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Louis Coroller - One of the best experts on this subject based on the ideXlab platform.
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suboptimal Bacillus licheniformis and Bacillus Weihenstephanensis spore incubation conditions increase heterogeneity of spore outgrowth time
Applied and Environmental Microbiology, 2020Co-Authors: Clément Trunet, Florence Postollec, Ivan Leguérinel, Narjes Mtimet, Olivier Couvert, Frederic Carlin, Annegabrielle Mathot, Veronique Broussolle, Louis CorollerAbstract: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 from 4.7 to 7.4, and temperature from 10°C to 37°C for B. Weihenstephanensis and from 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, 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 during 12 min or at 95°C during 2 min did not have the same impact on spore germination and outgrowth kinetics of B. Weihenstephanensis despite they both led to a tenfold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth by 1.2 fold and lowered the proportion of spores able to germinate and outgrow by 1.4 fold. Interestingly, we showed by a proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 compared to 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 sub-lethal 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 sp. 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 sub-lethal heat-treatment, a common situation in food processing and a concern for food preservation and safety.
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trustworthy identification of resistance biomarkers of Bacillus Weihenstephanensis workflow of the quality assurance procedure
Food Analytical Methods, 2018Co-Authors: Noemie Desriac, Florence Postollec, Louis Coroller, Sonia Pavan, Jerome Combrisson, Sylvie Halliersoulier, Danièle SohierAbstract:Omics databases have exploded, opening the avenue to take strain diversity or physiological variability into consideration in microbiological risk assessment (MRA). However, one obstacle to the integration of omics data in MRA is the production of quantitative data that may be used to build mathematical models. Gene expression is recognized as relevant biomarker to describe bacterial behavior and reverse transcription quantitative PCR (RT-qPCR) is considered as the gold standard for accurate, sensitive, and fast measurement of gene expression. However, numerous critical points may arise throughout the entire workflow of RT-qPCR data acquisition influencing accuracy of the results and reliability of the conclusions. Although recommendations about the minimum information that should be found in publications about quantitative real-time PCR experiments, heterogeneity in the reporting of RT-qPCR quality controls in publications remains. Herein, the step-by step RT-qPCR quality controls established for the selection of Bacillus Weihenstephanensis resistance biomarkers were described. Throughout this example, appropriate quality procedures and quality controls that shall be set up and carefully assessed to ensure reliable interpretations in RT-qPCR were depicted.
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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
International Journal of Food Microbiology, 2018Co-Authors: Clément Trunet, Florence Postollec, Ivan Leguérinel, Narjes Mtimet, A.-g. Mathot, Olivier Couvert, Frederic Carlin, Louis CorollerAbstract: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 ± 0.7 min with recovery at pH 7.40, and was tremendously shortened 5.1 ± 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 asparameters. 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.
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Dispersed phase volume fraction, weak acids and Tween 80 in a model emulsion: Effect on the germination and growth of Bacillus Weihenstephanensis KBAB4 spores
Food Research International, 2018Co-Authors: L. Léonard-akkari, Noemie Desriac, Ivan Leguérinel, A.-g. Mathot, S. Guégan, F. Courand, O. Couvert, J.f. Lepage, C. Rondeau, Louis CorollerAbstract:In foodstuffs, physico-chemical interactions and/or physical constraints between spores, inhibitors and food components may exist. Thus, the objective of this study was to investigate such interactions using a model emulsion as a microbial medium in order to improve bacterial spore control with better knowledge of the interactions in the formulation. Emulsions were prepared with hexadecane mixed with nutrient broth using sonication and were stabilized by Tween 80 and Span 80. The hexadecane ratio was either 35% (v/v) or 50% (v/v) and each emulsion was studied in the presence of organic acid (acetic, lactic or hexanoic) at two pH levels (5.5 and 6). Self-diffusion coefficients of emulsion components and the organic acids were measured by Pulsed Field Gradient-Nuclear Magnetic Resonance (PFG-NMR). The inhibition effect on the spore germination and cell growth of Bacillus Weihenstephanensis KBAB4 was characterized by the measure of the probability of growth using the most probable number methodology, and the measure of the time taken for the cells to germinate and grow using a single cell Bioscreen® method and using flow cytometry. The inhibition of spore germination and growth in the model emulsion depended on the dispersed phase volume fraction and the pH value. The effect of the dispersed phase volume fraction was due to a combination of (i) the lipophilicity of the biocide, hexanoic acid, that may have had an impact on the distribution of organic acid between hexadecane and the aqueous phases and (ii) the antimicrobial activity of the emulsifier Tween 80 detected at the acidic pH value. The interface phenomena seemed to have a major influence. Future work will focus on the exploration of these phenomena at the interface.
