Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells

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 ( ) and maximum specific growth rate ( max) 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 (N0). 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

Modelling biofilm formation of Salmonella enterica ser. Newport as a function of pH and water activity

The effect of pH and water activity (aw) on the formation of biofilm by Salmonella enterica ser. Newport, previously identified as a strong biofilm producer, was assessed. Biofilm formation was evaluated in tryptone soy broth at 37 C and at different combinations of pH (3.3e7.8) and aw (0.894e0.997). In total, 540 biofilm formation tests in 108 pH and aw combinations were carried out in polystyrene microtiter plates using crystal violet staining and optical density (OD; 580 nm) measurements. Since the individual effects of pH and aw on biofilm formation had a similar pattern to that observed for microbial growth rate, cardinal parameter models (CPMs) were used to describe these effects. CPMs described successfully the effects of these two environmental parameters, with the estimated cardinal values of pHmin, pHopt, pHmax, awmin and awopt being 3.58, 6.02, 9.71, 0.894 and 0.994, respectively. The CPMs assumption of the multiplicative inhibitory effect of environmental factors was validated in the case of biofilm formation using additional independent data (i.e. 430 OD data at 86 different combinations of pH and aw). The validation results showed a good agreement (r2 ¼ 0.938) between observed and predicted OD with no systematic error. In the second part of this study, a probabilistic model predicting the pathogen's biofilm formation boundaries was developed, and the degree of agreement between predicted probabilities and observations was as high as 99.8%. Hence, the effect of environmental parameters on biofilm formation can be quantitatively expressed using mathematical models, with the latter models, in turn, providing useful information for biofilm control in food industry environments

Fungal contaminants of Pyrus communis var. bambinella : macroscopic and molecular characterisation

Produce such as pear fruits are prone to post-harvest diseases caused by fungi or bacteria and can occur at any stage from harvest to consumption. Such organisms may cause soft spots or light brown lesions on fruit and this may lead to advanced fungal invasion, which is clearly seen by a variety of coloured mouldy growths on fruit. Fungal pathogens cause premature fruit spoilage of ‘Bambinella’, known as Maltese June Pear, a fruit endemic to the Maltese Islands.peer-reviewe

Development of a Microbial Time/Temperature Indicator Prototype for Monitoring the Microbiological Quality of Chilled Foods▿

A time/temperature indicator (TTI) system based on the growth and metabolic activity of a Lactobacillus sakei strain was developed for monitoring food quality throughout the chilled-food chain. In the designed system, an irreversible color change of a chemical chromatic indicator (from red to yellow) progressively occurs due to the pH decline that results from microbial growth and metabolism in a selected medium. The relation of the TTI response (color change) to the growth and metabolic activity (glucose consumption, lactic acid production, pH decrease) of L. sakei was studied. In addition, the temperature dependence of the TTI kinetics was investigated isothermally in the range of 0 to 16°C and modeled with a system of differential equations. At all temperatures tested, the pH and color changes of the TTI system followed closely the growth of L. sakei, with the endpoint (the time at which a distinct visual color change to the final yellow was observed) of the TTI coinciding with a population level of 107 to 108 CFU/ml. The endpoint decreased from 27 days at 0°C to 2.5 days at 16°C, yielding an activation energy of 97.7 kJ/mol, which was very close to the activation energy of the L. sakei growth rate in the TTI substrate (103.2 kJ/mol). Furthermore, experiments conducted on the effect of the inoculum level showed a negative linear relationship between the level of L. sakei inoculated in the system medium and the endpoint of the TTI. For example, the endpoint at 8°C ranged from 6 to 2 days for inoculum levels of 101 and 106 CFU/ml, respectively. This relationship allows the easy adjustment of the TTI endpoint at a certain temperature according to the shelf life of the food product of concern by using an appropriate inoculum level of L. sakei. The microbial TTI prototype developed in the present study could be used as an effective tool for monitoring shelf life during the distribution and storage of food products that are spoiled primarily by lactic acid bacteria or other bacteria exhibiting similar kinetic responses and spoilage potentials. Apart from the low cost, the main advantage of the proposed TTI is that its response closely matches the loss of the quality of a food product by simulating the microbial spoilage process in particular environments

