Population Genetic Structure of Listeria monocytogenes Strains as Determined by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing

Listeria monocytogenes is a ubiquitous bacterium that may cause the foodborne illness listeriosis. Only a small amount of data about the population genetic structure of strains isolated from food is available. This study aimed to provide an accurate view of the L. monocytogenes food strain population in France. From 1999 to 2014, 1,894 L. monocytogenes strains were isolated from food at the French National Reference Laboratory for L. monocytogenes and classified according to the five risk food matrices defined by the European Food Safety Authority (EFSA). A total of 396 strains were selected on the basis of different pulsed-field gel electrophoresis (PFGE) clusters, serotypes, and strain origins and typed by multilocus sequence typing (MLST), and the MLST results were supplemented with MLST data available from Institut Pasteur, representing human and additional food strains from France. The distribution of sequence types (STs) was compared between food and clinical strains on a panel of 675 strains. High congruence between PFGE and MLST was found. Out of 73 PFGE clusters, the two most prevalent corresponded to ST9 and ST121. Using original statistical analysis, we demonstrated that (i) there was not a clear association between ST9 and ST121 and the food matrices, (ii) serotype IIc, ST8, and ST4 were associated with meat products, and (iii) ST13 was associated with dairy products. Of the two major STs, ST121 was the ST that included the fewest clinical strains, which might indicate lower virulence. This observation may be directly relevant for refining risk analysis models for the better management of food safety. IMPORTANCE This study showed a very useful backward compatibility between PFGE and MLST for surveillance. The results enabled better understanding of the population structure of L. monocytogenes strains isolated from food and management of the health risks associated with L. monocytogenes food strains. Moreover, this work provided an accurate view of L. monocytogenes strain populations associated with specific food matrices. We clearly showed that some STs were associated with food matrices, such as meat, meat products, and dairy products. We opened the way to source attribution modeling in order to quantify the relative importance of the main food matrices

Understanding and characterization of physiological mechanisms involved in Lactococcus Lactis reducing activities

Parmi les bactéries lactiques, Lactococcus lactis est la plus utilisée en fabrication fromagère. Actuellement, les ferments lactiques sont majoritairement choisis pour leurs propriétés acidifiantes, protéolytiques et aromatiques. Un autre paramètre majeur est le potentiel redox (Eh). En effet, un Eh réducteur est souvent associé à une bonne qualité aromatique. L’activité réductrice de L. lactis pourrait donc être un nouveau paramètre à prendre en compte dans la maitrise du Eh dans la fabrication des produits laitiers fermentés. Néanmoins, les mécanismes impliqués dans l’activité réductrice de L. lactis demeurent encore inconnus. L’objectif de ce présent travail de thèse a été de les découvrir. Tout d’abord, nous avons développé des milieux de culture gélosé de discrimination redox utilisant des sels de tétrazolium pour cribler une banque de mutants aléatoires de L. lactis. Ceci a permis de démontrer la participation partielle de la chaine de transport d’électrons (Ménaquinones) dans l’activité réductrice de L. lactis. Ensuite, l’approche biochimique nous a permis de déterminer les composés biochimiques principaux contribuant à la diminution du Eh vers des valeurs très réductrices. La présence de groupements thiols exofaciaux est responsable du Eh réducteur atteint par L. lactis. Enfin, l’analyse protéomique utilisant un marquage spécifique des protéines thiols de surface a mis en évidence la présence d’une dizaine de protéines exposant des groupements thiols exofaciaux potentiellement impliquées dans l’activité réductrice de L. lactis. Les thiols sont connus pour être de très puissants antioxydants ce qui confère à L. lactis un intérêt supplémentaire à prendre en considération dans l’élaboration des produits laitiers fermentés.Among the Lactic Acid Bacteria, Lactococcus lactis is the most used in cheese making. Nowadays, starters are used mainly for their acidifying, proteolytic and flavor properties. Another important parameter is the redox potential (Eh). Indeed, reducing Eh is often related to good flavor properties. The reducing activity of L.lactis should be therefore a new parameter to take into account in the monitoring of Eh during dairy fermented products making. Nevertheless, the mechanisms involved in the reducing activity of L.lactis are still unknown. The aim of this work was to understand them. First of all, we have developed tetrazolium salts agar plate media in order to screen a random bank of mutants of L. lactis on their redox capacities. These media allowed us to demonstrate the partial implication of the electron transport chain (Menaquinone) in the reducing activities of L. lactis. Secondly, we have determined the biochemical compounds involved in the decrease of Eh to very reducing values thanks to a biochemical approach. Exofacial thiol groups are mainly responsible for the reducing Eh reached by L.lactis. Lastly, a proteomical analysis using a specific staining of thiols surface proteins revealed the presence of about ten proteins displaying thiols exofacials groups. These proteins might be involved in the reducing activity of L.lactis. Thiols are known to be very strong antioxidants which confer to L. lactis an additional interest to consider in dairy products making

