This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Alicyclobacillus acidoterrestris is a sporulating, acidophilic bacterial species which spoils aci... more Alicyclobacillus acidoterrestris is a sporulating, acidophilic bacterial species which spoils acidic beverages such as fruit juices. This work aims to quantify the heat resistance of Alicyclobacillus acidoterrestris spores and their recovery potential as a function of heating and recovery media pH. Methods and Results The heat treatments were carried out with the strain of Alicyclobacillus acidoterrestris Ad 746 in Bacillus acidoterrestris thermophilic (BAT) medium. The pH of the heating medium from pH 7 to pH 2 non significantly reduced the heat resistance. However, the pH levels of the recovery media strongly affected the apparent heat resistance of this strain. The maximum heat resistance was found when the pH was 4.70 and decreased when the pH decreased to pH 2.8, close to the minimum growth pH and when the recovery medium pH increased to pH 5.3. Conclusion The heating medium pH has a slight effect on the spore heat resistances of this acidophilic specie. However, the pH of the recovery media strongly affected the apparent heat resistance of this strain. Significance and impact of the study. The obtained parameters quantifying the heat resistance of Alicyclobacillus acidoterrestris spores are tools to optimize the heat treatments and to control its development.
Large-scale multivariate dataset on the characterization of microbiota diversity, microbial growt... more Large-scale multivariate dataset on the characterization of microbiota diversity, microbial growth dynamics, metabolic spoilage volatilome and sensorial profiles of two industrially produced meat products subjected to changes in lactate concentration and packaging atmosphere.
HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Omics databases have exploded, opening the avenue to take strain diversity or physiological varia... more 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.
The capacity of two primary growth models to describe Pseudomonas fluorescens and Pseudomonas gri... more The capacity of two primary growth models to describe Pseudomonas fluorescens and Pseudomonas grimontii biofilms' development was assessed. The Baranyi and the 'Logistic with Breaking Delay' models were applied for biofilms grown in various laboratory and pilot-plant devices, including pipes or mock-ups mimicking vegetables washing tanks in the fresh-cut food industry. An initial short transitional period not described by the growth models was observed during which cells rapidly attached to pilot-plant devices' surfaces. The following observed surface contamination growth patterns were consistent with both growth models. However, only the Baranyi model was relevant to the occurrence of wide variability and/or growth curves with no lag or stationary phases. Both surface design and hydrodynamics in pilot-plant devices strongly influenced biofilm growth curves. Based on fitted parameters, it was possible to differentiate between areas and relate these to design parameters such as sharp corners, welds or specific hydrodynamics as 2D, 3D or near-static flow conditions. 15-25 h lag phases reflecting equilibrium between cell attachment and release were observed for wall shear stresses exceeding 0.5 Pa under dynamic flow conditions. Consequently, flow pattern design improvements by increasing the shear rate would greatly reduce food cross-contamination risk, as cleaning operations could start before any significant biofilm developments.
The heat resistance of the bacterial spores of Moorella thermoacetica, Clostridium sporogenes, Ge... more The heat resistance of the bacterial spores of Moorella thermoacetica, Clostridium sporogenes, Geobacillus stearothermophilus and Bacillus coagulans was determined over a wide range of temperatures using the capillary method and thermoresistometer Mastia. The results showed that the two experimental methods gave similar heat resistance values excepted for Geobacillus stearothermophilus. The effect of temperature on thermal resistance was evaluated using the Arrhenius and Bigelow models. The fit of the heat sensitivity parameters of the Arrhenius and Bigelow models on the heat resistance parameter values obtained over a wide temperature range was equally good. Despite the apparent mathematical incompatibility of the two equations, it is recognized that they yield the same goodness of fit. This paper finds a mathematical reason for this convergence and explains why inside a temperature range of at least 100°C, no significant difference in the quality of fit between these two models can be found. * * D D T T z log log. (3) Concerning the kinetics of inactivation, in 1943 Katzin et al. quantified the heat resistance by the parameter D, which is the decimal reduction time (Katzin et al., 1943). In 2002, Mafart and collaborators quantified the heat resistance of bacteria when the inactivation kinetics is not of order 1. The Weibull model was re-parameterized using the
Microorganisms are able to adapt to different environments and evolve rapidly, allowing them to c... more Microorganisms are able to adapt to different environments and evolve rapidly, allowing them to cope with their new environments. Such adaptive response and associated protections toward other lethal stresses, is a crucial survival strategy for a wide spectrum of microorganisms, including food spoilage bacteria, pathogens, and organisms used in functional food applications. The growing demand for minimal processed food yields to an increasing use of combination of hurdles or mild preservation factors in the food industry. A commonly used hurdle is low pH which allows the decrease in bacterial growth rate but also the inactivation of pathogens or spoilage microorganisms. Bacillus cereus is a well-known food-borne pathogen leading to economical and safety issues in food industry. Because survival mechanisms implemented will allow bacteria to cope with environmental changes, it is important to provide understanding of B. cereus stress response. Thus this review deals with the adaptive ...
This study aims at the characterisation of growth behaviour of three strains of Bacillus amyloliq... more This study aims at the characterisation of growth behaviour of three strains of Bacillus amyloliquefaciens, isolated from ropy bread (ATCC8473), wheat grain (ISPA-S109.3) and semolina (ISPA-N9.1) to estimate rope spoilage risk in pan bread during shelf-life using the Sym'Previus tool. Cardinal values and growth/no growth boundaries were determined in broth, while artificial spore inoculations were performed in dough for various pan bread recipes to compare experimental counts with in silico growth simulations. Finally, two storage scenarios were tested to determine the probability to reach a spoilage threshold during bread shelf-life. Similarly to the safety criteria fixed for L. monocytogenes contamination in foodstuff complying with EC regulation, a potential rope spoilage threshold was arbitrary fixed at 5 log CFU/g for B. amyloliquefaciens. This study further underlines a higher rope spoilage potential of the ISPA strains as compared to the ATCC strain, thus emphasizing the interest to characterise both wild strains and reference strain to account for biological variability. In conclusion, this study showed that available decision making tools which are largely recognized to predict behaviour of pathogenic strains, shall also be used with spoilage strains to help maintain food quality and extend shelf-life.
Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-re... more 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.
This study aims to quantify the effect of salt and acid preliminary exposure on acid resistance o... more 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 midexponentially 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 (z pH). 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.
The food industry widely uses the F-value which considers microbial log-linear inactivation, whil... more 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.
Microorganisms are able to adapt to different environments and evolve rapidly, allowing them to c... more Microorganisms are able to adapt to different environments and evolve rapidly, allowing them to cope with their new environments. Such adaptive response and associated protections toward other lethal stresses, is a crucial survival strategy for a wide spectrum of microorganisms, including food spoilage bacteria, pathogens, and organisms used in functional food applications. The growing demand for minimal processed food yields to an increasing use of combination of hurdles or mild preservation factors in the food industry. A commonly used hurdle is low pH which allows the decrease in bacterial growth rate but also the inactivation of pathogens or spoilage microorganisms. Bacillus cereus is a well-known food-borne pathogen leading to economical and safety issues in food industry. Because survival mechanisms implemented will allow bacteria to cope with environmental changes, it is important to provide understanding of B. cereus stress response. Thus this review deals with the adaptive ...
Convergence of Bigelow and Arrhenius models over a wide range of heating temperatures
International Journal of Food Microbiology
The heat resistance of the bacterial spores of Moorella thermoacetica, Clostridium sporogenes, Ge... more The heat resistance of the bacterial spores of Moorella thermoacetica, Clostridium sporogenes, Geobacillus stearothermophilus and Bacillus coagulans was determined over a wide range of temperatures using the capillary method and thermoresistometer Mastia. The results showed that the two experimental methods gave similar heat resistance values excepted for Geobacillus stearothermophilus. The effect of temperature on thermal resistance was evaluated using the Arrhenius and Bigelow models. The fit of the heat sensitivity parameters of the Arrhenius and Bigelow models on the heat resistance parameter values obtained over a wide temperature range was equally good. Despite the apparent mathematical incompatibility of the two equations, it is recognized that they yield the same goodness of fit. This paper finds a mathematical reason for this convergence and explains why inside a temperature range of at least 100 °C, no significant difference in the quality of fit between these two models c...
Identification d'ARNm en tant que biomarqueurs de résistance acide chez Bacillus weihenstephanensis : vers une intégration de données transcriptomiques dans la prévision du comportement bactérien
In order to integrate omics data to quantitative microbiological risk assessment in foods, gene e... more 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.
In this study, species taxonomy, heat resistance and growth potential of mesophilic aerobic spore... more In this study, species taxonomy, heat resistance and growth potential of mesophilic aerobic sporeforming Bacillus strains isolated from commercially available couscous were determined. Methods and results: Aerobic spore-forming bacteria were isolated from three Algerian retail couscous samples heated at 80 C for 10 min. Plate counting of spore-forming bacteria showed a mean concentration of 20 CFU/g. By monitoring 16S gene sequencing, ten Bacillus strains were identified, belonging to 3 different species: five Bacillus licheniformis strains, four Bacillus cereus group strains sensu lato and one Bacillus subtilis strain. According to the panC gene sequencing, the four B. cereus strains were assigned to the group IV (mesophilic and heat resistant group, associated with cases of foodborne illness). B. cereus cells growth kinetics in moistened couscous semolina showed a specific growth rate of 0.33 h À1 at 30 C, confirming their growth ability in this media. The heat resistance (d value i.e. the first decimal reduction time) and heat sensitivity (z T values i.e. the temperature increase leading a tenfold reduction of the d value) of spores of B. cereus and B. subtilis strains were determined using Weibull and Bigelow models, respectively. d 100 C values are ranged from 0.14 to 7.90 min and estimated z T values ranged from 7.52 C to 10.38 C. Moreover, the estimate four decimal reduction times at 90 C (t4D) of spores of isolated B. cereus strains were from 0.58 h to 3.73 h. Conclusions: B. cereus strains isolated from retail packaged couscous semolina are resistant to heat treatment both during the industrial food process and can easily grow in moistened couscous semolina. These observations could explain numerous B. cereus outbreaks associated with couscous semolina consumption.
The aim of this study was to define an integrative approach to identify resistance biomarkers usi... more The aim of this study was to define an integrative approach to identify resistance biomarkers using gene expression quantification and mathematical modelling. Mid-exponentially growing cells were transferred into acid conditions (BHI, pH 4.6) to obtain inactivation kinetics, performed in triplicate. The inactivation curve was fitted with a mixed Weibull model. This model allowed to differentiate two subpopulations with various acid resistances among the initial population. In parallel, differential gene expression was quantified by RT-qPCR. While narL was down-regulated throughout acid inactivation, sigB and katA were up-regulated. sigB expression up-regulation peak was correlated to the less resistant subpopulation when katA up-regulation, was correlated to the more resistant subpopulation. Moreover, differences in population structure were highlighted between each replicate. The higher proportion of the more resistant subpopulation was linked to a higher katA gene expression. These results suggest that sigB and katA might be used as different types of biomarkers, for instance to track moderate and high acid-resistance, respectively. The use of this approach combining RT-qPCR and predictive modelling to track cellular biomarker variations appears as an interesting tool to take into account physiological cell responses into mathematical modelling, allowing an accurate prediction of microbial behaviour.
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