Germination and outgrowth of Bacillus mycoides KBAB4 spores are impacted by environmental pH, quantitatively analyzed at single cell level with sporetracker.

Quantifying spore germination and outgrowth heterogeneity is challenging. Single cell level analysis should provide supplementary knowledge regarding the impact of unfavorable conditions on germination and outgrowth dynamics. This work aimed to quantify the impact of pH on spore germination and outgrowth, investigating the behavior of individual spore crops, produced under optimal and suboptimal conditions. Bacillus mycoides (formerly B. weihenstephanensis) KBAB4 spores, produced at pH 7.4 and at pH 5.5 were incubated at different pH values, from pH 5.2 to 7.4. The spores were monitored by microscopy live imaging, in controlled conditions, at 30 °C. The images were analyzed using SporeTracker, to determine the state of single cells. The impact of pH on germination and outgrowth times and rates was estimated and the correlation between these parameters was quantified. The correlation between germination and outgrowth times was significantly higher at low pH. These results suggest that an environmental pressure highlights the heterogeneity of spore germination and outgrowth within a spore population. Results were consistent with previous observations at population level, now confirmed and extended to single cell level. Therefore, single cell level analyses can be used to quantify the heterogeneity of spore populations, which is of interest in order to control the development of spore-forming bacteria, responsible for food safety issues.

Are Bacillus thuringiensis strains like any other Bacillus cereus strains? Phenotypic-based tools to locate Bacillus thuringiensis in the diversity of the Bacillus cereus sensu lato group.

Some Bacillus thuringiensis (Bt) strains are used as pesticide agent. This species belongs to Bacillus cereus (Bc) group which contains many species with a high phenotypic diversity, and could be pathogenic like B. cereus. The aim of this study was to characterize the phenotype of 90 strains belonging to Bc group, half of which were Bt. Knowing that Bt strains belong to different phylogenetic Bc groups, do Bt strains have the same phenotype than other Bc group strains? Five phenotypic parameters were estimated for 90 strains in the Bc group, of which 43 were Bt strains: minimal, maximal and optimal growth temperature, cytotoxicity on Caco-2 cells, heat resistance of spores. The dataset was processed by principal component analysis, showing that 53% of the variance of the profiles corresponded to factors linked to growth, heat resistance and cytotoxicity. The phenotype followed the phylogenetic groups based on panC. Bt strains showed similar behavior to other strains in the Bc group, in our experimental conditions. Commercial bio-insecticide strains were mesophilic with low heat resistance.

Suboptimal Bacillus licheniformis and Bacillus weihenstephanensis Spore Incubation Conditions Increase Heterogeneity of Spore Outgrowth Time.

Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges of 4.7 to 7.4 and temperatures of 10°C to 37°C for B. weihenstephanensis and 18°C to 59°C for B. licheniformis Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, a lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C for 12 min or at 95°C for 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite the fact that they both led to a 10-fold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth 1.2-fold and lowered the proportion of spores able to germinate and outgrow 1.4-fold. Interestingly, we showed by proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 than in those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH.IMPORTANCE Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of Bacillus spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.

Quantifying permeabilization and activity recovery of Bacillus spores in adverse conditions for growth.

Heat treatment is the main hurdle used to eliminate spores in foods but the pH conditions which spores encounter after the treatment have a tremendous impact on the spore ability to germinate, outgrow and grow. The aim of this work was to discriminate the inactive permeable spores and the active spores in unfavorable acidic conditions, after a heat treatment. In this study, Bacillus weihenstephanensis KBAB4 was used as model micro-organism for psychrotrophic Bacillus. The spores were heat treated to inactivate 90% of the population, 12 min at 85 °C, or 2 min at 95 °C. After each treatment the spores were incubated at pH 5.50 or pH 7.40. The evolution of dormant spores, permeable spores, germinated and vegetative cells was monitored by flow cytometry using a double staining. LDS 751, stains in red all the permeable cells, and CFDA stains in green cells presenting an esterase activity. Dormant spores did not show neither red fluorescence nor green fluorescence. Permeabilized spores which did not recover metabolic activity were red fluorescent but not green fluorescent. Germinated spores (permeabilized and having an esterase activity) appeared red fluorescent and green fluorescent due to their permeability and their metabolic activity. This method allowed the differentiation of the impact of heat treatment and post-treatment incubation pH on the two first steps of germination: spore permeabilization and activity recovery. Having a better understanding of spore germination at unfavorable post-treatment pH allows a better control of spore forming bacteria in foods.