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mrna biomarkers selection based on partial least square algorithm in order to further predict Bacillus Weihenstephanensis acid resistance
Food Microbiology, 2015Co-Authors: Noemie Desriac, Florence Postollec, Louis Coroller, Frederic Jannic, Danièle SohierAbstract:In order to integrate omics data to quantitative microbiological risk assessment in foods, gene expressions may serve as bacterial behaviour biomarkers. In this study an integrative approach encompassing predictive modelling and mRNAs quantifications, was followed to select molecular biomarkers to further predict the acid resistance of Bacillus Weihenstephanensis. A multivariate analysis was performed to correlate the acid bacterial resistance and the gene expression of vegetative cells with or without exposure to stressing conditions. This mathematical method provides the advantage to take gene expressions and their interactions into account. The use of the Partial Least Squares algorithm allowed the selection of nine genes as acid resistance biomarkers among thirty targeted genes. According to their involvement in the general acid stress response of Bacillus, these genes were assigned to three different biological modules namely, metabolic rearrangements, general stress response and oxidative stress response. The oxidative stress response appeared as the major activated biological module in B. Weihenstephanensis cells submitted to acid stress conditions. Furthermore, as a firstly described model, the developed concept showed promising results to further be used to predict bacterial resistance using gene expression. Thus, this study underlines the possibility to integrate the bacterial physiology state, using omics biomarkers, into bacterial behaviour modelling and provide mechanistic understanding in acid bacterial resistance mechanisms.
Florence Postollec - One of the best experts on this subject based on the ideXlab platform.
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suboptimal Bacillus licheniformis and Bacillus Weihenstephanensis spore incubation conditions increase heterogeneity of spore outgrowth time
Applied and Environmental Microbiology, 2020Co-Authors: Clément Trunet, Florence Postollec, Ivan Leguérinel, Narjes Mtimet, Olivier Couvert, Frederic Carlin, Annegabrielle Mathot, Veronique Broussolle, Louis CorollerAbstract: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 from 4.7 to 7.4, and temperature from 10°C to 37°C for B. Weihenstephanensis and from 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, 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 during 12 min or at 95°C during 2 min did not have the same impact on spore germination and outgrowth kinetics of B. Weihenstephanensis despite they both led to a tenfold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth by 1.2 fold and lowered the proportion of spores able to germinate and outgrow by 1.4 fold. Interestingly, we showed by a proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 compared to 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 sub-lethal 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 sp. 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 sub-lethal heat-treatment, a common situation in food processing and a concern for food preservation and safety.
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trustworthy identification of resistance biomarkers of Bacillus Weihenstephanensis workflow of the quality assurance procedure
Food Analytical Methods, 2018Co-Authors: Noemie Desriac, Florence Postollec, Louis Coroller, Sonia Pavan, Jerome Combrisson, Sylvie Halliersoulier, Danièle SohierAbstract:Omics databases have exploded, opening the avenue to take strain diversity or physiological variability into consideration in microbiological risk assessment (MRA). However, one obstacle to the integration of omics data in MRA is the production of quantitative data that may be used to build mathematical models. Gene expression is recognized as relevant biomarker to describe bacterial behavior and reverse transcription quantitative PCR (RT-qPCR) is considered as the gold standard for accurate, sensitive, and fast measurement of gene expression. However, numerous critical points may arise throughout the entire workflow of RT-qPCR data acquisition influencing accuracy of the results and reliability of the conclusions. Although recommendations about the minimum information that should be found in publications about quantitative real-time PCR experiments, heterogeneity in the reporting of RT-qPCR quality controls in publications remains. Herein, the step-by step RT-qPCR quality controls established for the selection of Bacillus Weihenstephanensis resistance biomarkers were described. Throughout this example, appropriate quality procedures and quality controls that shall be set up and carefully assessed to ensure reliable interpretations in RT-qPCR were depicted.
