Growth Of Clostridium Perfringens Research Articles

Estimation of growth of Clostridium perfringens in cooked beef under fluctuating temperature conditions

A new concept for estimating the bacterial growth under temperature fluctuations was hypothesized and validated using Clostridium perfringens as a test organism. This new methodology was based on the Gompertz models to calculate the equivalent growth times under different temperatures, and estimated the bacterial population under temperature fluctuations. The new concept was tested in ground beef maintained under fluctuating temperature conditions. The estimation accuracy of this methodology was generally within 1.0 log 10 (cfu/g). Although the methodology was based on C. perfringens, it can potentially be applied to other foodborne pathogens to predict the bacterial growth under temperature fluctuations.

Impact of Cooking, Cooling, and Subsequent Refrigeration on the Growth or Survival of Clostridium perfringens in Cooked Meat and Poultry Products

In January 1999, the Food Safety and Inspection Service (FSIS) finalized performance standards for the cooking and chilling of meat and poultry products in federally inspected establishments. More restrictive chilling (stabilization)requirements were adopted despite the lack of strong evidence of a public health risk posed by industry practices employing the original May 1988 guidelines (U.S. Department of Agriculture FSIS Directive 7110.3). Baseline data led the FSIS to estimate a “worst case” of 104Clostridium perfringens cells per g in raw meat products. The rationale for the FSIS performance standards was based on this estimate and the assumption that the numbers detected in the baseline study were spores that could survive cooking. The assumptions underlying the regulation stimulated work in our laboratory to help address why there have been so few documented outbreaks of C. perfringens illness associated with the consumption of commercially processed cooked meat and poultry products. Our research took into account the numbers of C. perfringens spores in both raw and cooked products. One hundred ninety-seven raw comminuted meat samples were cooked to 73.9°C and analyzed for C. perfringens levels. All but two samples had undetectable levels (<3 spores per g). Two ground pork samples contained 3.3 and 66 spores per g. Research was also conducted to determine the effect of chilling on the outgrowth of C. perfringens spores in cured and uncured turkey. Raw meat blends inoculated with C. perfringens spores, cooked to 73.9°C, and chilled according to current guidelines or under abuse conditions yielded increases of 2.25 and 2.44 log10 CFU/g for uncured turkey chilled for 6 h and an increase of 3.07 log10 CFU/g for cured turkey chilled for 24 h. No growth occurred in cured turkey during a 6-h cooling period. Furthermore, the fate of C. perfringens in cooked cured and uncured turkey held at refrigeration temperatures was investigated. C. perfringens levels decreased by 2.52, 2.54, and 2.75 log10 CFU/g in cured turkey held at 0.6, 4.4, and 10°C, respectively, for 7 days. Finally, 48 production lots of ready-to-eat meat products that had deviated from FSIS guidelines were analyzed for C. perfringens levels. To date, 456 samples have been tested, and all but 25 (ranging from 100 to 710 CFU/g) of the samples contained C. perfringens at levels of <100 CFU/g. These results further support historical food safety data that suggest a very low public health risk associated with C. perfringens in commercially processed ready-to-eat meat and poultry products.

Dynamic computer simulation of Clostridium perfringens growth in cooked ground beef

The objective of this study was to develop a computer simulation algorithm to dynamically estimate and predict the growth of Clostridium perfringens in cooked ground beef. The computational algorithm was based on the implicit form of the Gompertz model, the growth kinetics of C. perfringens in cooked ground beef, and the fourth-order Runge–Kutta numerical method. This algorithm was validated using a cocktail of three strains of C. perfringens spores grown under isothermal, square-waved, linear cooling, and exponential cooling temperature profiles. In general, the results of computer simulation matched closely with the experimental data with the absolute errors less than 0.5 log 10 CFU/g. This method may be a useful tool for the food industry, regulatory agencies, distributors, and retailers to predict the effect of temperature abuse on the microbial safety of C. perfringens and other foodborne pathogens in processed meat products.

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.

