Evaluation of different approaches for modeling Escherichia coli O157:H7 survival on field lettuce.

The ability to predict the behavior of Escherichia coli O157:H7 on contaminated field lettuce is essential for the development of accurate quantitative microbial risk assessments. The survival pattern of the species was assessed from several data sets derived from field-based experiments, which were analyzed by regression analysis fitting one monophasic model (log-linear) and two biphasic (Weibull and Cerf's model) models. Probabilistic models were also simulated with @RISK™, integrating the fitted monophasic and biphasic models in order to analyze their impact on the estimate of the extent of die-off subsequent to a contamination event in the field. Regression analysis indicated that E. coli O157:H7 followed a biphasic decay pattern in most cases, with the Weibull and Cerf's model showing similar good fit to individual and pooled survival data. Furthermore, results from the stochastic analysis demonstrated that using the log-linear model could lead to different risk estimates from those obtained with biphasic models, with a lower prevalence in the former scenario as no tailing is assumed in this model. The models and results derived from this work provide the first suitable mathematical base upon which to build probabilistic models to predict the fate of E. coli O157:H7 on field-grown leafy green vegetable.

Simulation of Escherichia coli O157:H7 behavior in fresh-cut lettuce under dynamic temperature conditions during distribution from processing to retail.

The temperature of packaged lettuce was recorded throughout a retail supply chain in Canada during the various stages of storage and shipping from the processor to retail. Temperatures were monitored in 27 cases of lettuce destined for three stores in three replicate trials conducted during the winter. A dynamic model that predicts the effect of temperature on the growth or die-off of Escherichia coli O157:H7 in packaged fresh-cut lettuce was applied to simulate the behavior of E. coli O157:H7 in the system. Simulations were carried out using distributions to account for variation in the temperature parameter and the die-off coefficient of the dynamic growth/death model. The results indicate that there was a predicted overall mean decline in cell numbers of 0.983 log cfu g⁻¹ and that the extent of cell death was proportional to the total time spent in the cold chain. Slight growth was predicted in a few instances when the dynamic temperature was above the permissive temperature of 5°C. These results suggest that generally there would be little or no growth of E. coli O157:H7 in product maintained at the proper temperature in the chain. Moreover, the predicted decline in cell numbers at refrigeration temperatures suggests that storage at 5°C or below prior to consumption would reduce populations of the pathogen in fresh-cut lettuce.

Development of a dynamic growth-death model for Escherichia coli O157:H7 in minimally processed leafy green vegetables.

Escherichia coli O157:H7, an occasional contaminant of fresh produce, can present a serious health risk in minimally processed leafy green vegetables. A good predictive model is needed for Quantitative Risk Assessment (QRA) purposes, which adequately describes the growth or die-off of this pathogen under variable temperature conditions experienced during processing, storage and shipping. Literature data on behaviour of this pathogen on fresh-cut lettuce and spinach was taken from published graphs by digitization, published tables or from personal communications. A three-phase growth function was fitted to the data from 13 studies, and a square root model for growth rate (µ) as a function of temperature was derived: µ=(0.023*(Temperature-1.20))(2). Variability in the published data was incorporated into the growth model by the use of weighted regression and the 95% prediction limits. A log-linear die-off function was fitted to the data from 13 studies, and the resulting rate constants were fitted to a shifted lognormal distribution (Mean: 0.013; Standard Deviation, 0.010; Shift, 0.001). The combined growth-death model successfully predicted pathogen behaviour under both isothermal and non-isothermal conditions when compared to new published data. By incorporating variability, the resulting model is an improvement over existing ones, and is suitable for QRA applications.

Examination of stress and virulence gene expression in Escherichia coli O157:H7 using targeted microarray analysis.