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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
International Journal of Food Microbiology, 2018Co-Authors: Clément Trunet, Florence Postollec, Ivan Leguérinel, Narjes Mtimet, A.-g. Mathot, Olivier Couvert, Frederic Carlin, Louis CorollerAbstract: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 ± 0.7 min with recovery at pH 7.40, and was tremendously shortened 5.1 ± 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 asparameters. 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.
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mrna biomarkers selection based on partial least square algorithm in order to further predict Bacillus Weihenstephanensis acid resistance
Food Microbiology, 2015Co-Authors: Noemie Desriac, Florence Postollec, Louis Coroller, Frederic Jannic, Danièle SohierAbstract:In order to integrate omics data to quantitative microbiological risk assessment in foods, gene expressions may serve as bacterial behaviour biomarkers. In this study an integrative approach encompassing predictive modelling and mRNAs quantifications, was followed to select molecular biomarkers to further predict the acid resistance of Bacillus Weihenstephanensis. A multivariate analysis was performed to correlate the acid bacterial resistance and the gene expression of vegetative cells with or without exposure to stressing conditions. This mathematical method provides the advantage to take gene expressions and their interactions into account. The use of the Partial Least Squares algorithm allowed the selection of nine genes as acid resistance biomarkers among thirty targeted genes. According to their involvement in the general acid stress response of Bacillus, these genes were assigned to three different biological modules namely, metabolic rearrangements, general stress response and oxidative stress response. The oxidative stress response appeared as the major activated biological module in B. Weihenstephanensis cells submitted to acid stress conditions. Furthermore, as a firstly described model, the developed concept showed promising results to further be used to predict bacterial resistance using gene expression. Thus, this study underlines the possibility to integrate the bacterial physiology state, using omics biomarkers, into bacterial behaviour modelling and provide mechanistic understanding in acid bacterial resistance mechanisms.
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modeling the recovery of heat treated Bacillus licheniformis ad978 and Bacillus Weihenstephanensis kbab4 spores at suboptimal temperature and ph using growth limits
Applied and Environmental Microbiology, 2015Co-Authors: Clément Trunet, Florence Postollec, Ivan Leguérinel, Danièle Sohier, Narjes Mtimet, Olivier Couvert, Frederic Carlin, Annegabrielle Mathot, Louis CorollerAbstract: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.
Noemie Desriac - One of the best experts on this subject based on the ideXlab platform.
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trustworthy identification of resistance biomarkers of Bacillus Weihenstephanensis workflow of the quality assurance procedure
Food Analytical Methods, 2018Co-Authors: Noemie Desriac, Florence Postollec, Louis Coroller, Sonia Pavan, Jerome Combrisson, Sylvie Halliersoulier, Danièle SohierAbstract:Omics databases have exploded, opening the avenue to take strain diversity or physiological variability into consideration in microbiological risk assessment (MRA). However, one obstacle to the integration of omics data in MRA is the production of quantitative data that may be used to build mathematical models. Gene expression is recognized as relevant biomarker to describe bacterial behavior and reverse transcription quantitative PCR (RT-qPCR) is considered as the gold standard for accurate, sensitive, and fast measurement of gene expression. However, numerous critical points may arise throughout the entire workflow of RT-qPCR data acquisition influencing accuracy of the results and reliability of the conclusions. Although recommendations about the minimum information that should be found in publications about quantitative real-time PCR experiments, heterogeneity in the reporting of RT-qPCR quality controls in publications remains. Herein, the step-by step RT-qPCR quality controls established for the selection of Bacillus Weihenstephanensis resistance biomarkers were described. Throughout this example, appropriate quality procedures and quality controls that shall be set up and carefully assessed to ensure reliable interpretations in RT-qPCR were depicted.