Effect of Food Processing-Related Stresses on Acid Tolerance of Listeria monocytogenes

Stationary-phase cells of Listeria monocytogenes grown in glucose-free or glucose-containing media were exposed for 90 min to various stresses, including acid stress (pH 4.0 to 7.0), osmotic stress (10.5 to 20.5% NaCl), and various temperatures (−5 to 50°C), and were further exposed to pH 3.5. Exposure to a mildly acidic (pH 5.0 to 6.0) environment provided protection of the pathogen against acid upon subsequent exposure. This adaptive response, however, was found to be strongly dependent on other environmental conditions during the shock, such as temperature or the simultaneous presence of a second stress factor (NaCl). Growth of L. monocytogenes in the presence of glucose resulted in enhanced survival of the pathogen at pH 3.5. Sublethal stresses other than acidic stresses, i.e., osmotic, heat, and low-temperature stresses, did not affect the acid resistance of L. monocytogenes (P > 0.5). More-severe levels of these stresses, however, resulted in sensitization of the pathogen to acid

Effect of chlorine stress on the subsequent growth behavior of individual salmonella cells

The present work investigates the effect of chlorine stress on the subsequent growth behavior of individual Salmonella cells. A time-lapse microscopy method was used which allowed to evaluate the effect of chlorine on the division times of Salmonella individual cells and the percentage of cells able to divide after the treatment. The results showed that the percentage of cells able to divide after the chlorine treatment decreased from 92.7% for untreated cells to 43.12% and 22% for cell exposed to 127 and 150 mg/l chlorine for 3 min, respectively. The first division time of Salmonella cells was not affected by the chlorine stress at the lower tested concentration of 11 mg/l. Exposure at higher chlorine concentrations however, resulted in significantly longer and more variable division times. The mean first division times were 1.46 +/- 0.61, 1.41 +/- 0.53, 1.69 +/- 0.59, 5.34 +/- 4.03 and 19.2 +/- 8.71 h after 3 min treatments with 0, 11, 61, 127 and 150 mg/l chlorine, respectively. The effect of chlorine on the second division time of the cells was milder compared to the first division. Exposure of cells to chlorine concentrations up to 61 mg/l did not affect the second division. These results indicate that the daughter cells have no "memory" of the chlorine treatment at these concentrations. For cells exposed to the highest tested chlorine concentration of 150 mg/l the mean second division time was almost 3.5 times longer compared to untreated cells indicating that potential damages of the cells caused by the chlorine treatment are not fully repaired in the second generation. The quantitative data provided by this study at the level of individual cell may lead to a better understanding of microbial resistance to chlorine and improve sanitation and decontamination procedures in the food industry123311316CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP302763/2014-7; 305804/2017-0não tem2014/03791-1, 2016/20821-

Effect of storage temperature on the lag time of Geobacillus stearothermophilus individual spores

The lag times (lambda) of Geobacillus stearothermophilus single spores were studied at different storage temperatures ranging from 45 to 59 degrees C using the Bioscreen C method. A significant variability of lambda was observed among individual spores at all temperatures tested. The storage temperature affected both the position and the spread of the lambda distributions. The minimum mean value of lambda (i.e. 10.87 h) was observed at 55 degrees C, while moving away from this temperature resulted in an increase for both the mean and standard deviation of lambda, A Cardinal Model with Inflection (CMI) was fitted to the reverse mean lambda, and the estimated values for the cardinal parameters T-min, T-max, T-opt and the optimum mean lambda of G. stearothermophilus were found to be 38.1, 64.2, 53.6 degrees C and 10.3 h, respectively. To interpret the observations, a probabilistic growth model for G. stearothermophilus individual spores, taking into account lambda variability, was developed. The model describes the growth of a population, initially consisting of No spores, over time as the sum of cells in each of the N-0 imminent subpopulations originating from a single spore. Growth simulations for different initial contamination levels showed that for low N-0 the number of cells in the population at any time is highly variable. An increase in N-0 to levels exceeding 100 spores results in a significant decrease of the above variability and a shorter lambda of the population. Considering that the number of G. stearothermophilus surviving spores in the final product is usually very low, the data provided in this work can be used to evaluate the probability distribution of the time-to-spoilage and enable decision-making based on the "acceptable level of risk".Understanding the impact of manufacturing processes in the ecology / European Social Fund (ESF

