Fate of Listeria monocytogenes during the manufacture and ripening of Parmesan cheese

Parmesan cheese was made from a mixture of pasteurized whole and skim milk that was inoculated to contain ca. 10(4) to 10(5) cells of Listeria monocytogenes/ml. Curd was cooked at 51 degrees C (124 degrees F) for ca. 45 min. During cheese making, maximum numbers of L. monocytogenes appeared just before cooking; at this point, the increase over initial numbers was a .61 to 1.0 order of magnitude. During cooking of curd, the average decrease in numbers of L. monocytogenes was a .22 order of magnitude. During cheese ripening, numbers of L. monocytogenes decreased almost linearly and faster than reported for other hard cheeses. Listeria monocytogenes strain California died faster than did strain V7. Listeria monocytogenes were not detected in cheese after 2 to 16 wk of ripening, depending on the strain of the pathogen and the lot of cheese. Parmesan cheese made in this study was not a favorable medium for survival of L. monocytogenes.     

Fate of Listeria monocytogenes in orally dosed chicks

The fate of Listeria monocytogenes in chicks perorally dosed with these bacteria at 2 days of age was determined by bacterial enumeration, immunoperoxidase staining and histological examination of the liver, muscle and gastrointestinal tract. Results revealed listerial egress from the digestive tract and elimination of the organism from the body in most of the chicks within 9 days post-inoculation. L. monocytogenes was isolated from the caecum of only one of 10 chicks examined at 4 weeks post-inoculation. Results indicate that chickens are not likely to be common reservoirs of L. monocytogenes. Intestinal carriage of L. monocytogenes by poultry may frequently be transient, resulting from ingestion of Listeria-contaminated feed and soil.     

Fate of Listeria monocytogenes in bovine manure-amended soil

The survival and growth of Listeria monocytogenes in soil amended with bovine manure was studied under different environmental conditions of temperature, nutrients, and soil microflora. Autoclaved soil was compared with unautoclaved soil for assessing the influence of competitive soil microflora on the survival of L. monocytogenes. Initial L. monocytogenes cell numbers of 5 to 6 log CFU/g survived for up to 43, 43, and 14 days in manure-amended autoclaved soil at 5, 15, and 21 degrees C, respectively. In manure-amended unautoclaved soil, the pathogen was detectable for up to 43, 21, and 21 days at 5, 15, and 21 degrees C, respectively. L. monocytogenes was inactivated more rapidly in autoclaved soil amended with manure at a manure/soil ratio of 1:10 than in the more dilute (1:100) manure in soil samples at both 15 and 21 degrees C. However, in manure-amended unautoclaved soil, L. monocytogenes survived longer in samples with ratios of 1:10 than in the more dilute (1:100) manure-amended soil. The persistence of L. monocytogenes for several weeks in manure-amended soil suggests listeriae could be transmitted through soil to fresh produce or to shoes, clothing, and hands of field workers, especially during the cold months.     

Fate of Listeria monocytogenes in experimentally contaminated French sausages

Listeria monocytogenes has been recognized as one of the most important foodborne pathogens dealt with by the food. The bacterium has been found in every part along the pork processing industry from the slaughterhouse to the cutting room and the delicatessen factories. During the fermentation and drying of sausages, L. monocytogenes tends to decrease substantially. However, despite the various hurdles in the dry sausage manufacturing process, L. monocytogenes is able to survive and is detected in the final products. The present study has evaluated growth and survival of eight different L. monocytogenes strains (originating from sausage, sausage industry environment and from clinical cases of listeriosis) in experimentally inoculated French sausages with 10(4) cfu g(-1). This study points out the fact that the decrease of L. monocytogenes contamination rate during the manufacturing process of sausages is strain dependent (p < 0.001) and mainly due to the drying and maturation step than to the fermentation itself. Whatever the strains studied, almost no decrease of the contamination rate was noted during the fermentation step. However hurdle-adapted strains (those isolated from sausages or sausage industry environment) were more difficult to cure from sausages (decrease by 1.5 log10) than non-adapted strains (decrease by 3 log10) at the end of the drying period (day 35), when sausages were ready for consumption. These sausages became safe only at the best before date. As a consequence, L. monocytogenes and more particularly those "adapted" strains might represent a very important issue for hygienists since these strains originating from sausages or production environment themselves are likely to contaminate sausages during manufacturing and remain in the final products. However, the high inoculum levels used in the study (10(4) cfu g(-1)) are not representative of the natural contamination of L. monocytogenes commonly encountered in the raw material for sausages. If such contamination happened to be inferior to 100 cfu g(-1), then the manufacturing process used in this study would be able to produce "safe" sausages according to the European regulation requiring the absence of L. monocytogenes in 25 g of food with a tolerance of below 100 cfu g(-1) at the best before date.     