Description of growth of Clostridium perfringens in cooked beef with multiple linear models

The traditional linear model used in food microbiology employs three linear segments to describe the process of food spoilage and categorize a growth curve into three phases — lag, exponential, and stationary. The linear model is accurate only within certain portions of each phase of a growth process, and can underestimate or overestimate the transitional phases. While sigmoid functions (such as the Gompertz and logistic equations) can be used to fit the experimental growth data more accurately, they fail to indicate the physiological state of bacterial growth. The objective of this paper was to develop a new methodology to describe and categorize accurately the bacterial growth as a process using Clostridium perfringens as a test organism. This methodology utilized five linear segments represented by five linear models to categorize a bacterial growth process into lag, first transitional, exponential, second transitional, and stationary phases. Growth curves described in this paper using multiple linear models were more accurate than the traditional three-segment linear models, and were statistically equivalent to the Gompertz models. With the growth rates of transitional phases set to 1/3 of the exponential phase, the durations of the lag, first transitional, exponential, and second transitional phases in a growth curve described by the new method were correlated linearly. Since this linear relationship was independent of temperature, a complete five-segment growth curve could be generated from the maximum growth rate and a known duration of the first four growth phases. Moreover, the lag phase duration defined by the new method was a linear function of the traditional lag phase duration calculated from the Gompertz equation. With this relationship, the two traditional parameters (lag phase and maximum growth rate) used in a three-segment linear model can be used to generate a more accurate five-segment linear growth curve without involving complicated mathematical calculations.

Inhibition of Growth, Enterotoxin Production, and Spore Formation of Clostridium perfringens by Extracts of Medicinal Plants

The extracts of 14 plants used in the traditional medicine of Mexico were evaluated for their effects on the growth, spore formation, and enterotoxin production of Clostridium perfringens type A. The extracts of Psidium guajava L., Haemotoxylon brasiletto, and Euphobia prostata were the most effective inhibitors of growth, spore formation, and enterotoxin production. No enterotoxins were detected when extracts were added to the media at less than the MIC for growth.

Predictive model for growth of Clostridium perfringens during cooling of cooked cured chicken

Estimates of the growth kinetics of Clostridium perfringens from spores at temperatures applicable to the cooling of cooked cured chicken products are presented. A model for predicting relative growth of C. perfringens from spores during cooling of cured chicken is derived using a nonlinear mixed effects analysis of the data. This statistical procedure has not been used in the predictive microbiology literature that has been written for microbiologists. However, recently software systems have been including this statistical procedure. The primary growth curves, based on the stages of cell development, identify two parameters: (1) germination, outgrowth, and lag (GOL) time, or lag phase time; and (2) exponential growth rate, egr. The mixed effects model does not consider GOL and egr as constants, but as random variables that would, in all likelihood, differ for different cooling events with the same temperature. As such, it is estimated that the egr, for a given temperature, has a CV of approximately 19%. The model obtained by the mixed effects model is compared to the one obtained by the more traditional two-stage approach. The estimated parameters from the derived models are virtually the same. The model predicts, for example, a geometric mean relative growth of about 9·4 with an upper 95% confidence limit of 21·3 when cooling the product from 51°C to 12°C in 8 h, assuming log linear decline in temperature with time. C. perfringens growth from spores was not observed at a temperature of 12°C for up to 3 weeks.

A probabilistic analysis of Clostridium perfringens growth during food service operations

The purpose of this study was threefold: first, the study was designed to illustrate the use of data and information collected in food safety surveys in a quantitative risk assessment. In this case, the focus was on the food service industry; however, similar data from other parts of the food chain could be similarly incorporated. The second objective was to quantitatively describe and better understand the role that the food service industry plays in the safety of food. The third objective was to illustrate the additional decision-making information that is available when uncertainty and variability are incorporated into the modelling of systems.

Growth of Clostridium perfringens from spore inocula in cooked cured beef: development of a predictive model

The objective of this study was to develop a model to predict the growth of C. perfringens from spores at temperatures applicable to the cooling of cooked cured meat products. C. perfringens growth from spores was not observed at a temperature of 12 °C for up to 3 weeks. The two parameters: germination, outgrowth, and lag (GOL) time and exponential growth rate, EGR, were determined using a function derived from mechanistic and stochastic considerations and the observed relative growths at specified times. A general model to predict the amount of relative growth for arbitrary temperature was determined by fitting the exponential growth rates to a square root Ratkowsky function, and assuming a constant ratio of GOL and generation times. The predicted relative growth is sensitive to the value of this ratio. A closed form equation was developed that can be used to estimate the relative growth for a general cooling scenario and determine a standard error of the estimate. The equation depends upon microbiological assumptions of the effect of history of the GOL times for gradual changes in temperature. Applying multivariate statistical procedures, a confidence interval was computed on the prediction of the amount of growth for a given temperature. The model predicts, for example, a relative growth of 3.17 with an upper 95% confidence limit of 8.50 when cooling the product from 51 to 11 °C in 8 h, assuming a log linear decline in temperature with time.

