Alexandra Lianou

A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments.

Contamination of ready-to-eat products with Listeria monocytogenes may occur at several stages before consumption. Accessibility to the public and relatively limited control interventions at retail and food service establishments (compared with the processing sector of the food industry) and the lack of a specific regulatory framework increase the likelihood of introduction of this pathogen into some foods in these establishments. This review is a compilation of available information on the incidence and transmission of L. monocytogenes through ready-to-eat products at the retail and food service level. The potential transmission of L. monocytogenes within retail and food service operations has been indicated in epidemiological investigations and by survey data. Potential sources of the organism in these operations include the environment, food handlers, and incoming raw ingredients or processed products that have become contaminated after the lethality treatment at the manufacturing facility. L. monocytogenes may be present at retail and food service establishments in various ready-to-eat products, both prepackaged and those packaged in the store, and occasionally at high concentrations. This issue dictates the need for development and application of effective control measures, and potential control approaches are discussed here. Good manufacturing practices, appropriate cleaning, sanitation and hygiene programs, and temperature control required for prevention or inhibition of growth of the pathogen to high levels are critical for control of L. monocytogenes in the retail and food service sector. A comprehensive food safety system designed to be functional in retail and food service operations and based on the philosophy of hazard analysis and critical control point systems and a series of sound prerequisite programs can provide effective control of L. monocytogenes in these environments. However, competent delivery of food safety education and training to retail and food service managers and food handlers must be in place for successful implementation of such a system.

Growth and Stress Resistance Variation in Culture Broth among Listeria monocytogenes Strains of Various Serotypes and Origins

Twenty-five Listeria monocytogenes strains of various serotypes and sources, including clinical and food isolates associated with the same outbreaks, were characterized and compared based on growth rates and heat and acid death rates. Growth was monitored in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) at 4 and 30 degrees C for 32 days and 20 h, respectively. Heat and acid stress responses in TSBYE heated to 55 degrees C or acidified to pH 3.0 with lactic acid were evaluated for 240 or 120 min, respectively. Extensive variation in growth and stress resistance was observed among the tested strains. Growth rate differences were less evident at 30 than at 4 degrees C, where growth rates (log CFU per milliliter per day) ranged from 0.28 to 0.43. Thermal and acid death rates (log CFU per milliliter per minute) ranged from -0.023 to -0.052 and from -0.012 to -0.134, respectively. Serotype appeared to play a significant role (P < 0.05) only with respect to the heat resistance of the organism. Serotype 4b isolates as a group had lower heat resistance than did isolates representing all other serotypes combined. Although no clear origin-related (food versus clinical) trends were observed under the tested conditions, outbreak-related isolates of serotype 4b had lower acid death rates (higher acid resistance) (P < 0.05) than did the rest of the strains belonging to this serotype. Strain Scott A exhibited slow growth at 4 degrees C and low acid resistance, behavior that was distinct among both clinical and serotype 4b isolates. The results of this study highlight the risks associated with extrapolation to other strains of findings obtained with only one strain of L. monocytogenes. This information should be useful when test strains are to be selected for the evaluation of antimicrobial alternatives in ready-to-eat meat and other food products and when risk assessments are to be conducted.

Strain variability of the behavior of foodborne bacterial pathogens: A review

Differences in phenotypic responses among strains of the same microbial species constitute an important source of variability in microbiological studies, and as such they need to be assessed, characterized and taken into account. This review provides a compilation of available research data on the strain variability of four basic behavioral aspects of foodborne bacterial pathogens including: (i) virulence; (ii) growth; (iii) inactivation; and (iv) biofilm formation. A particular emphasis is placed on the foodborne pathogens Listeria monocytogenes and Salmonella enterica. The implications of strain variability for food safety challenge studies and microbial risk assessment are discussed also. The information provided indicates that the variability among strains of foodborne bacterial pathogens with respect to their behavior can be significant and should not be overlooked. However, in order for the mechanisms underlying the observed strain variability to be elucidated and understood, phenotypic variability data, such as those reviewed here, should be evaluated in conjunction with corresponding findings of studies assessing the molecular/physiological basis of this variability.

Stochasticity in colonial growth dynamics of individual bacterial cells

ABSTRACT Conventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells of Salmonella enterica serotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μmax) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (N 0). For bacterial populations with N 0 of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.