Temperature-assisted pressure inactivation of Listeria monocytogenes in turkey breast meat

Ready-to-eat turkey breast meat samples were surface-inoculated with a five-strain cocktail of Listeria monocytogenes cultures to a final concentration of approximately 10(7) CFU/g. The inoculated meat samples were vacuum-packaged and pressure treated at 300 MPa for 2 min, 400 MPa for 1 min, and 500 MPa for 1 min at initial sample temperatures of 1, 10, 20, 30, 40, 50, and 55 degrees C. L. monocytogenes was most resistant to pressure at temperatures between 10 and 30 degrees C. As temperature decreased below 10 degrees C or increased over 30 degrees C, its pressure sensitivity increased. This enhanced inactivation effect was more pronounced when meat samples were treated at higher temperature than at lower temperature. For example, a 1-min treatment of 500 MPa at 40 degrees C reduced the counts by 3.8 log(10), while at 1 and 20 degrees C the same treatment reduced counts by 1.4 and 0.9 log(10), respectively (P<0.05). The survival curves of L. monocytogenes were obtained at 300 MPa and 55 degrees C, 400 MPa and 50 degrees C, and 500 MPa and 40 degrees C. With increasing treatment time, the three survival curves showed a rapid initial drop in bacteria counts with a diminishing inactivation rate or tailing effect. The survival data were fitted with a linear and a nonlinear, Weibull, models. The Weibull model consistently produced better fit to the survival data than the linear model.     

Thermal inactivation, growth, and survival studies of Listeria monocytogenes strains belonging to three distinct genotypic lineages

Twenty-one Listeria monocytogenes strains belonging to three different genotypic lineages were evaluated for differences between lineages and between individual strains with respect to thermal inactivation, growth, and survival. Three sets of heat inactivation conditions (60 degrees C, pH 6.0, and 0.5 M lactate; 55 degrees C, pH 6.0, and 0.5 M lactate; and 50 degrees C, pH 4.0, and 0.5 M lactate) were used on strains grown in modified brain heart infusion (BHI) broth with and without glucose. Two sets of growth conditions (35 degrees C, pH 6.5, and 0.1 M lactate and 5 degrees C, pH 6.5, and 0.1 M lactate) were used with modified BHI broths to determine lag phases and exponential growth rates. Two sets of conditions (28 degrees C, pH 4.0, and 1 M lactate and 28 degrees C, pH 4.5, and 0.5 M lactate) were used with modified BHI broth to determine survival times (D-values). Thermal inactivation D-values were consistently lowest for lineage III, but differences were not significant for any set of conditions tested. Some significant differences were observed between lineages with respect to some of the growth and survival conditions tested. Extensive strain-to-strain variation was observed for all parameters tested. Average coefficients of variation for the thermal inactivation, growth, and survival studies were 0.31, 0.18, and 0.26, respectively. Strain-to-strain variations were approximately equal to the uncertainties associated with the analytical procedures. The results obtained indicate a diversity among strains encountered in food processing that must be accounted for in process calculations and risk assessments.     