Clostridium perfringens β2-toxin in an African elephant (Loxodonta africana) with ulcerative enteritis

A 22-year-old female African elephant (Loxodonta africana) developed diarrhoea of unknown cause which lasted for two days. The animal was euthanased after it remained recumbent and refused to get up....

Survival and growth of enterotoxin-positive and enterotoxin-negative Clostridium perfringens in laboratory media

The survival and growth characteristics of enterotoxin-positive and enterotoxin-negative Clostridium perfringens were compared. Spores of C. perfringens were heated and incubated in laboratory media to simulate the cooking and inadequate storage of cooked food. In our experiment, enterotoxin-positive and enterotoxin-negative spores were heated and incubated individually and it was found that spores of enterotoxin-positive strains were more heat-resistant than spores of the enterotoxin-negative strains. In another experiment, cocktails of enterotoxin-positive and enterotoxin-negative spores were heated and incubated. At the time of inoculation, the ratio of enterotoxin-positive spores to enterotoxin-negative spores was about 1:100. In the case of high-temperature heat treatment, the number of enterotoxin-negative spores decreased to below that of enterotoxin-positive spores after heating, and enterotoxin-positive strains grew to high levels during incubation. In the case of low-temperature heat treatment, the number of enterotoxin-negative spores remained almost unchanged after heating and enterotoxin-positive strains did not grow to high CFU levels during incubation.

Cooling Rate Effect on Outgrowth of Clostridium Perfringens in Cooked, Ready-to-Eat Turkey Breast Roasts

The potential for Clostridium perfringens spores to germinate and grow in cooked, ready-to-eat turkey products was evaluated to determine a safe cooling rate within the critical temperatures of 48.9 C (120 F) through 12.8 C (55 F). Raw turkey deli breast roasts were inoculated with a cocktail of C. perfringens spores (NCTC 8238, NCTC 8239, and NCTC 10388) and cooked in a steam oven to an internal temperature of 72 C. The sample roasts were then cooled through the critical cooling range at rates yielding cooling times of 6, 8, and 10 h. Turkey roasts were analyzed for spore growth and multiplication using tryptose-sulfite-cycloserine agar and anaerobic incubation at 37 C for 48 h. Cooling times of 6 and 8 h showed no proliferation of C. perfringens that would violate the USDA/Food Safety Inspection Service safe cooling standard criteria, which would allow no more than a 1 log10 multiplication between 48.9 and 12.8 C. A 9.6-h cooling period between the designated temperatures at a 95% confidence interval was determined to be adequate for nonproliferation of C. perfringens. On the other hand, a 95% tolerance interval would be more stringent in that it suggests no more than an 8.9-h cooling period. Tolerance intervals required that 95% of all our observations did not exceed the limit of 1 log10 increase in C. perfringens. This study indicated that in cooked, ready-to-eat turkey deli breasts, a cooling period between 48.9 C (120 F) and 12.8 C (55 F) of no greater than 8.9 h should be utilized to prevent possible C. perfringens foodborne outbreaks.

In vitro study of Prebiotic Properties of Levan-type Exopolysaccharides from Lactobacilli and Non-digestible Carbohydrates Using Denaturing Gradient Gel Electrophoresis

Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.

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.

Feasibility of using food-grade additives to control the growth of Clostridium perfringens