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Dispersed phase volume fraction, weak acids and Tween 80 in a model emulsion: Effect on the germination and growth of Bacillus Weihenstephanensis KBAB4 spores
Food Research International, 2018Co-Authors: L. Léonard-akkari, Noemie Desriac, Ivan Leguérinel, A.-g. Mathot, S. Guégan, F. Courand, O. Couvert, J.f. Lepage, C. Rondeau, Louis CorollerAbstract:In foodstuffs, physico-chemical interactions and/or physical constraints between spores, inhibitors and food components may exist. Thus, the objective of this study was to investigate such interactions using a model emulsion as a microbial medium in order to improve bacterial spore control with better knowledge of the interactions in the formulation. Emulsions were prepared with hexadecane mixed with nutrient broth using sonication and were stabilized by Tween 80 and Span 80. The hexadecane ratio was either 35% (v/v) or 50% (v/v) and each emulsion was studied in the presence of organic acid (acetic, lactic or hexanoic) at two pH levels (5.5 and 6). Self-diffusion coefficients of emulsion components and the organic acids were measured by Pulsed Field Gradient-Nuclear Magnetic Resonance (PFG-NMR). The inhibition effect on the spore germination and cell growth of Bacillus Weihenstephanensis KBAB4 was characterized by the measure of the probability of growth using the most probable number methodology, and the measure of the time taken for the cells to germinate and grow using a single cell Bioscreen® method and using flow cytometry. The inhibition of spore germination and growth in the model emulsion depended on the dispersed phase volume fraction and the pH value. The effect of the dispersed phase volume fraction was due to a combination of (i) the lipophilicity of the biocide, hexanoic acid, that may have had an impact on the distribution of organic acid between hexadecane and the aqueous phases and (ii) the antimicrobial activity of the emulsifier Tween 80 detected at the acidic pH value. The interface phenomena seemed to have a major influence. Future work will focus on the exploration of these phenomena at the interface.
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mrna biomarkers selection based on partial least square algorithm in order to further predict Bacillus Weihenstephanensis acid resistance
Food Microbiology, 2015Co-Authors: Noemie Desriac, Florence Postollec, Louis Coroller, Frederic Jannic, Danièle SohierAbstract:In order to integrate omics data to quantitative microbiological risk assessment in foods, gene expressions may serve as bacterial behaviour biomarkers. In this study an integrative approach encompassing predictive modelling and mRNAs quantifications, was followed to select molecular biomarkers to further predict the acid resistance of Bacillus Weihenstephanensis. A multivariate analysis was performed to correlate the acid bacterial resistance and the gene expression of vegetative cells with or without exposure to stressing conditions. This mathematical method provides the advantage to take gene expressions and their interactions into account. The use of the Partial Least Squares algorithm allowed the selection of nine genes as acid resistance biomarkers among thirty targeted genes. According to their involvement in the general acid stress response of Bacillus, these genes were assigned to three different biological modules namely, metabolic rearrangements, general stress response and oxidative stress response. The oxidative stress response appeared as the major activated biological module in B. Weihenstephanensis cells submitted to acid stress conditions. Furthermore, as a firstly described model, the developed concept showed promising results to further be used to predict bacterial resistance using gene expression. Thus, this study underlines the possibility to integrate the bacterial physiology state, using omics biomarkers, into bacterial behaviour modelling and provide mechanistic understanding in acid bacterial resistance mechanisms.
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sensitivity of Bacillus Weihenstephanensis to acidic changes of the medium is not dependant on physiological state
Food Microbiology, 2013Co-Authors: Noemie Desriac, Florence Postollec, Ivan Leguérinel, Danièle Sohier, D Durand, Louis CorollerAbstract: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.