Survival of Listeria monocytogenes after cleaning and sanitation of wooden shelves used for cheese ripening

The effect of cleaning and heat disinfection processes of wooden shelves used for cheese ripening on the survival of Listeria monocytogenes was examined. The cut boards were inoculated with a suspension containing 5.5?×?10⁷ colony forming units (CFU)/ml of L.??monocytogenes. Survival of L.??monocytogenes was investigated in the wood shavings. During the 24-hour incubation period in a humidity chamber, Listeria counts increased by 0.8 log units on average (n?=?9), compared to results 1?h after incubation, indicating the absence of antimicrobial properties of the wood in use. Additionally, after incubation for 24?h at room temperature, the boards were cleaned by soaking them for 15?min in a solution of hot alkaline detergent followed by brushing and rinsing with warm water. Some of the cleaned boards were subsequently heat treated at 80°C for 5?min and at 65°C for 15?min, respectively. The cleaning procedure alone was not sufficient to render L.??monocytogenes from the upper 2?mm wood layer inactive. In the case of both temperature-time combinations for heat disinfection, however, L.??monocytogenes was not detectable. The present study shows that the use of wooden shelves does not affect the hygienic safety of cheeses if such shelves are in good repair and are thoroughly cleaned and sanitized by heat treatment. Therefore, there is no reason to replace wood employed in cheese ripening processes with other materials.     

Dynamic modeling of Listeria monocytogenes growth in pasteurized vanilla cream after postprocessing contamination

A product-specific model was developed and validated under dynamic temperature conditions for predicting the growth of Listeria monocytogenes in pasteurized vanilla cream, a traditional milk-based product. Model performance was also compared with Growth Predictor and Sym'Previus predictive microbiology software packages. Commercially prepared vanilla cream samples were artificially inoculated with a five-strain cocktail of L. monocytogenes, with an initial concentration of 102 CFU g(-1), and stored at 3, 5, 10, and 15 degrees C for 36 days. The growth kinetic parameters at each temperature were determined by the primary model of Baranyi and Roberts. The maximum specific growth rate (mu(max)) was further modeled as a function of temperature by means of a square root-type model. The performance of the model in predicting the growth of the pathogen under dynamic temperature conditions was based on two different temperature scenarios with periodic changes from 4 to 15 degrees C. Growth prediction for dynamic temperature profiles was based on the square root model and the differential equations of the Baranyi and Roberts model, which were numerically integrated with respect to time. Model performance was based on the bias factor (B(f)), the accuracy factor (A(f)), the goodness-of-fit index (GoF), and the percent relative errors between observed and predicted growth. The product-specific model developed in the present study accurately predicted the growth of L. monocytogenes under dynamic temperature conditions. The average values for the performance indices were 1.038, 1.068, and 0.397 for B(f), A(f), and GoF, respectively for both temperature scenarios assayed. Predictions from Growth Predictor and Sym'Previus overestimated pathogen growth. The average values of B(f), A(f), and GoF were 1.173, 1.174, and 1.162, and 1.267, 1.281, and 1.756 from Growth Predictor and Sym'Previus, respectively.     

Influence of temperature variations on growth,injury survival and inactivation of Listeria monocytogenes in goat milk samples at laboratory scale

Transmission of the thermo‐tolerant pathogen Listeria monocytogenes via contaminated milk and its products, can lead to serious food‐borne illness. In this study, the effects of selected temperatures on survival, percentage injury and inactivation of L. monocytogenes in goat milk samples collected from two different farms were evaluated. Low temperature ranges (0, 5, 10 °C) had a bacteriostatic effect; while at temperatures of 25 and 45 °C, this pathogen grew luxuriantly. However, growth was comparatively slow at 15 °C throughout a 12‐h stress period. Furthermore, a high temperature range (50, 55, 60 and 65 °C) resulted in the elimination of this pathogen within 4 h of stress. Results of Scanning Electron Microscopy showed morphological changes in the cells upon induction of stress temperatures.     

