John S. Novak

Quorum sensing: A primer for food microbiologists

Quorum sensing is a signaling mechanism through which bacteria modulate a number of cellular functions (genes), including sporulation, biofilm formation, bacteriocin production, virulence responses, as well as others. Quorum sensing is a mechanism of cell-to-cell communication and is mediated by extracellular chemical signals generated by the bacteria when specific cell densities are reached. When the concentration of the signal (and cell population) is sufficiently high, the target gene or genes are either activated or repressed. Quorum sensing increases the ability of the bacteria to have access to nutrients or to more favorable environmental niches and enhances bacterial defenses against eukaryotic hosts, competing bacteria, and environmental stresses. The physiological and clinical aspects of quorum sensing have received considerable attention and have been studied at the molecular level. Little is known, however, on the role of quorum sensing in food spoilage or in the growth and/or toxin production of pathogens present in food. A number of compounds have been isolated or synthesized that antagonize quorum sensors, and application of these antagonists may potentially be useful in inhibiting the growth or virulence mechanisms of bacteria in different environments, including food. It is important that food microbiologists have an awareness and an understanding of the mechanisms involved in bacterial quorum sensing, since strategies targeting quorum sensing may offer a means to control the growth of undesirable bacteria in foods.

Microbial safety of minimally processed foods.

VARIABLE FOOD ENVIRONMENTS Microbial Safety of Bakery Products, James P. Smith, Daphne Phillips Daifas, Wassim El-Khoury, and John W. Austin Concerns with Minimal Processing in Apple, Citrus, and Vegetable Products, Kathleen T. Rajkowski and Elizabeth A. Baldwin The Microbial Safety of Minimally Processed Seafood with Respect to Listeria Monocytogenes, Adam D. Hoffman, Kenneth L. Gall, and Martin Wiedmann, Fate of Clostridium Perfringens in Cook-Chill Foods, John S. Novak Sous-Vide Processed Foods: Safety Hazards and Control of Microbial Risks, Vijay K. Juneja PATHOGEN DETECTION AND ASSESSMENT HACCP and Regulations Applied to Minimally Processed Foods, O. Peter Snyder, Jr. Rapid Methods for Microbial Detection in Minimally Processed Foods, Karl R. Matthews Quantitative Risk Assessment of Minimally Processed Foods, Siobain Duffy, Yuhuan Chen, and Donald W. Schaffner CURRENT AND FUTURE INNOVATIONS Microbial Safety during Nonthermal Preservation of Foods, Gaurav Tewari Modified Atmosphere Packaging for Shelf-Life Extension, James T. C. Yuan Washing and Sanitizing Raw Materials for Minimally Processed Fruit and Vegetable Products, Gerald M. Sapers Microbial Safety, Quality, Extended Shelf-Life, and Sensory Aspects of Fresh-Cut Fruits and Vegetables, Hong Zhuang, M. Margaret Barth, and Thomas R. Hankinson Irradiation of Fresh and Minimally Processed Fruits, Vegetables, and Juices, Brendan A. Niemira Irradiation of Minimally Processed Meats, Christopher H. Sommers Biological Control on Minimally Processed Fruits and Vegetables, Britta Leverentz, Wojciech Janisiewicz, and William S. Conway INDEX

An assessment of pasteurization treatment of water, media, and milk with respect to Bacillus spores

This study evaluated the ability of spore-forming Bacillus spp. to resist milk pasteurization conditions from 72 to 150 degrees C. Spores from the avirulent surrogate Sterne strain of Bacillus anthracis, as well as a representative strain of a common milk contaminant that is also a pathogen, Bacillus cereus ATCC 9818, were heated at test temperatures for up to 90 min in dH2O, brain heart infusion broth, or skim milk. In skim milk, characteristic log reductions (log CFU per milliliter) for B. anthracis spores were 0.45 after 90 min at 72 degrees C, 0.39 after 90 min at 78 degrees C, 8.10 after 60 min at 100 degrees C, 7.74 after 2 min at 130 degrees C, and 7.43 after 0.5 min at 150 degrees C. Likewise, log reductions (log CFU per milliliter) for viable spores of B. cereus ATCC 9818 in skim milk were 0.39 after 90 min at 72 degrees C, 0.21 after 60 min at 78 degrees C, 7.62 after 60 min at 100 degrees C, 7.37 after 2 min at 130 degrees C, and 7.53 after 0.5 min at 150 degrees C. No significant differences (P < 0.05) in thermal resistance were observed for comparisons of spores heated in dH2O or brain heart infusion broth compared with results observed in skim milk for either strain tested. However, spores from both strains were highly resistant (P < 0.05) to the pasteurization temperatures tested. As such, pasteurization alone would not ensure complete inactivation of these spore-forming pathogens in dH2O, synthetic media, or skim milk.

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 degrees 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 degrees 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 degrees 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.

An ultrastructural comparison of spores from various strains of Clostridium perfringens and correlations with heat resistance parameters

It has been shown that Clostridium perfringens isolates associated with food poisoning carry a chromosomal cpe gene, whereas nonfood-borne human gastrointestinal disease isolates carry a plasmid cpe gene. In addition, the chromosomal cpe gene isolates exhibit greater heat resistance as compared with the plasmid cpe strains. Therefore, the current study conducted ultrastructural measurements of spores from several plasmid and chromosomal cpe-positive C. perfringens isolates. In support of the dehydration mechanism of spore heat resistance, the C. perfringens spore core average size was found to show a negative correlation with D-values for spores obtained at 100 degrees C. Dipicolinic acid (DPA) concentrations assayed for the spores did not correlate well with C. perfringens spore core averages nor with D(10)-values at 100 degrees C. Spore core thickness might be a distinguishing phenotypic characteristic used to identify heat resistance and survival potential of C. perfringens in improperly cooked foods.