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identification of mrna as biomarker of Bacillus Weihenstephanensis acid resistance toward the integration of omic data into predictive microbiology
2013Co-Authors: Noemie DesriacAbstract:Predictive microbiology is a tool to assess food microbiological risks and optimise food processes. However predictive microbiology which predicts the bacterial behaviour does not take bacterial physiological state into consideration. The aim ofthis work is to identify molecular biomarkers to assess the impact of bacterial adaptation on the subsequent acid resistance. In this study, the acid resistance of Bacillus Weihenstephanensis wasinvestigated and mRNAs were identified as acid resistance biomarkers.(i) The bacterial acid resistance of different mildstress adapted cells was quantified using cultural methods. Mild salt-adapted cells were less resistant than cells grown in optimal conditions;and the latter less resistant than acid-adaptedcells. However, the bacterial resistance of B.Weihenstephanensis followed the same patternwhen facing acidic changes of the environment and that, whatever the environmental condition previously encountered.(ii) For RT-qPCR gene expression quantifications a specific rotative PCR device based on the PallGeneDisc® Technology was developed.(iii) Omic data and bacterial acid resistances correlation allows the selection of biomarkers to track the more resistant and the more sensitive cells present within the bacterial population.(iv) Both linear and non linear correlations allowed to define two types of biomarkers: ‘Directbiomarker’ for which expression patterns uponmild stress treatment were linearly correlated to the subsequent acid resistance and ‘long-actingbiomarkers’ which were transiently up-regulatedduring mild stress exposure and correlated to increased acid resistance over time.(v) A multivariate analysis was performed to correlate the acid bacterial resistance and the gene expression of vegetative cells. This mathematical method provides the advantage to take gene expressions and their interactions into account and allowed the selection of 9 genes as acid resistance biomarkers of B. Weihenstephanensis. Finally, some promising results were also obtained. There by, it would be feasible to use gene expression at a given time to predict the bacterialsurvival behaviour in lethal acid conditions.
Jacques Mahillon - One of the best experts on this subject based on the ideXlab platform.
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complete genome sequence of bacteriophage deep purple a novel member of the family siphoviridae infecting Bacillus cereus
Archives of Virology, 2018Co-Authors: Louise Hock, Annika Gillis, Jacques MahillonAbstract:Bacteriophage Deep-Purple, isolated from an agricultural soil in Belgium, lyses the emetic Bacillus Weihenstephanensis strain LH002 and exhibits a lytic activity against 55% of emetic Bacillus cereus and B. Weihenstephanensis strains. Deep-Purple is able to complete its lytic cycle within 45 min and is stable to a large range of pHs and temperatures below 60 °C. It possesses an icosahedral head of about 63 nm in diameter and a non-contractile tail of approximately 165 nm in length. The genome of this newly classifiable Siphoviridae family member is 36,278 bp long, with a G+C content of 38.36% and 40 putative CDSs. Most CDSs do not display similarity with other B. cereus group phages supporting the idea that Deep-Purple belongs to a new and currently uncharacterised Siphoviridae subfamily.
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Role of plasmid plasticity and mobile genetic elements in the entomopathogen Bacillus thuringiensis serovar israelensis
FEMS Microbiology Reviews, 2018Co-Authors: Annika Gillis, Nancy Fayad, Lionel Makart, Alexandre Bolotine, Alexei Sorokine, Mireille Kallassy, Jacques MahillonAbstract:Bacillus thuringiensis is a well-known biopesticide that has been used for more than 80 years. This spore-forming bacterium belongs to the group of Bacillus cereus that also includes, among others, emetic and diarrheic pathotypes of B. cereus, the animal pathogen Bacillus anthracis and the psychrotolerant Bacillus Weihenstephanensis. Bacillus thuringiensis is rather unique since it has adapted its lifestyle as an efficient pathogen of specific insect larvae. One of the peculiarities of B. thuringiensis strains is the extent of their extrachromosomal pool, with strains harbouring more than 10 distinct plasmid molecules. Among the numerous serovars of B. thuringiensis, 'israelensis' is certainly emblematic since its host spectrum is apparently restricted to dipteran insects like mosquitoes and black flies, vectors of human and animal diseases such as malaria, yellow fever, or river blindness. In this review, the putative role of the mobile gene pool of B. thuringiensis serovar israelensis in its pathogenicity and dedicated lifestyle is reviewed, with specific emphasis on the nature, diversity, and potential mobility of its constituents. Variations among the few related strains of B. thuringiensis serovar israelensis will also be reported and discussed in the scope of this specialised insect pathogen, whose lifestyle in the environment remains largely unknown.