Modelling the growth of Listeria monocytogenes in dynamic conditions

A recurrent neural network for the prediction of Listeria monocytogenes growth under pH and a(w) variable conditions was developed. The use of this model offered the possibility to take into account the consequences of the variations of the factors on L. monocytogenes growth. The effects of solutions, such as NaCl, acetic acid and NaOH, and their interactions on the response of L. monocytogenes cells were studied. Furthermore, the results showed the capacity of the recurrent neural network to predict growths carried out in different experimental conditions without using those used for its elaboration.     

Effect of high pressure, in combination with antilisterial agents, on the growth of Listeria monocytogenes during extended storage of cooked chicken

A cocktail of Listeria monocytogenes strains was inoculated into cooked chicken (∼2.2 × 10³ CFU g⁻¹) which was then pressure-treated (600 MPa/2 min/20 °C) and stored for up to 105 days at 8 °C. In addition, sodium lactate (2% w/w) or a pressure-resistant Weissella viridescens strain, known to have antilisterial activity, were added to the meat prior to inoculation with the pathogen and pressure treatment, to investigate the effect on Listeria survival.Pressure treatment alone was not sufficient to eliminate all of the Listeria. Numbers of survivors were initially below the level of detection (50 CFU g⁻¹) but increased during storage to reach >10⁸ CFU g⁻¹ by day 21. The presence of W. viridescens significantly extended the lag phase of any Listeria that survived the initial pressure treatment by ∼35 days, but numbers then increased to reach ∼10⁷ CFU g⁻¹ by day 105. The addition of 2% sodium lactate in combination with pressure treatment was most effective at inhibiting the growth of L. monocytogenes and numbers remained below the limit of detection throughout the 105 day storage.The addition of antimicrobial agents, in combination with pressure, could be used to give additional food safety assurance without increasing pressure hold time.     

Modelling thermal inactivation of Listeria monocytogenes in sucrose solutions of various water activities

The heat resistance of Listeria monocytogenes was determined in sucrose solutions with water activity (a(w)) ranging from 0.99 to 0.90. At all temperatures investigated shape of the survival curves depended on the a(w) of the treatment medium. The survival curves for a(w)=0.99 appeared to be linear, for a(w)=0.96 were slightly upwardly concaved and for a(w)=0.93 and 0.90 were markedly concave upward. A mathematical model based on the Weibull distribution provided a good fit for all the survival curves obtained in this investigation. The effect of the temperature and a(w) on the Weibull model parameters was also studied. The shape parameter (p) depended on the a(w) of the treatment medium but in each medium of different a(w) the temperature did not have a significant effect on this parameter. The p parameter followed a linear relationship with a(w). The scale parameter (delta) decreased with the temperature following an exponential relationship and increased by decreasing the a(w) in the range from 0.99 to 0.93. However the delta parameter of survival curves obtained at a(w)=0.90 were lower than those obtained at a(w)=0.93. A mathematical model based on the Weibull parameters was built to describe the joint effect of temperature and a(w) on thermal inactivation of L. monocytogenes. This model provides a more complete information on the influence of the a(w) on the L. monocytogenes than the data initially generated. The model developed indicated that the effect of the a(w) on the thermal resistance of L. monocytogenes varied depending upon the temperature of treatment.     

Effect of prior growth conditions on the thermal inactivation of 13 strains of Listeria monocytogenes in two heating menstrua

The thermal tolerance of 13 Listeria monocytogenes strains was tested using a submerged heating coil apparatus. The strains were grown individually for 18 h at 37 degrees C in acidogenic tryptic soy broth (without dextrose) supplemented with 1% glucose and 1% glutamine (TSB+G) or nonacidogenic tryptic soy broth supplemented with 1% glutamine but containing no glucose (dextrose) (TSB-G). The former medium results in cells induced for pH-dependent, stationary-phase acid resistance, whereas the latter medium allows L. monocytogenes to grow to high numbers in the absence of glucose, yielding cells that are not induced for pH-dependent, stationary-phase acid resistance. The average final pH values of the 18-h TSB+G and the TSB-G cultures were 4.7 and 6.7, respectively. The cells grown in the acid resistance-inducing and non-acid resistance-inducing media were then tested in two heating menstrua that consisted of brain heart infusion broth adjusted to pH 3.0 and water activity (a(w)) of 0.987 or pH 7.0 and a(w) 0.970. In 14 of the 26 menstruum-strain combinations tested, the acid resistance-induced strains were more heat resistant then the equivalent noninduced cultures. No difference in the pattern of thermal resistance in response to induction of acid resistance was apparent among the different serovars tested. The results suggest that the ability of prior induction of acid resistanceto enhance thermal resistance can vary substantially among L. monocytogenes strains.     