Predicting heat process efficiency in thermal processes when bacterial inactivation is not log-linear.

The food industry widely uses the F-value which considers microbial log-linear inactivation, while microbial heat inactivation may result in a non-log-linear inactivation pattern due to genetic or phenotypical heterogeneity. This may yield discrepancies in predicting microbial heat inactivation under dynamic conditions of heat treatment. In this paper, we suggest the calculation of the equivalent time of heat treatment at a given temperature to overcome these constraints. To validate our proposal, the heat inactivation of Bacillus pumilus, showing non-log-linear behavior, was predicted for 4 different heat inactivation profiles and bacterial enumeration was performed to determine whether prediction errors were acceptable. When the proportion of residuals in an acceptable zone from 1 log (fail safe) to 0.5 log (fail dangerous) was greater or equal to 70%, the model was considered as acceptable for predictions of the tested data. The new approach gave four different temperature profiles, with 96, 85, 85 and 100% of the residuals in the acceptable zone, indicating satisfactory prediction. Thus the proposed practical alternative to simulate microbial heat inactivation kinetics is able to extend the F-value to non-log-linear inactivation patterns.

Effect of incubation temperature and pH on the recovery of Bacillus weihenstephanensis spores after exposure to a peracetic acid-based disinfectant or to pulsed light.

The recovery at a range of incubation temperatures and pH of spores of Bacillus weihenstephanensis KBAB4 exposed to a peracetic acid-based disinfectant (PABD) or to pulsed light was estimated. Spores of B. weihenstephanensis were produced at 30 °C and pH 7.00, at 30 °C and pH 5.50, or at 12 °C and pH 7.00. The spores were treated with a commercial peracetic acid-based disinfectant at 80 mg·mL-1 for 0 to 200 min at 18 °C or by pulsed light at fluences ranging between 0.4 and 2.3 J·cm-2 for pulsed light treatment. After each treatment, the spores were incubated on nutrient agar at 12 °C, 30 °C or 37 °C, or at pH 5.10, 6.00 or 7.40. Incubation temperature during recovery had a significant impact only near the recovery limits, beyond which surviving spores previously exposed to a PABD or to pulsed light were not able to form colonies. In contrast, a decrease in pH of the recovery nutrient agar had a progressive impact on the ability of spores to form colonies. The time to first log reduction after PABD treatment was 29.5 ±â€¯0.7 min with recovery at pH 7.40, and was tremendously shortened 5.1 ±â€¯0.2 min with recovery at pH 5.10. Concerning the fluence necessary for the first log reduction, it was 1.5 times higher when the spores were recovered at pH 6.00 compared to a recovery at pH 5.10. The impact of recovery temperature and pH can be described with a mathematical model using cardinal temperature and pH as parameters. These effects of temperature and pH on recovery of Bacillus weihenstephanensis spores exposed to a disinfectant combining peracetic acid and hydrogen peroxide, or pulsed light are similar, although these treatments are of different natures. Sporulation temperature or pH did not impact resistance to the peracetic acid-based disinfectant or pulsed light.

Modeling the recovery of heat-treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 spores at suboptimal temperature and pH using growth limits.

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.

Sensitivity of Bacillus weihenstephanensis to acidic changes of the medium is not dependant on physiological state.