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cereulide production by Bacillus Weihenstephanensis strains during growth at different ph values and temperatures
Food Microbiology, 2017Co-Authors: Alizee Guerin, Helene Thorsen Ronning, Va Ronique Broussolle, Jacques Mahillon, Claire Dargaignaratz, Per Einar Granum, Thierry Clavel, Christophe NguyentheAbstract:Besides Bacillus cereus, some strains of the psychrotolerant, potentially foodborne pathogen Bacillus Weihenstephanensis can produce the emetic toxine (cereulide). This toxin is a heat- and acid-stable cyclic dodecadepsipeptide that causes food intoxication with vomiting. However, some severe clinical cases with lethal outcomes have been described. If cereulide can be produced during refrigerated storage, it will not be inactivated by reheating food, representing an important risk of food intoxication for consumers. In this paper, we determined the capacity of the B. Weihenstephanensis strains BtB2-4 and MC67 to grow and produce cereulide on agar media at temperatures from 8 °C to 25 °C and at a pH from 5.4 to 7.0. At 8 °C, strain BtB2-4 produced quantifiable amounts of cereulide, whereas the limit of detection was reached for strain MC67. For BtB2-4, cereulide production increased 5-fold between 8 °C and 10–15 °C and by more than 100-fold between 15 °C and 25 °C. At temperatures of 10 °C and higher, cereulide concentrations were within the range of those reported by previous works in foods implicated in emetic poisoning. At 25 °C, decreasing the pH to 5.4 reduced cereulide production by strain BtB2-4 by at least 20-fold.
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the genetic diversity of cereulide biosynthesis gene cluster indicates a composite transposon tnces in emetic Bacillus Weihenstephanensis
BMC Microbiology, 2014Co-Authors: Kai Xu, Zhiming Yuan, Jacques Mahillon, Lingling Yang, Xiaomin HuAbstract:Background: Cereulide is a cyclic dodecadepsipeptide ionophore, produced via non-ribosomal peptide synthetases (NRPS), which in rare cases can lead to human death. Early studies had shown that emetic toxin formation belongs to a homogeneous group of Bacillus cereus sensu stricto and the genetic determinants of cereulide (a 24-kb gene cluster of cesHPTABCD) are located on a 270-kb plasmid related to the Bacillus anthracis virulence plasmid pXO1. Results: The whole genome sequences from seven emetic isolates, including two B. cereus sensu stricto and five Bacillus Weihenstephanensis strains, were compared, and their inside and adjacent DNA sequences of the cereulide biosynthesis gene clusters were analyzed. The sequence diversity was observed, which classified the seven emetic isolates into three clades. Different genomic locations of the cereulide biosynthesis gene clusters, plasmid-borne and chromosome-borne, were also found. Potential mobile genetic elements (MGEs) were identified in the flanking sequences of the ces gene cluster in all three types. The most striking observation was the identification of a putative composite transposon, Tnces, consisting of two copies of ISces element (belonging to IS6 family) in opposite orientations flanking the ces gene cluster in emetic B. Weihenstephanensis. The mobility of this element was tested by replacing the ces gene cluster by a Km R gene marker and performing mating-out transposition assays in Escherichia coli. The results showed that Tnces::km transposes efficiently (1.04 × 10 �3 T/R) and produces 8-bp direct repeat (DR) at the insertion sites. Conclusions: Cereulide biosynthesis gene clusters display sequence diversity, different genomic locations and association with MGEs, in which the transposition capacity of a resistant derivative of the composite transposon Tnces in E. coli was demonstrated. Further study is needed to look for appropriate genetic tools to analysis the transposition of Tnces in Bacillus spp. and the dynamics of other MGEs flanking the ces gene clusters.
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diversity of pulsed field gel electrophoresis patterns of cereulide producing isolates of Bacillus cereus and Bacillus Weihenstephanensis
Fems Microbiology Letters, 2014Co-Authors: Virginie Castiaux, Katelijne Dierick, Elise Nguessan, Izabela Swiecicka, Laurence Delbrassinne, Jacques MahillonAbstract:Bacillus cereus is an important foodborne pathogen causing diarrhoea, emesis and in, rare cases, lethal poisonings. The emetic syndrome is caused by cereulide, a heat‐stable toxin. Originally considered as a rather homogenous group, the emetic strains have since been shown to display some diversity, including the existence of two clusters of mesophilic B. cereus and psychrotolerant Bacillus Weihenstephanensis. Using Pulsed‐Field Gel Electrophoresis (PFGE) analysis, this research aimed to better understand the diversity and spatio‐temporal occurrence of emetic strains originating from environmental or food niches versus those isolated from foodborne cases. The diversity was evaluated using a set of 52 B. cereus and B. Weihenstephanensis strains isolated between 2000 and 2011 in ten countries. PFGE analysis could discriminate 17 distinct profiles (pulsotypes). The most striking observations were: i) more than one emetic pulsotype can be observed in a single outbreak, ii) the number of distinct isolates involved in emetic intoxications is limited and these potentially clonal strains frequently occurred in successive and independent food poisoning cases, iii) isolates from different countries displayed identical profiles, and iv) the cereulide‐producing psychrotolerant B. Weihenstephanensis were, so far, only isolated from environmental niches.