Universal Primer-Multiplex PCR Approach for Simultaneous Detection of Escherichia coli, Listeria monocytogenes, and Salmonella spp. in Food Samples

ABSTRACT:  Escherichia coli ,  Listeria monocytogenes , and  Salmonella  spp. are 3 kinds of the most important food-borne human pathogens. Traditional microbiological analysis is labor-intensive, time-consuming, and easily contaminated, thus producing false positive signals; it also involves much subjectivity judgments. Multiplex-PCR could be applied to detect multiple target organisms simultaneously to save time and labor, but there is always disproportionate amplification resulting from the disparity of different primers. To gain a rapid and sensitive method, a universal primer-multiplex PCR system (UP-M-PCR) was developed and applied for simultaneous detection of the 3 organisms. This method simplified traditional multiplex-PCR reaction system and overcame its amplification disparities among different primers; moreover, it got a high specificity and sensitivity (85, 155, and 104 copies/reaction for  E. coli  O157,  L. monocytogenes , and  Salmonella  spp., respectively). Compared with the time-consuming and laborious microbiological analysis, UP-M-PCR had a lower risk of cross-contamination without inoculation and incubation. Test results for 36 food samples showed that UP-M-PCR method got a relative accuracy of 91.77% when compared with traditional microbiological analysis. It could serve as a rapid screening method for pathogen detection and could detect target genes even in dead pathogenic cells. In addition, it has the potential to be performed in an automation mode and might find broader application in simultaneous detection of other multiple pathogens.     

Modelling the competitive growth of Listeria monocytogenes and Listeria innocua in enrichment broths

The overgrowth of Listeria innocua in enrichment broths designed for the isolation of Listeria monocytogenes is believed to result from two factors: a selective growth advantage of L. innocua, and/or an inhibitory interspecies interaction. The generation times of 13 isolates of L. innocua and L. monocytogenes were determined in Brain Heart Infusion (BHI) and a variety of enrichment media. No significant differences were found in growth characteristics between either species in the various media, suggesting that the growth advantage of L. innocua in enrichment media was not as significant as previously described. Kinetic analysis of mixed cultures of L. monocytogenes and isolates of L. innocua producing a variety of inhibitory activities demonstrated the possibility of an inhibitory interaction between these two species resulting in the overgrowth of the enrichment culture with L. innocua. Modelling the evolution of the ratio between two populations in an enrichment process was used to analyze the impact of a selective growth advantage in L. innocua in an enrichment process for growth of L. monocytogenes. These findings support the widely held view that an overgrowth of L. innocua in the enrichment process can result from both a selective growth advantage as well as the production of inhibitory compounds. From a practical perspective, these interactions can result in an increase in false negatives.     

The effects of growth temperature and growth phase on the inactivation of Listeria monocytogenes in whole milk subject to high pressure processing