Compréhension et caractérisation des mécanismes physiologiques impliqués dans l'activité réductrice de Lactococcus Lactis

Parmi les bactéries lactiques, Lactococcus lactis est la plus utilisée en fabrication fromagère. Actuellement, les ferments lactiques sont majoritairement choisis pour leurs propriétés acidifiantes, protéolytiques et aromatiques. Un autre paramètre majeur est le potentiel redox (Eh). En effet, un Eh réducteur est souvent associé à une bonne qualité aromatique. L activité réductrice de L. lactis pourrait donc être un nouveau paramètre à prendre en compte dans la maitrise du Eh dans la fabrication des produits laitiers fermentés. Néanmoins, les mécanismes impliqués dans l activité réductrice de L. lactis demeurent encore inconnus. L objectif de ce présent travail de thèse a été de les découvrir. Tout d abord, nous avons développé des milieux de culture gélosé de discrimination redox utilisant des sels de tétrazolium pour cribler une banque de mutants aléatoires de L. lactis. Ceci a permis de démontrer la participation partielle de la chaine de transport d électrons (Ménaquinones) dans l activité réductrice de L. lactis. Ensuite, l approche biochimique nous a permis de déterminer les composés biochimiques principaux contribuant à la diminution du Eh vers des valeurs très réductrices. La présence de groupements thiols exofaciaux est responsable du Eh réducteur atteint par L. lactis. Enfin, l analyse protéomique utilisant un marquage spécifique des protéines thiols de surface a mis en évidence la présence d une dizaine de protéines exposant des groupements thiols exofaciaux potentiellement impliquées dans l activité réductrice de L. lactis. Les thiols sont connus pour être de très puissants antioxydants ce qui confère à L. lactis un intérêt supplémentaire à prendre en considération dans l élaboration des produits laitiers fermentés.Among the Lactic Acid Bacteria, Lactococcus lactis is the most used in cheese making. Nowadays, starters are used mainly for their acidifying, proteolytic and flavor properties. Another important parameter is the redox potential (Eh). Indeed, reducing Eh is often related to good flavor properties. The reducing activity of L.lactis should be therefore a new parameter to take into account in the monitoring of Eh during dairy fermented products making. Nevertheless, the mechanisms involved in the reducing activity of L.lactis are still unknown. The aim of this work was to understand them. First of all, we have developed tetrazolium salts agar plate media in order to screen a random bank of mutants of L. lactis on their redox capacities. These media allowed us to demonstrate the partial implication of the electron transport chain (Menaquinone) in the reducing activities of L. lactis. Secondly, we have determined the biochemical compounds involved in the decrease of Eh to very reducing values thanks to a biochemical approach. Exofacial thiol groups are mainly responsible for the reducing Eh reached by L.lactis. Lastly, a proteomical analysis using a specific staining of thiols surface proteins revealed the presence of about ten proteins displaying thiols exofacials groups. These proteins might be involved in the reducing activity of L.lactis. Thiols are known to be very strong antioxidants which confer to L. lactis an additional interest to consider in dairy products making.DIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Growth potential assessment of listeria in milk fat products by challenge testing.

Milk fat products (MFP), including butter and low-fat dairy spreads, are a specific type of ready-to-eat food known as water-in-fat emulsions, in which the behavior of microbial foodborne pathogens such as Listeria onocytogenes is not clearly known. This study investigated the growth and survival of L. monocytogenes, and of Listeria innocua as a surrogate for L. monocytogenes, in these foods using challenge testing. Three commercial MFPs with various fat contents (butter, half butter and low-fat dairy spread) and two samples of traditional churned butter with various water droplet sizes were artificially contaminated with Listeria. Total mesophilic microflora including lactic acid bacteria, pH and Listeria were monitored throughout the shelf life. The growth potential of Listeria was calculated in the course of the shelf life and remains below the limit value of 0.5 log cfu/g during the whole shelf life in any of thebutter. However, the concentration of Listeria remained stable during the shelf life in the tested MFPs (commercial and churned) except in the commercial low-fat dairy spread in which Listeria decreased gradually