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

Abstract 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.

Viability of Clostridium perfringens, Escherichia coli, and Listeria monocytogenes surviving mild heat or aqueous ozone treatment on beef followed by heat, alkali, or salt stress.

The threat of pathogen survival following ozone treatment of meat necessitates careful evaluation of the microorganisms surviving under such circumstances. The objective of this study was to determine whether sublethal aqueous ozone treatment (3 ppm of O3 for 5 min) of microorganisms on beef surfaces would result in increased or decreased survival with respect to subsequent heat, alkali, or NaCl stress. A mild heat treatment (55 degrees C for 30 min) was used for comparison. Reductions in three-strain cocktails of Clostridium perfringens, Escherichia coli O157:H7, and Listeria monocytogenes on beef following the heat treatment were 0.14, 0.77, and 1.47 log10 CFU/g, respectively, whereas reductions following ozone treatment were 1.28, 0.85, and 1.09 log10 CFU/g, respectively. C. perfringens cells exhibited elevated heat resistance at 60 degrees C (D60 [time at 60 degrees C required to reduce the viable cell population by 1 log10 units or 90%] = 17.76 min) following heat treatment of beef (55 degrees C for 30 min) but exhibited reduced viability at 60 degrees C following ozone treatment (D60 = 7.64 min) compared with the viability of untreated control cells (D60 = 13.84 min). The D60-values for L. monocytogenes and E. coli O157:H7 following heat and ozone exposures were not significantly different (P > 0.05). C. perfringens cells that survived ozone treatment did not exhibit increased resistance to pH (pH 6 to 12) relative to non-ozone-treated cells when grown at 37 degrees C for 24 h. The heat treatment also resulted in decreased numbers of surviving cells above and below neutral pH values for both E. coli O157:H7 and L. monocytogenes relative to those of non-heat-treated cells grown at 37 degrees C for 24 h. There were significant differences (P < 0.05) in C. perfringens reductions with increasing NaCl concentrations. The effects of NaCl were less apparent for E. coli and L. monocytogenes survivors. It is concluded that pathogens surviving ozone treatment of beef are less likely to endanger food safety than are those surviving sublethal heat treatments.

Effects of refrigeration or freezing on survival of Listeria monocytogenes Scott A in under-cooked ground beef

Abstract Listeria monocytogenes Scott A, inoculated into ground beef, was heat-shocked at 46 °C for 60 min to enhance stress adaptations and simulate sublethal minimal cooking conditions. The effects of refrigeration at 4 °C or freezing at −20 °C were examined on pathogen survival prior to or following mild cooking at 60 °C. D 10 -values for heat-shocked samples were elevated as compared to nonheat-shocked controls. Refrigerated and frozen storage did not influence the observed effects. Cellular injury of survivors increased with timed exposure to 60 °C. The effects of refrigerated and frozen storage on heat-adapted L. monocytogenes in ground beef did not decrease the potential of the food-borne pathogen to survive additional low temperature cooking of food and raised concerns over the food safety of contaminated, temperature-abused foods.

Clostridium perfringens: hazards in new generation foods

The retail increase in refrigerated ready-to-eat foods provides an exploitable environment suitable for an opportunistic pathogen such as Clostridium perfringens. The microorganism, in spite of a requirement for 13 essential amino acids, an optimal growth range of 43–45 °C, pH range 5–8, and a substrate water activity range of 0.93–0.97, can be found to be ubiquitously viable in food, water and air. Survival under extreme conditions is largely a factor of differentiation from metabolically active vegetative cells to highly resistant (100 °C for 60 min), dormant spores. It is the sporulation of large numbers of vegetative cells (106/g of food) and the associated release of enterotoxin in the human intestine that results in typical gastrointestinal symptoms of acute abdominal pain and diarrhea in conjunction with a loss of membrane ionic balance. As a consequence of the inevitable presence of C. perfringens in foods, the Food Safety Inspection Service (FSIS) of the US Department of Agriculture (USDA) has established performance standards for the pathogen in meat products. This review examines the current knowledge base of specific characteristics and attributes that make this microorganism a food safety concern. In addition, suggestions are made to reduce the likelihood of food-borne infection and transmission.

Increased thermotolerance of Clostridium perfringens spores following sublethal heat shock

Abstract Beef gravy samples inoculated with Clostridium perfringens spores were heat shocked at 75 °C for 20 min, and then thermotolerance at 100 °C was assessed using a submerged-coil heating apparatus. Survivors were enumerated on Shahidi Ferguson Perfringens agar. An association of the heat resistance with the origin of the C. perfringens could not be established due to significant variations in the heat resistance among strains. Interestingly, deviations from classical logarithmic linear declines in the log numbers with time were not observed in both control and heat shocked samples. D -values at 100 °C for C. perfringens spores ranged from 15.5 to 21.4 min. Heat shocked spores of 9 out of 10 strains had significantly higher ( p D -values at 100 °C than unstressed spores. Proteins with epitopic and size similarity to Escherichia coli GroEL and Bacillus subtilis small acid-soluble protein, SspC, were present in spores. However, heat shock treated spores did not appear to significantly increase expression of these proteins. Acquired thermotolerance is of substantial practical importance to food processors and should provide useful information for designing thermal treatments to eliminate C. perfringens spores in ready-to-eat foods.