This study aims to quantify the effect of salt and acid preliminary exposure on acid resistance of vegetative cells of Bacillus weihenstephanensis. The psychrotolerant strain KBAB4 was cultured until the mid-exponentially phase (i) in BHI, (ii) in BHI supplemented with 2.5% salt or (iii) in BHI acidified at pH 5.5 with HCl. The growing cells were subsequently inactivated in lethal acid conditions ranging from 4.45 to 4.70. Based on statistical criteria, a primary mixed-Weibull model was used to fit the acid inactivation kinetics. The acid resistance was enhanced for acid-adapted cells and decreased for salt-adapted cells. The secondary modelling of the bacterial resistance allowed the quantification of the change in pH leading to a ten folds variation of the bacterial resistance, i.e. cells sensitivity (zpH). This sensitivity was not significantly affected whatever the preliminary mild exposure and the presence of sub-populations with different acid resistances. These results highlighted that pre-incubation conditions influence bacterial acid resistance without affecting the sensitivity to acidic modifications, with a 10 fold reduction of Bacillus acid resistance observed for a reduction of 0.37 pH unit. Quantification of such adaptive stress response might be instrumental in quantitative risk assessment more particularly in food formulation, particularly for low-acid minimally processed foods.

Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers.

Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-resistance towards otherwise lethal stresses. In this study, an approach was followed to select molecular biomarkers (quantitative gene expressions) to predict induced acid resistance after exposure to various mild stresses, i.e. exposure to sublethal concentrations of salt, acid and hydrogen peroxide during 5 min to 60 min. Gene expression patterns of unstressed and mildly stressed cells of Bacillus weihenstephanensis were correlated to their acid resistance (3D value) which was estimated after exposure to lethal acid conditions. Among the twenty-nine candidate biomarkers, 12 genes showed expression patterns that were correlated either linearly or non-linearly to acid resistance, while for the 17 other genes the correlation remains to be determined. The selected genes represented two types of biomarkers, (i) four direct biomarker genes (lexA, spxA, narL, bkdR) for which expression patterns upon mild stress treatment were linearly correlated to induced acid resistance; and (ii) nine long-acting biomarker genes (spxA, BcerKBAB4_0325, katA, trxB, codY, lacI, BcerKBAB4_1716, BcerKBAB4_2108, relA) which were transiently up-regulated during mild stress exposure and correlated to increased acid resistance over time. Our results highlight that mild stress induced transcripts can be linearly or non-linearly correlated to induced acid resistance and both approaches can be used to find relevant biomarkers. This quantitative and systematic approach opens avenues to select cellular biomarkers that could be incremented in mathematical models to predict microbial behaviour.

Modelling the effect of temperature, water activity and pH on the growth of Serpula lacrymans.

AIMS: To predict the risk factors for building infestation by Serpula lacrymans, which is one of the most destructive fungi causing timber decay in buildings. METHODS AND RESULTS: The growth rate was assessed on malt extract agar media at temperatures between 1.5 and 45°C, at water activity (a(w)) over the range of 0.800-0.993 and at pH ranges from 1.5 to 11.0. The radial growth rate (µ) and the lag phase (λ) were estimated from the radial growth kinetics via the plots radius vs time. These parameters were then modelled as a function of the environmental factors tested. Models derived from the cardinal model (CM) were used to fit the experimental data and allowed an estimation of the optimal and limit values for fungal growth. Optimal growth rate occurred at 20°C, at high a(w) level (0.993) and at a pH range between 4.0 and 6.0. The strain effect on the temperature parameters was further evaluated using 14 strains of S. lacrymans. The robustness of the temperature model was validated on data sets measured in two different wood-based media (Quercus robur L. and Picea abies). CONCLUSIONS: The two-step procedure of exponential model with latency followed by the CM with inflection gives reliable predictions for the growth conditions of a filamentous fungus in our study. The procedure was validated for the study of abiotic factors on the growth rate of S. lacrymans. SIGNIFICANCE AND IMPACT OF THE STUDY: This work describes the usefulness of evaluating the effect of physico-chemical factors on fungal growth in predictive building mycology. Consequently, the developed mathematical models for predicting fungal growth on a macroscopic scale can be used as a tool for risk assessment of timber decay in buildings.

The wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temperature but not on previous cell history.

While bacterial spores are mostly produced in a continuous process, this study reports a two-step sporulation methodology. Even though spore heat resistance of numerous spore-forming bacteria is known to be dependent on sporulation conditions, this approach enables the distinction between the vegetative cell growth phase in nutrient broth and the sporulation phase in specific buffer. This study aims at investigating whether the conditions of growth of the vegetative cells, prior to sporulation, could affect spore heat resistance. For that purpose, wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores, produced via a two-step sporulation process, was determined from vegetative cells harvested at four different stages of the growth kinetics, i.e. early exponential phase, late exponential phase, transition phase or early stationary phase. To assess the impact of the temperature on spore heat resistance, sporulation was performed at 10 °C, 20 °C and 30 °C from cells grown during a continuous or a discontinuous temperature process, differentiating or not the growth and sporulation temperatures. Induction of sporulation seems possible for a large range of growth stages. Final spore concentration was not significantly affected by the vegetative cell growth stage while it was by the temperature during growing and sporulation steps. The sporulation temperature influences the heat resistance of B. weihenstephanensis KBAB4 spores much more than growth temperature prior to sporulation. Spores produced at 10 °C were up to 3 times less heat resistant than spores produced at 30 °C.

Quantification of spore resistance for assessment and optimization of heating processes: a never-ending story.

The assessment and optimization of food heating processes require knowledge of the thermal resistance of target spores. Although the concept of spore resistance may seem simple, the establishment of a reliable quantification system for characterizing the heat resistance of spores has proven far more complex than imagined by early researchers. This paper points out the main difficulties encountered by reviewing the historical works on the subject. During an early period, the concept of individual spore resistance had not yet been considered and the resistance of a strain of spore-forming bacterium was related to a global population regarded as alive or dead. A second period was opened by the introduction of the well-known D parameter (decimal reduction time) associated with the previously introduced z-concept. The present period has introduced three new sources of complexity: consideration of non log-linear survival curves, consideration of environmental factors other than temperature, and awareness of the variability of resistance parameters. The occurrence of non log-linear survival curves makes spore resistance dependent on heating time. Consequently, spore resistance characterisation requires at least two parameters. While early resistance models took only heating temperature into account, new models consider other environmental factors such as pH and water activity ("horizontal extension"). Similarly the new generation of models also considers certain environmental factors of the recovery medium for quantifying "apparent heat resistance" ("vertical extension"). Because the conventional F-value is no longer additive in cases of non log-linear survival curves, the decimal reduction ratio should be preferred for assessing the efficiency of a heating process.

Physiological traits of Penicillium glabrum strain LCP 08.5568, a filamentous fungus isolated from bottled aromatized mineral water.

Penicillium glabrum is a ubiquitous fungus distributed world wide. This fungus is a frequent contaminant in the food manufacturing industry. Environmental factors such as temperature, water activity and pH have a great influence on fungal development. In this study, a strain of P. glabrum referenced to as LCP 08.5568, has been isolated from a bottle of aromatized mineral water. The effects of temperature, a(w) and pH on radial growth rate were assessed on Czapeck Yeast Agar (CYA) medium. Models derived from the cardinal model with inflection [Rosso et al., 1993 An unexpected correlation between cardinal temperatures of microbial growth highlighted by a new model. J. Theor. Bio. 162, 447-463.] were used to fit the experimental data and determine for each factor, the cardinal parameters (minimum, optimum and maximum). Precise characterisation of the growth conditions for such a fungal contaminant, has an evident interest to understand and to prevent spoilage of food products.

Modelling pH evolution and lactic acid production in the growth medium of a lactic acid bacterium: application to set a biological TTI.

