nexile, C. sphenoides, C. indolis, C. ramosum, C. cocleatum, C. butyricum, C. sordellii, and C. innocuum) easily and rapidly. The PCR annealing temperature was set at a uniform 60 C for application to all strains at the same time. To confirm the specificities of these primers, 85 intestinal bacteria in total, including type strains, reference strains, and isolates were used. Ten primers (including those for C. perfringens to C. cocleatum) indicated high specificities. Although there were some cross-reactions with the other three primers, the target species were distinguishable from other bacteria by the different sizes of PCR products.     

Biochemical differentiation between enterotoxigenic heat-sensitive and heat-resistant Clostridium perfringens strains

Some biochemical characteristics of 37 enterotoxigenic Clostridium perfringens strains isolated from human feces, ground beef, and soil samples by heat-selection methods and of two NCTC strains were studied. Two different biochemical patterns closely related to the heat resistance of the strains were found. The strains placed into group 1 were trehalose, inositol, and sorbitol negative and synthesized heat-resistant spores, while those placed into group 2 were trehalose and inositol positive and synthesized heat-sensitive spores. Sorbitol fermentation was variable among the strains of this last group. The strains of group 1 were more cellobiose, melibiose, and salicin fermentative than those of group 2. Only the strains placed into group 2 synthesized toxins of sufficient levels for typing. In spite of having been isolated by mild heat treatment of the specimens, two strains showed the same biochemical and toxigenic characteristics of the strains of group 1. The heating of these two strains did not modify their characteristics. We conclude that enterotoxigenic C. perfringens strains showing the two different toxigenic and biochemical patterns are present in the human gut, ground beef, and, probably, in soil. These strains may be differentiated on the basis of their capacity to produce acid from trehalose, inositol, and sorbitol, heat resistance of the spores and grade of toxigenicity. The heat-selection methods used for isolation of C. perfringens strains from different sources exerted a selection of strains from one or another group, but had no influence on their toxigenic and biochemical properties.     

Evaluation of acid phosphatase as a confirmation test for Clostridium perfringens isolated from water

AIMS: To evaluate testing for acid phosphatase as an alternative method for the confirmation of Clostridium perfringens isolated from water. METHODS AND RESULTS: Sixty-two reference strains of Clostridium were tested for their ability to produce acid phosphatase, as well as reduction of sulfite on tryptose sulfite cycloserine agar (TSC) and production of fluorescence in TSC supplemented with 4-methylumbelliferylphosphate (MUP). Additionally 155 environmental presumptive C. perfringens isolates from TSC incubated at 44 degrees C were identified and tested for acid phosphatase production and by the conventional MNLG (testing for motility, nitrate reduction, lactose fermentation and gelatin liquefaction) confirmation procedure. Twenty-seven strains from 15 species of Clostridium-reduced sulfite to some extent on TSC incubated at 44 degrees C, with a significant number of species being able to grow well at this temperature, indicating that a confirmation step is needed for the enumeration of C. perfringens on this medium. All 10 strains of C. perfringens tested, together with one strain each of Clostridium baratii and Clostridium rectum produced acid phosphatase. These also produced fluorescence on MUP supplemented TSC, as did 13 strains of acid phosphatase negative, sulfite-reducing clostridia, representing nine species. Of the environmental isolates, 114 were identified as C. perfringens of which 108 (94.7%) were confirmed by the acid phosphatase test compared with 104 (91.2%) by the MNLG tests. CONCLUSIONS: Testing for acid phosphatase production is at least as reliable, and much simpler to perform, than the current standard confirmation MNLG procedure. Incorporation of MUP into TSC does not reliably improve the identification of presumptive C. perfringens. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of testing for acid phosphatase as a confirmation test for C. perfringens would substantially simplify the analysis for this bacterium from water samples, and reduce the analysis time to confirmed counts.