Escherichia coli O157:H7 poses a threat to humans through food- and water-borne transmission. To investigate how environmental stresses affect the Escherichia coli O157:H7 transcriptome, we designed a targeted microarray consisting of stress response and virulence genes (n = 125) to analyze the impact of acidified (pH 3.5), cold (7.5 degrees C), and fresh tryptic soy broth (TSB) (37 degrees C) on E. coli O157:H7 stress response and virulence gene expression. Nutrient replenishment with fresh TSB resulted in 72 differentially expressed genes (> or = 1.5-fold change; p < 0.05), with 65 induced. All queried global and specific stress regulators were affected, as were 12 virulence genes. Cold-shocked cells displayed 17 differentially expressed genes, with 10 being induced. Induction of rpoS, members of the sigma(H) regulon (clpB, dnaK, ftsH), and acid resistance (AR) genes (gadA, gadX) was observed. Porin transcript (ompC, ompF) and gapA and tufA ancillary genes were repressed. Acid shock resulted in 24 differentially expressed genes, with 21 induced. No induction of any stationary phase AR system was observed, though acid-coping mechanisms were recruited, including mar and phoB induction, and repression of ompC and ompF. Stress regulators were induced, including relA, soxS, rpoE, and rpoH. The microarray data were validated by quantitative real-time polymerase chain reaction. Exposure to sublethal stress events led to the induction of diverse stress response networks. In the food chain, sublethal events may render cells increasingly resistant to future stresses, potentially leading to increased survival.

Predictive modeling of siderphore production by Pseudomonas fluorescens under iron limitation.

Iron is required by many microorganisms for growth. Although it is the most abundant transition metal on earth, its solubility is very low and therefore its bioavailability is poor. To overcome this limitation, many microorganisms have developed iron chelating mechanisms that enable them to bind the metal to organic molecules from which they are later released. In particular, pseudomonads are prominent producers of the chelator pyoverdine that has a high iron binding capability. We present a mathematical model for pyoverdine production by Pseudomonas fluorescens. It is a nonlinear and non-autonomous system of four ordinary differential equations for the dependent variables size of bacterial population, pyoverdine, dissolved iron and chelated iron. The transient adaptation of the average physiological state of the population to the environmental condition is explicitly included in the model formulation. A complete qualitative description of the model solution is given, based on analytical techniques. The model is quantitatively validated against experimental data of pyoverdine and population size. To this end we conduct and discuss a parameter identification study. It is found that the model, if calibrated using pyoverdine data alone is able to predict the population size and vice versa, with some restrictions. Thus the model can be used as an indirect experimental tool.

Correlation between the change in the kinetics of the ribosomal RNA rrnB P2 promoter and the transition from lag to exponential phase with Pseudomonas fluorescens.

Developing accurate mathematical models to describe the pre-exponential lag phase in food-borne pathogens presents a considerable challenge to food microbiologists. While the growth rate is influenced by current environmental conditions, the lag phase is affected in addition by the history of the inoculum. A deeper understanding of physiological changes taking place during the lag phase would improve accuracy of models, and in earlier studies a strain of Pseudomonas fluorescens containing the Tn7-luxCDABE gene cassette regulated by the rRNA promoter rrnB P2 was used to measure the influence of starvation, growth temperature and sub-lethal heating on promoter expression and subsequent growth. The present study expands the models developed earlier to include a model which describes the change from exponential to linear increase in promoter expression with time when the exponential phase of growth commences. A two-phase linear model with Poisson weighting was used to estimate the lag (LPDLin) and the rate (RLin) for this linear increase in bioluminescence. The Spearman rank correlation coefficient (r=0.830) between the LPDLin and the growth lag phase (LPDOD) was extremely significant (P

Effect of sub-lethal heating and growth temperature on expression of the ribosomal RNA rrnB P(2) promoter during the lag phase of Pseudomonas fluorescens.