Quantifying the effect of water activity and storage temperature on single spore lag times of three moulds isolated from spoiled bakery products

9th International Conference on Predictive Modelling in Food (ICPMF); Rio de Janeiro, BRAZILThe inhibitory effect of water activity (a(w)) and storage temperature on single spore lag times of Aspergillus niger, Eurotium repens (Aspergillus pseudoglaucus) and Penicillium corylophilum strains isolated from spoiled bakery products, was quantified. A full factorial design was set up for each strain. Data were collected at levels of a(w) varying from 0.80 to 0.98 and temperature from 15 to 35 degrees C. Experiments were performed on malt agar, at pH 5.5. When growth was observed, ca 20 individual growth kinetics per condition were recorded up to 35 days. Radius of the colony vs time was then fitted with the Buchanan primary model. For each experimental condition, a lag time variability was observed, it was characterized by its mean, standard deviation (sd) and 5th percentile, after a Normal distribution fit. As the environmental conditions became stressful ( e.g. storage temperature and a(w) lower), mean and sd of single spore lag time distribution increased, indicating longer lag times and higher variability. The relationship between mean and sd followed a monotonous but not linear pattern, identical whatever the species. Next, secondary models were deployed to estimate the cardinal values (minimal, optimal and maximal temperatures, minimal water activity where no growth is observed anymore) for the three species. That enabled to confirm the observation made based on raw data analysis: concerning the temperature effect, A. niger behaviour was significantly different from E. repens and P. corylophilum: Tops of 37.4 degrees C (standard deviation 1.4 degrees C) instead of 27.1 degrees C (1.4 degrees C) and 25.2 degrees C (1.2 degrees C), respectively. Concerning the a(w) effect, from the three mould species, E. repens was the species able to grow at the lowest a(w) (aw(min), estimated to 0.74 (0.02)). Finally, results obtained with single spores were compared to findings from a previous study carried out at the population level (Dagnas et al., 2014). For short lag times days), there was no difference between lag time of the population (ca 2000 spores inoculated in one spot) and mean (nor 5th percentile) of single spore lag time distribution. In contrast, when lag time was longer, i.e. under more stressful conditions, there was a discrepancy between individual and population lag times (population lag times shorter than 5th percentiles of single spore lag time distribution), confirming a stochastic process. Finally, the temperature cardinal values estimated with single spores were found to be similar to those obtained at the population level, whatever the species. All these findings will be used to describe better mould spore lag time variability and then to predict more accurately bakery product shelf-life

Applicability of an Arrhenius Model for the Combined Effect of Temperature and CO(2) Packaging on the Spoilage Microflora of Fish

The temperature behavior of the natural microflora on the Mediterranean fish red mullet (Mullus barbatus) was examined as a case study. The growth of the spoilage bacteria Pseudomonas spp., Shewanella putrefaciens, Brochothrix thermosphacta, and lactic acid bacteria was modeled as a function of temperature and the concentration of carbon dioxide in modified atmosphere packaging. Combined models were developed and comparatively assessed based on polynomial, Belehradek, and Arrhenius equations. The activation energy parameter of the Arrhenius model, E(A), was independent of the packaging atmosphere and ranged from 75 to 85 kJ/mol for the different bacteria, whereas the preexponential constant decreased exponentially with the packaging CO(2) concentration. We evaluated the applicability of the models developed by using experimental bacterial growth rates obtained from 42 independent experiments performed with three Mediterranean fish species and growth rates predicted from the models under the same temperature and packaging conditions. The accuracy factor and bias factor were used as statistical tools for evaluation, and the developed Arrhenius model and the Belehradek model were judged satisfactory overall