Time temperature integrators or indicators (TTIs) are effective tools making the continuous monitoring of the time temperature history of chilled products possible throughout the cold chain. Their correct setting is of critical importance to ensure food quality. The objective of this study was to develop a model to facilitate accurate settings of the CRYOLOG biological TTI, TRACEO. Experimental designs were used to investigate and model the effects of the temperature, the TTI inoculum size, pH, and water activity on its response time. The modelling process went through several steps addressing growth, acidification and inhibition phenomena in dynamic conditions. The model showed satisfactory results and validations in industrial conditions gave clear evidence that such a model is a valuable tool, not only to predict accurate response times of TRACEO, but also to propose precise settings to manufacture the appropriate TTI to trace a particular food according to a given time temperature scenario.

Quantifying the effects of heating temperature, and combined effects of heating medium pH and recovery medium pH on the heat resistance of Salmonella typhimurium.

The influence of heating treatment temperature, pH of heating and recovery medium on the survival kinetics of Salmonella typhimurium ATCC 13311 is studied and quantified. From each non-log linear survival curve, Weibull model parameters were estimated. An average shape parameter value of 1.67 was found, which is characteristic of downward concavity curves and is in agreement with values estimated from other S. typhimurium strains. Bigelow type models quantifying the heating temperature, heating and recovery medium pH influences are fitted on scale parameter delta data (time of first decimal reduction), which reflects the bacterial heat resistance. The estimate of z(T) (4.64 degrees C) is in the range of values given in the literature for this species. The influence of pH of the heating medium on the scale parameter (z(pH): 8.25) is lower than that of the recovery pH medium influence (z(')(pH): 3.65).

General model, based on two mixed weibull distributions of bacterial resistance, for describing various shapes of inactivation curves.

Cells of Listeria monocytogenes or Salmonella enterica serovar Typhimurium taken from six characteristic stages of growth were subjected to an acidic stress (pH 3.3). As expected, the bacterial resistance increased from the end of the exponential phase to the late stationary phase. Moreover, the shapes of the survival curves gradually evolved as the physiological states of the cells changed. A new primary model, based on two mixed Weibull distributions of cell resistance, is proposed to describe the survival curves and the change in the pattern with the modifications of resistance of two assumed subpopulations. This model resulted from simplification of the first model proposed. These models were compared to the Whiting's model. The parameters of the proposed model were stable and showed consistent evolution according to the initial physiological state of the bacterial population. Compared to the Whiting's model, the proposed model allowed a better fit and more accurate estimation of the parameters. Finally, the parameters of the simplified model had biological significance, which facilitated their interpretation.

Effect of water activities of heating and recovery media on apparent heat resistance of Bacillus cereus spores.

Spores of Bacillus cereus were heated and recovered in order to investigate the effect of water activity of media on the estimated heat resistance (i.e., the D value) of spores. The water activity (ranging from 0.9 to 1) of the heating medium was first successively controlled with three solutes (glycerol, glucose, and sucrose), while the water activity of the recovery medium was kept near 1. Reciprocally, the water activity of the heating medium was then kept at 1, while the water activity of the recovery medium was controlled from 0.9 to 1 with the same depressors. Lastly, in a third set of experiments, the heating medium and the recovery medium were adjusted to the same activity. As expected, added depressors caused an increase of the heat resistance of spores with a greater efficiency of sucrose with respect to glycerol and glucose. In contrast, when solutes were added to the recovery medium, under an optimal water activity close to 0.98, a decrease of water activity caused a decrease in the estimated D values. This effect was more pronounced when sucrose was used as a depressor instead of glycerol or glucose. When the heating and the recovery media were adjusted to the same water activity, a balancing effect was observed between the protective influence of the solutes during heat treatment and their negative effect during the recovery of injured cells, so that the overall effect of water activity was reduced, with an optimal value near 0.96. The difference between the efficiency of depressors was also less pronounced. It may then be concluded that the overall protective effect of a decrease in water activity is generally overestimated.