Current models for the lag phase of food-borne pathogens are limited by our poor understanding of the physiological changes taking place as bacterial cells prepare for exponential growth. In a previous paper in this series, a strain of Pseudomonas fluorescens containing the Tn7-luxCDABE gene cassette regulated by the rRNA promoter rrnB P(2) was used to measure the influence of starvation on the lag phase duration (LPD(OD)) and growth rate (R(OD)). rrnB P(2) promoter activity increased exponentially during the lag phase, and was characterized by lag (LPD(Exp)) and rate (R(Exp)) parameters. In the present study, this work was expanded to include the influence of growth temperature (10 to 30 degrees C) and exposure to sub-lethal heating at 47 degrees C. With these additional datasets, the LPD(Exp) was often more pronounced than had been noted with starvation, so the original exponential association model (EXP) was compared to logistic and Gompertz (GOM) models. Based on root mean square error, the GOM model gave the better fit for some of the sub-lethal heating and growth temperature datasets; however, the EXP model was assessed as best overall. Increased growth temperature and decreased time of sub-lethal heating produced significant decreases in LPD(OD) and LPD(Exp) and increases in R(OD) and R(Exp). The results suggest that different stressors have differential effects on gene expression and subsequent growth.

Effect of starvation on expression of the ribosomal RNA rrnB P2 promoter during the lag phase of Pseudomonas fluorescens.

Mathematical modelling of food-borne pathogen survival and growth is an important and expanding area of food microbiology. Effective models have been developed for growth rate as influenced by the environment; however, reliable models which describe the lag phase prior to exponential growth are more difficult to obtain. In order to improve our understanding of the physiological changes that take place in the microbial cell during this adaptation period, the effect of starvation on the expression of a gene for ribosomal RNA (rRNA) synthesis-an important step in preparing the cells for growth-was examined. A strain of Pseudomonas fluorescens containing the Tn7-luxCDABE gene cassette regulated by the rRNA promoter rrnB P(2) was used as a model system. Growth was measured as optical density at 600 nm (OD(600)), and fitting was achieved with a two-phase linear model to obtain the parameters growth rate (R(OD)) and lag phase duration (LPD(OD)). The increase in bioluminescence (measured as natural log [ln] relative light units per unit OD(600)) after inoculation of stationary phase cells into fresh tryptic soy broth (TSB) followed an exponential association model, with lag (LPD(Exp)) and rate (R(Exp)) parameters. Starvation of cells in either spent TSB or in MOPS buffer resulted in time-dependent linear increases in both lag parameters and, in the case of TSB, a decrease in the R(Exp) parameter. The results show that models can be developed for expression of genes during the lag phase, which will improve our ability to make accurate predictions of food-borne pathogen growth.

Effect of environmental stresses on the mean and distribution of individual cell lag times of Escherichia coli O157:H7.

The effect of starvation, heat or acid stress on duration of individual cell lag time (tau) and standard deviation (SD) of tau was investigated using Escherichia coli O157:H7. Cells were stressed by exposure to acid (pH 3.5), heat (50 degrees C), or starvation in either glucose-free mineral medium (MOPS), tryptic soy broth (TSB) or Luria broth (LB). Stressed cells were then diluted into wells of a Bioscreen plate to obtain single cells per well. Replicate time to detection (td) values were obtained using the Bioscreen and used to calculate the tau and SD. Significant (P< or =0.05) increases in tau over untreated controls were found for the following treatments: 14 days in acid; 2 h of heating; 3 days starvation in MOPS; and 2 days starvation in either TSB or LB. The largest increase in tau was >2-fold from 2.5 to 5.6 h observed with the heat treatment. MOPS starvation was more detrimental to the cells than was acid treatment over the same time period. A significant increase in SD was found with 21 days acid treatment, and 2 days starvation in either TSB or LB. No significant increase in SD was found for MOPS starvation or heat treatment. Lognormal, Gamma, ExtremeValue and Weibull distributions were fitted to the tau data using BestFit. The results suggest that the Lognormal distribution is suitable for fitting tau data from either stressed or unstressed cells.

Assessment of distributions for fitting lag times of individual cells in bacterial populations.