Strain variability of the biofilm-forming ability of Salmonella enterica under various environmental conditions

The biofilm-forming ability of 60 Salmonella enterica strains was assessed at different pH values (3.8-7.0), NaCl concentrations (0.5-8.0%) and temperatures (4-37°C). A total of 4320 biofilm formation tests (60 strains×12 different environmental conditions×6 replicates) were carried out. Biofilm formation was evaluated in tryptone soy broth after 48h of incubation in polystyrene microtiter plates using crystal violet staining, and its quantification was based on the difference between the optical density measurements of the test and negative control (uninoculated) samples (ΔOD(580nm)). The tested strains formed biofilms under a wide range of environmental conditions, while extensive strain variability was observed with the mean ΔOD(580nm) values ranging from 0 to 2.388 depending on the strain and the condition evaluated. The strain variability of biofilm formation was affected by all three of the environmental parameters tested, and appeared to increase as the environmental conditions became less favorable for the organism. In addition, the increase in the strain variability caused by pH was found to be much greater than that caused by NaCl or temperature. For example, the coefficient of variation (CV=standard deviation/mean∗100) of ΔOD(580nm) among the tested strains at pH 7.0-0.5% NaCl-37°C was 104%, while at pH 3.8-0.5% NaCl-37°C, pH 7.0-8.0% NaCl-37°C and pH 7.0-0.5% NaCl-8°C was 351.5%, 204.1% and 175.6%, respectively. The optimum conditions for biofilm formation, providing the maximum ΔOD(580nm), varied significantly among the tested strains. Among the evaluated conditions, most of the S. enterica strains were clustered as forming their highest amount of biofilm at pH 5.5 (35 strains; 58.3%), at 0.5% NaCl (29 strains; 48.3%) and at 25°C (32 strains; 53.3%). No relationships were observed between the biofilm-forming ability of the strains and their serotype or their growth kinetic behavior as this was evaluated in a previous study. The findings of this study provide useful information in advancing the current understanding of strain variability, as well as in strain selection for the evaluation of the efficacy of disinfection/sanitation procedures against biofilm formation.

Effect of the growth environment on the strain variability of Salmonella enterica kinetic behavior.

Intra-species variability of microbial growth kinetic behavior is an event with important implications for food safety research. Aiming at the evaluation of the growth variability among Salmonella enterica strains as affected by the growth environment, the kinetic behavior of 60 isolates of the pathogen was assessed at 37 °C in tryptone soy broth of different pH values (4.3-7.0) and NaCl concentrations (0.5-6.0%). Maximum specific growth rate (μ(max)) values corresponding to each strain and growth condition were estimated by means of absorbance detection times of serially decimally diluted cultures using the automated turbidimetric system Bioscreen C. A total of 9600 optical density curves were generated for the strains and the growth conditions tested. The variability of μ(max) among the S. enterica strains was important and greater than that observed within the strains (i.e. among replicates). Moreover, strain variability increased as the growth conditions became more stressful both in terms of pH and NaCl. The coefficient of variation of μ(max) among the tested strains at pH 7.0-0.5% NaCl was 6.1%, while at pH 4.3-0.5% NaCl and pH 7.0-6.0% NaCl was 11.8% and 23.5%, respectively. Beyond the scientific interest in understanding strain variability, the findings of this study should be useful in strain selection for exploitation in food safety challenge studies as well as in incorporating strain variability in predictive microbiology and microbial risk assessment.

Changes in the microbial composition of raw milk induced by thermization treatments applied prior to traditional Greek hard cheese processing.

The microbiological quality, safety, and composition of mixtures of ewes and goats milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67 degrees C for 30 s. Such mild thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60 degrees C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67 degrees C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.

A stochastic approach for integrating strain variability in modeling Salmonella enterica growth as a function of pH and water activity.

