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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., (Copyright © 2020 American Society for Microbiology.)

Spore-forming bacteria are natural contaminants of food raw materials, and sporulation can occur in many environments from farm to fork. In order to characterize and to predict spore formation over time, we developed a model that describes both the kinetics of growth and the differentiation of vegetative cells into spores. The model is based on a classical growth model and enables description of the kinetics of sporulation with the addition of three parameters specific to sporulation. Two parameters are related to the probability of each vegetative cell to commit to sporulation and to form a spore, and the last one is related to the time needed to form a spore once the cell is committed to sporulation. The goodness of fit of this growth-sporulation model was assessed using growth-sporulation kinetics at various temperatures in laboratory medium or in whey for Bacillus subtilis , Bacillus cereus , and Bacillus licheniformis The model accurately describes the kinetics in these different conditions, with a mean error lower than 0.78 log 10 CFU/ml for the growth and 1.08 log 10 CFU/ml for the sporulation. The biological meaning of the parameters was validated with a derivative strain of Bacillus subtilis 168 which produces green fluorescent protein at the initiation of sporulation. This model provides physiological information on the spore formation and on the temporal abilities of vegetative cells to differentiate into spores and reveals the heterogeneity of spore formation during and after growth. IMPORTANCE The growth-sporulation model describes the progressive transition from vegetative cells to spores with sporulation parameters describing the sporulation potential of each vegetative cell. Consequently, the model constitutes an interesting tool to assess the sporulation potential of a bacterial population over time with accurate parameters such as the time needed to obtain one resistant spore and the probability of sporulation. Further, this model can be used to assess these data under various environmental conditions in order to better identify the conditions favorable for sporulation regarding the time to obtain the first spore and/or the concentrations of spores which could be reached during a food process., (Copyright © 2019 American Society for Microbiology.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)