To develop mathematical models describing lag times of individual bacterial cells (tau), experimental tau data were fitted to a variety of continuous distributions using BestFit. Six strains of Escherichia coli O157:H7 were used, and serial dilutions were made in Bioscreen multi-well plates to get single cells per well. Detection times (td) for individual wells were converted to tau using the maximum specific growth rate (mu) for each strain. All strains were subject to 25 trials, with up to 100 replicate wells per trial. Some strains had significantly longer t(d), and lower mu, but the tau values were not significantly different. Distributions were best fit in the order Pearson V>Pearson VI>Extreme Value>Lognormal>Lognormal2>Inverse Gaussian based on the Anderson-Darling statistic. The Lognormal distribution was selected because there was less variability in the parameter values, and parameters have specific biological meanings. Distributions could be fit to sample populations as low as six, with fittings and parameter values comparable to those obtained with larger samples (up to 89). Extreme Value, Pearson V, and Pearson VI distributions were more suitable for fitting tau values generated from a Lognormal distribution when the numbers of sample points were few, which suggested that there are similarities between the distributions. The results suggest that a Lognormal distribution can be used successfully to characterize tau.

Application of the electronic nose to the classification of resistance to Western flower thrips in chrysanthemums.

A metal oxide sensor-based electronic nose was tested for its ability to discriminate among chrysanthemum cultivars with varying degrees of resistance to western flower thrips (WFT), based on volatile chemicals released from cut leaves. Cultivars that were susceptible, intermediate, or resistant to WFT [based on mean cultivar rank (MCR)] were used as standards, and were correctly classified (> 90%) by using discriminant function analysis. Several cultivars with unknown resistance were classified based on the standards, and were used as standards in a subsequent trial to classify other unknowns. The results of this study demonstrate some agreement between the WFT resistance categories as designated by the electronic nose and results of feeding bioassays (MCR), suggesting that this technique may serve as a useful screening tool for WFT resistance.

Factors Influencing the Survival and Growth of Listeria monocytogenes on the Surface of Canadian Retail Wieners.

A study of survival and growth of Listeria monocytogenes on Canadian retail wieners was undertaken to assess the potential hazard of this product. All-beef, poultry or beef/pork wieners from a total of six processing plants were surface-inoculated with a three-strain cocktail of L. monocytogenes , and stored under vacuum at 5°C for up to 28 days. Of a total of 61 samples tested, 40 (65.6%) supported growth of the pathogen. A model was derived for 35 samples with stepwise multiple regression analysis, which used initial pH (pH0), initial lactic acid bacteria counts (LAB0), and LAB after 14 days (LAB14) to explain 48.1% of the variation in the L monocytogenes counts after 14 days. The observed times to 1-log increase in counts for 25 samples in which growth occurred were compared to times predicted by the U. S. Dept. of Agriculture (USDA) Pathogen Modelling Program. The latter model, based on storage temperature, and initial pH, nitrite and salt concentrations, explained only 12.4% of the variation in the observed times. Scanning electron microscopy showed that the wiener surface was composed of a dense layer of coagulated protein. This may alter the ability of L. monocytogenes to develop relative to growth conditions in the meat formulation. These results suggest that retail wieners may support growth of L monocytogenes , which could present a health hazard to target groups of consumers if subjected to post-processing contamination. They also suggest that further work is needed to derive adequate models to allow an accurate prediction of the growth of L. monocytogenes on ready-to-eat meat products.

Effect of Preservatives and Growth Factors on Secretion of Listeriolysin O by Listeria monocytogenes.

The influence of selected preservatives and growth factors on listeriolysin O (LLO) secretion by Listeria monocytogenes was examined. Secretion of LLO was maximal in tryptic soy broth medium. LLO secretion paralleled growth at 10 and 30°C in buffered proteose peptone medium. LLO secretion was supported by a number of carbohydrates and was inhibited by long-chain (P13-18) polyphosphates. Cell yield but not LLO secretion was increased by agitation. Both growth and LLO secretion were inhibited by nitrite, while LLO secretion was selectively inhibited by sorbate and NaCl. These results suggest that LLO secretion is more sensitive than growth to the inhibitory action of preservatives.