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trustworthy identification of resistance biomarkers of Bacillus Weihenstephanensis workflow of the quality assurance procedure
Food Analytical Methods, 2018Co-Authors: Noemie Desriac, Florence Postollec, Louis Coroller, Sonia Pavan, Jerome Combrisson, Sylvie Halliersoulier, Danièle SohierAbstract:Omics databases have exploded, opening the avenue to take strain diversity or physiological variability into consideration in microbiological risk assessment (MRA). However, one obstacle to the integration of omics data in MRA is the production of quantitative data that may be used to build mathematical models. Gene expression is recognized as relevant biomarker to describe bacterial behavior and reverse transcription quantitative PCR (RT-qPCR) is considered as the gold standard for accurate, sensitive, and fast measurement of gene expression. However, numerous critical points may arise throughout the entire workflow of RT-qPCR data acquisition influencing accuracy of the results and reliability of the conclusions. Although recommendations about the minimum information that should be found in publications about quantitative real-time PCR experiments, heterogeneity in the reporting of RT-qPCR quality controls in publications remains. Herein, the step-by step RT-qPCR quality controls established for the selection of Bacillus Weihenstephanensis resistance biomarkers were described. Throughout this example, appropriate quality procedures and quality controls that shall be set up and carefully assessed to ensure reliable interpretations in RT-qPCR were depicted.
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mrna biomarkers selection based on partial least square algorithm in order to further predict Bacillus Weihenstephanensis acid resistance
Food Microbiology, 2015Co-Authors: Noemie Desriac, Florence Postollec, Louis Coroller, Frederic Jannic, Danièle SohierAbstract:In order to integrate omics data to quantitative microbiological risk assessment in foods, gene expressions may serve as bacterial behaviour biomarkers. In this study an integrative approach encompassing predictive modelling and mRNAs quantifications, was followed to select molecular biomarkers to further predict the acid resistance of Bacillus Weihenstephanensis. A multivariate analysis was performed to correlate the acid bacterial resistance and the gene expression of vegetative cells with or without exposure to stressing conditions. This mathematical method provides the advantage to take gene expressions and their interactions into account. The use of the Partial Least Squares algorithm allowed the selection of nine genes as acid resistance biomarkers among thirty targeted genes. According to their involvement in the general acid stress response of Bacillus, these genes were assigned to three different biological modules namely, metabolic rearrangements, general stress response and oxidative stress response. The oxidative stress response appeared as the major activated biological module in B. Weihenstephanensis cells submitted to acid stress conditions. Furthermore, as a firstly described model, the developed concept showed promising results to further be used to predict bacterial resistance using gene expression. Thus, this study underlines the possibility to integrate the bacterial physiology state, using omics biomarkers, into bacterial behaviour modelling and provide mechanistic understanding in acid bacterial resistance mechanisms.
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modeling the recovery of heat treated Bacillus licheniformis ad978 and Bacillus Weihenstephanensis kbab4 spores at suboptimal temperature and ph using growth limits
Applied and Environmental Microbiology, 2015Co-Authors: Clément Trunet, Florence Postollec, Ivan Leguérinel, Danièle Sohier, Narjes Mtimet, Olivier Couvert, Frederic Carlin, Annegabrielle Mathot, Louis CorollerAbstract: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.
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sensitivity of Bacillus Weihenstephanensis to acidic changes of the medium is not dependant on physiological state
Food Microbiology, 2013Co-Authors: Noemie Desriac, Florence Postollec, Ivan Leguérinel, Danièle Sohier, D Durand, Louis CorollerAbstract: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.
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Prediction of Bacillus Weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers.
International journal of food microbiology, 2013Co-Authors: Noemie Desriac, Florence Postollec, Louis Coroller, Tjakko Abee, Danièle Sohier, H.m.w. Den BestenAbstract: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.