The aim of this study was to explore the effect of a wide range of growth temperatures, growth phases and plating media on the inactivation of Listeria monocytogenes by high pressure processing (HPP). In part one, L. monocytogenes was grown to mid-stationary phase at 4, 15, 25, 35 or 43 degrees C, inoculated into whole UHT milk at approximately 10(7) CFU/ml and high pressure processed at 400 MPa at room temperature (20-25 degrees C). Afterward, the HPP milk was plated on Tryptic Soy Yeast Extract Agar (TSYEA) and Modified Oxford Agar (MOX) to determine the degree of injury. For part two, cells were grown to mid-exponential, late-exponential or mid-stationary phase at 15 or 43 degrees C and processed in the same way. Time to reach a 5-log reduction was determined and data were analysed by ANOVA. The results from part one showed that both growth temperature and plating medium had a significant effect (P < 0.001) on the inactivation of stationary phase L. monocytogenes by HPP. Tukey's pairwise comparisons revealed that the effects of all temperatures, except 35 and 43 degrees C, were significantly different (P < 0.05). Cells grown at 15 degrees C were most sensitive to HPP, followed by cells grown at 4, 25 or 35 degrees C, with cells grown at 43 degrees C appearing to be the most resistant. Inactivation of cells grown at 4, 15 or 25 degrees C followed first order kinetics, whereas cells grown at 35 or 43 degrees C displayed non-linear inactivation kinetics due to tailing. In part two, both growth phase and plating medium had significant effects on the inactivation (P < or = 0.001) of L. monocytogenes by HPP. Cells grown at 15 degrees C to mid-stationary phase were the most pressure-resistant when tested on both media, and were significantly more resistant (P < 0.05) than cells grown at the same temperature to the other two phases of growth. There was no significant difference between mid- and late-exponential phase cells grown at 15 degrees C. When cells were grown at 43 degrees C, mid-exponential phase cells were significantly more sensitive (P < 0.05) than either late-exponential or mid-stationary phase cells, with no difference between late-exponential or mid-stationary phase cells. It was postulated that membrane composition, stationary phase proteins and/or stress proteins may affect pressure resistance.     

Thermal inactivation of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in breaded pork patties

ABSTRACT:  Decimal reduction times ( D -values) and thermal resistance constants ( z -values) for 3 foodborne pathogenic bacteria in formulated ready-to-eat breaded pork patties were determined with thermal inactivation studies. Meat samples, inoculated with Escherichia coli O157:H7, Salmonella , and Listeria monocytogenes cultures or uninoculated controls, were packaged in sterile bags, immersed in circulated water bath, and held at 55, 57.5, 60, 62.5, 65, 67.5, and 70 °C for different durations of time. The D - and z -values were determined by using a linear regression model. Average calculated D -values for E. coli O157:H7, Salmonella , and L . monocytogenes at a temperature range of 55 to 70 °C were 32.11 to 0.08 min, 69.48 to 0.29 min, and 150.46 to 0.43 min, respectively. Calculated z -values for E. coli O157:H7, Salmonella , and L. monocytogenes were 5.4, 6.2, and 5.9 °C, respectively. The results of this study will be useful to food processors to validate thermal lethality of the studied foodborne pathogens in ready-to-eat breaded pork patties.     

High-pressure processing of Gorgonzola cheese: influence on Listeria monocytogenes inactivation and on sensory characteristics

The presence of Listeria monocytogenes on the rind of Gorgonzola cheese is difficult to avoid. This contamination can easily occur as a consequence of handling during ripening. The aims of this study were to determine the efficiency of high-pressure processing (HPP) for inactivation of L. monocytogenes on cheese rind and to evaluate the influence of HPP treatments on sensory characteristics. Gorgonzola cheese rinds, after removal, were inoculated (about 7.0 log CFU/g) with L. monocytogenes strains previously isolated from other Gorgonzola cheeses. The inoculated cheese rinds were processed with an HPP apparatus under conditions of pressure and time ranging from 400 to 700 MPa for 1 to 15 min. Pressures higher than 600 MPa for 10 min or 700 MPa for 5 min reduced L. monocytogenes more than 99%. A reduction higher than 99.999% was achieved pressurizing cheese rinds at 700 MPa for 15 min. Lower pressure or time treatments were less effective and varied in effectiveness with the cheese sample. Changes in sensory properties possibly induced by the HPP were evaluated on four different Gorgonzola cheeses. A panel of 18 members judged the treated and untreated cheeses in a triangle test. Only one of the four pressurized cheeses was evaluated as different from the untreated sample. HPP was effective in the reduction of L. monocytogenes on Gorgonzola cheese rinds without significantly changing its sensory properties. High-pressure technology is a useful tool to improve the safety of this type of cheese.