Previously, it was demonstrated that the combination of sucrose laurate (SL) ethylenediaminetetraacetate (E) and butylated hydroxyl anisole (B) (SLEB) was an effective antimicrobial agent against both gram-negative (aerobes) and gram-positive (facultative anaerobes) foodborne bacteria. This investigation examines the sensitivity of Clostridium perfringens to SLEB relative to: (1) the minimum inhibitory concentration (MIC) of SLEB required to inhibit the growth of C. perfringens and (2) the antibacterial effectiveness of different combination ratios of SLEB in fluid thioglycollate medium (FTM). Results indicated that the MIC of SLEB (1:1:1, v/v/v) against C. perfringens on tryptose sulfite cycloserine (TSC) agar was >150 ppm at 37°C. However, in FTM, a SLEB (1:1:1, v/v/v) concentration of >100 ppm inhibited C. perfringens during an incubation (anaerobic) period of 196 h at 37°C. The sensitivity of C. perfringens to different combination ratios was also investigated in FTM. The results showed that, when the concentrations of SL and E were held at 75 ppm in the SLEB combination, and the concentration of B increased from 0 to 75 ppm, C. perfringens growth increased initially during the first 24 h of incubation (37°C) but remained constant during the next 48 h. Similarly, when concentrations of SL and E were held constant at 150 ppm in the SLEB combination and the B ratio increased from 50 to 150 ppm in FTM, C. perfringens viability decreased in all of the treated samples during 72-h incubation at 37°C. The results indicated that SLEB was an effective inhibitor of C. perfringens growth activities, and the ratios of the components of SLEB can be adjusted to meet specific preservation needs.

Effect of Antibiotic Growth Promoters and Anticoccidials on Growth of Clostridium perfringens in the Caeca and on Performance of Broiler Chickens

The effects of the growth promoters avoparcin and avilamycin and the ionophore anticoccidials maduramicin, narasin and monensin on the growth of Clostridium perfringens (Cp) in the caeca and on performance of broiler chickens were tested in 2 experiments. The supplements were fed as single feed additives or in some combinations. No clinical signs or lesions caused by coccidia were observed in any of the studies. All supplements had an antibacterial effect on Cp and improved growth rate significantly. Carcass yield of birds fed growth promoters avilamycin or avoparcin was significantly higher compared with birds fed anticoccidials. These data indicate that, what concerns bird performance, during good hygienic conditions supplementation with antibiotic growth promoters may not be necessary when the diet is supplemented with an anticoccidial with antibacterial effects.

Carbohydrate source and bifidobacteria influence the growth of Clostridium perfringens in vivo and in vitro

The purpose of this study was to test the effects of administering bifidobacteria with either 2% fructooligosaccharide (FOS), 2% wheat bran oligosaccharide (WBOS), 2% soybean oligosaccharide (SBOS) or no added oligosaccharide (control) on cecal populations of bifidobacteria and C. perfringens, a potential pathogen and normal inhabitant of the mammalian colon, in the rat. Rats were randomly assigned to one of four treatment groups: basal diet + 1 ml daily gavage of skim milk containing 10 8 bifidobacteria, 2% FOS + bifidobacteria, 2% SBOS + bifidobacteria or 2% WBOS + bifidobacteria. The rats were fed the diets for 4 wk, after which cecal concentrations of bifidobacteria and C. perfringens were assessed. WBOS and SBOS feeding resulted in higher concentrations of bifidobacteria and C. perfringens relative to FOS-feeding or control. However, no differences in either bifidobacteria or C. perfringens were detected between control and FOS groups. Parallel to this animal study, in vitro competition experiments with bifidobacteria and C. perfringens were performed in growth media containing either glucose, FOS or WBOS as the primary carbon source. The concentrations of bifidobacteria and C. perfringens were determined at each hour for 10 h and specific growth rates (μ) were calculated. The μ for C. perfringens, co-cultured with bifidobacteria in glucose-based or WBOS-based media was significantly decreased relative to C. perfringens alone. However, no significant difference was found between the μ for C. perfringens co-cultured in FOS-based media with bifidobacteria and the C. perfringens grown alone in FOS-based media. These findings indicate that different oligosaccharides have differential effects on the populations of bifidobacteria and C. perfringens in vivo and that certain oligosaccharides may potentiate an inhibitory action of bifidobacteria against C. perfringens.

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

The Effects of Nonautoclaved and Autoclaved Water-Soluble Wheat Extracts on the Growth of Clostridium perfringens

Clostridium perfringens is the causative agent of necrotic enteritis, a commonly diagnosed disease in chickens that is also observed in turkeys and geese. Two trials were conducted to determine the in vitro effect of filter-sterilized, water-soluble wheat extracts on the growth of C. perfringens. The extracts were either nonautoclaved or autoclaved at 121 C for 40 min and were used to reconstitute thioglycolate broth media.Results of this study suggest that growth of C. perfringens is suppressed in vitro by inclusion of either extract. Glycosyl composition analysis revealed no significant differences in arabinose, xylose, or mannose content between the nonautoclaved and autoclaved extracts. Galactose, glucose, and total glycosyl content were significantly higher in the nonautoclaved extract.