Strain variability of the growth behavior of foodborne pathogens has been acknowledged as an important issue in food safety management. A stochastic model providing predictions of the maximum specific growth rate (μ(max)) of Salmonella enterica as a function of pH and water activity (a(w)) and integrating intra-species variability data was developed. For this purpose, growth kinetic data of 60 S. enterica isolates, generated during monitoring of growth in tryptone soy broth of different pH (4.0-7.0) and a(w) (0.964-0.992) values, were used. The effects of the environmental parameters on μ(max) were modeled for each tested S. enterica strain using cardinal type and gamma concept models for pH and a(w), respectively. A multiplicative without interaction-type model, combining the models for pH and a(w), was used to describe the combined effect of these two environmental parameters on μ(max). The strain variability of the growth behavior of S. enterica was incorporated in the modeling procedure by using the cumulative probability distributions of the values of pH(min), pH(opt) and a(wmin) as inputs to the growth model. The cumulative probability distribution of the observed μ(max) values corresponding to growth at pH 7.0-a(w) 0.992 was introduced in the place of the models parameter μ(opt). The introduction of the above distributions into the growth model resulted, using Monte Carlo simulation, in a stochastic model with its predictions being distributions of μ(max) values characterizing the strain variability. The developed model was further validated using independent growth kinetic data (μ(max) values) generated for the 60 strains of the pathogen at pH 5.0-a(w) 0.977, and exhibited a satisfactory performance. The mean, standard deviation, and the 5th and 95th percentiles of the predicted μ(max) distribution were 0.83, 0.08, and 0.69 and 0.96h(-1), respectively, while the corresponding values of the observed distribution were 0.73, 0.09, and 0.61 and 0.85h(-1). The stochastic modeling approach developed in this study can be useful in describing and integrating the strain variability of S. enterica growth kinetic behavior in quantitative microbiology and microbial risk assessment.

Assuring Growth Inhibition of Listerial Contamination during Processing and Storage of Traditional Greek Graviera Cheese: Compliance with the New European Union Regulatory Criteria for Listeria monocytogenes

The current microbiological regulatory criteria in the European Union specify a maximum Listeria monocytogenes population of 100 CFU/g allowable in ready-to-eat foods provided the product will not exceed this limit throughout its shelf life. The aim of this study was to validate the manufacturing method for traditional Greek Graviera cheese produced from thermized milk. Initial challenge experiments evaluated the fate of inoculated L. monocytogenes (ca. 4 log CFU/ml, three-strain cocktail) in thermized Graviera cheese milk (TGCM; 63 degrees C for 30 s) in the presence and absence of a product-specific starter culture (SC) in vitro. Milk samples were incubated for 6 h at 37 degrees C and then for 66 h at 18 degrees C. Experiments were conducted to evaluate the fate of a cocktail of three nonpathogenic L. monocytogenes and L. innocua indicator strains inoculated (ca. 3 log CFU/g) in Graviera cheeses commercially manufactured from TGCM+SC. Cheeses were brined, ripened at 18 degrees C and 90% relative humidity for 20 days, and stored at 4 degrees C for up to day 60 under vacuum. In TGCM, L. monocytogenes increased by ca. 2 log units, whereas in TGCM+SC L. monocytogenes growth was retarded (P < 0.05) after a ca. 1-log increase within 6 h at 37 degrees C. Populations of Listeria indicator strains did not grow in TGCM+SC cheeses at any stage; they declined 10-fold in fresh cheeses within 5 days and then survived with little death thereafter. Thus, growth inhibition but not inactivation of potent natural Listeria contaminants at levels below 100 CFU/g occurs in the core of traditional Greek Graviera cheese during fermentation, ripening, and storage.

Evaluation of the effect of defrosting practices of ground beef on the heat tolerance of Listeria monocytogenes and Salmonella Enteritidis.

The effect of common defrosting practices of ground beef, including (i) defrosting in the refrigerator (5°C for 15h), (ii) defrosting at room temperature (25°C for 12h) and (iii) defrosting in the microwave, on the heat tolerance of artificially inoculated Listeria monocytogenes and Salmonella Enteritidis, was studied. The thermal inactivation of S. Enteritidis was not, overall, affected by defrosting practices. In contrast, defrosting at room temperature resulted, overall, in an increased heat tolerance of L. monocytogenes compared to the rest tested defrosting practices. Inactivation kinetics of the two pathogens for the different defrosting practices were determined by fitting the data to the Weibull model. The δ parameter of the Weibull model (heat challenge time (min) required for the first 1-log reduction) for S. Enteritidis and for defrosting at 25°C, microwave defrosting, defrosting at 5°C and for the control (fresh ground beef inoculated with the pathogens just before the heat challenge trials) was 1.13, 1.62, 1.60 and 0.96, respectively, while the corresponding values for L. monocytogenes were 20.13, 10.82, 9.95 and 9.47, respectively. The findings of this study should be useful in risk assessments and in developing food handling guidelines for the consumers.