Simultaneous Enumeration of the Characteristic Microorganisms in Yogurt Using the Hydrophobic Grid Membrane Filter System.

Utilization of the hydrophobic grid membrane system (HGMF) in conjunction with erioglaucine-supplemented tryptone-phytone-yeast extract (TPYE) agar permitted the differential enumeration of Streptococcus thermophilus and Lactobacillus bulgaricus in yogurt. An anaerobic incubation of 42°C resulted in conditions under which streptococcal colonies were light blue and the lactobacillus colonies were a distinct dark blue. Excellent correlation was achieved when the above method was compared with the International Dairy Federation Standard 117:1983 by coincident analysis of 12 commercial yogurts. The method allowed a reliable single plate determination of cocci to rod ratios from 20:1 to 1:5.

Acid-Coagulation of Evaporated Milk by a Coculture of Enterococcus faecium and Bacillus subtilis 1.

Two gram-positive microorganisms, a spore-forming rod and a coccus, were isolated from a can of evaporated milk which had coagulated after storage at 37°C for 3 months. The two strains were identified as Bacillus subtilis and Enterococcus faecium , respectively. Individually, neither strain produced acid in sterile skim milk; however, when cultured together, the pH dropped rapidly and the milk coagulated. Similar results were obtained with E. faecium and a proteolytic Pseudomonas sp. Acid production by E. faecium in sterile skim milk was closely related to proteolysis produced by addition of partially purified B. subtilis proteinase. Acid production was also stimulated by addition of enzymatic digests of casein. It was concluded that spoilage in canned evaporated milk had resulted from production of acid by E. faecium when supplied with small molecular weight nitrogenous compounds derived from milk proteins by the action of B. subtilis proteinase.

Comparison of the Hide Powder Azure and Casein-Trinitrobenzene Sulfonic Acid Methods for Determining Proteolysis in Skim Milk.

Three extracellular psychrotroph proteinases were assayed in a defined buffer system and in skim milk, using the insoluble protein-dye complex, hide powder azure (HPA), as well as by quantifying free amino groups released from casein with trinitrobenzene-sulfonic acid (TNBS). Apparent Km values for HPA of approximately 60 mg/ml were found for each enzyme while for sodium caseinate, the apparent Km of 9.14 mg/ml for enzyme ATCC 15456 was 3-fold greater than for enzymes 13 and 32A. Vmax values for enzyme ATCC 15456 were 3 to 3.5 and 1.6 to 2.8-fold lower than for the other two enzymes with HPA and sodium caseinate, respectively. When activities in skim milk were being determined, trichloroacetic acid (TCA) concentrations of 2 and 8% for HPA and TNBS, respectively, were chosen to maximize recovery of degradation products and minimize background. Proteolysis measurements in skim milk with the two methods were closely correlated (r = 0.903 to 0.920) for each of the three enzymes. Detectability values (obtained by dividing the standard error of estimate for each method by its slope ratio) of 0.098 and 0.093 for the HPA and TNBS methods, respectively, indicate that the two methods do not differ significantly (P>0.05) in reliability.

Evaluation of an 18°C/45-Hour Plate Count Technique for the Enumeration of Psychrotrophic Bacteria in Raw and Pasteurized Milk.

Bacterial counts were done at 7°C for 10 d and at 18°C for 45 h on 93 samples of raw and 185 samples of pasteurized milk. As an additional test, catalase-positive microorganisms were enumerated at 18°C/45 h. Close correlations were obtained between the numbers of microorganisms following 18°C/45-h and 7°C/10-d incubations in raw (r2=0.866) and pasteurized (r2=0.936) milk samples. Similar correlations (r2=0.860 and 0.946) were noted for the 18°C/45-h-catalase and the 7°C/10-d methods for raw and pasteurized milk, respectively. Results suggest that incubation at 18°C for 45 h provides a reliable estimate of the numbers of psychrotrophs in raw and pasteurized milk and that the use of catalase does not improve sensitivity of the test significantly.