Experimental conditions affect the site of tetrazolium violet reduction in the electron transport chain of Lactococcus lactis

International audienceThe reduction of tetrazolium salts to coloured formazans is often used as an indicator of cell metabolism during microbiology studies, although the reduction mechanisms have never clearly been established in bacteria. The objective of the present study was to identify the reduction mechanisms of tetrazolium violet (TV) in Lactococcus lactis using a mutagenesis approach, under two experimental conditions generally applied in microbiology: a plate test with growing cells, and a liquid test with non-growing (resting) cells. The results showed that in both tests, TV reduction resulted from electron transfer from an intracellular donor (mainly NADH) to TV via the electron transport chain (ETC), but the reduction sites in the ETC depended on experimental conditions. Using the plate test, menaquinones were essential for TV reduction and membrane NADH dehydrogenases (NoxA and/or NoxB) were partly involved in electron transfer to menaquinones. In this case, TV reduction mainly occurred outside the cells and in the outer part of the plasma membrane. During the liquid test, TV was directly reduced by NoxA and/or NoxB, probably in the inner part of the membrane, where NoxA and NoxB are localized. In this case, reduction was directly related to the intracellular NADH pool. Based on these findings, new applications for TV tests are proposed, such as NADH pool determination with the liquid test and the screening of mutants affected in menaquinone biosynthesis with the plate test. Preliminary results using other tetrazolium salts in the plate test showed that the reduction sites depended on the salt, suggesting that similar studies should be carried out with other tetrazolium salts so that the outcome of each test can be interpreted correctly

Screening of lactic acid bacteria for reducing power using a tetrazolium salt reduction method on milk agar.

WOS:000315703100020 ; www.elsevier.com/locate/jbioscInternational audienceReducing activity is a physiological property of lactic acid bacteria (LAB) of technological importance. We developed a solid medium with tetrazolium dyes enabling weakly and strongly reducing LAB to be discriminated. It was used to quantify populations in a mixed culture (spreading method) and screen strains (spot method)

Contribution of exofacial thiol groups in the reducing activity of Lactococcus lactis

International audienceLactococcus lactis can decrease the redox potential at pH 7 (E h7) from 200 to -200 mV in oxygen free Man-Rogosa-Sharpe media. Neither the consumption of oxidizing compounds or the release of reducing compounds during lactic acid fermentation were involved in the decrease in Eh7 by the bacteria. Thiol groups located on the bacterial cell surface appear to be the main components that are able to establish a greater exchange current between the Pt electrode and the bacteria. After the final Eh7 (-200 mV) was reached, only thiol-reactive reagents could restore the initial Eh7 value. Inhibition of the proton motive force showed no effect on maintaining the final Eh7 value. These results suggest that maintaining the exofacial thiol (-SH) groups in a reduced state does not depend on an active mechanism. Thiol groups appear to be displayed by membrane proteins or cell wall-bound proteins and may participate in protecting cells against oxidative stress

Green strategies to control redox potential in the fermented food industry

International audienceLactic acid bacteria (LAB) are important microorganisms in the food industry as functional starters for the manufacture of fermented food products and as probiotics. Redox potential (Eh) is a parameter of the physicochemical environment of foods that influences key oxidation-reduction reactions involved in process performances and product quality. Eh can be modified by different methods, using redox molecules, catalytic activity of enzymes or LAB themselves, technological treatments like electroreduction or heating, and finally gases. Nowadays new applications for food manufacture must undertake green process innovation. This paper presents the strategies for Eh modification in a sustainable manner for production of LAB biomass (starters, probiotics) and fermented food products (fermented milks, cheeses and others). While the use of chemical or enzymes may be subject to controversy, the use of gases offers new opportunities, in combination with LAB. Protection against food-borne microorganisms, an increasing growth and viability of LAB, and a positive impact on food flavour are expected

A procedure for reproducible measurement of redox potential (E h) in dairy processes

International audienceThe oxidoreduction potential (E h) is of growing interest in dairy research and the dairy industry. Lactic acid bacteria are able to decrease the E h to considerable reducing values, and it is an important feature of the technological characteristics of lactic starters and probiotics. Reducing E h is also necessary for the development of the characteristic flavour of certain fermented dairy products such as cheeses. Moreover, data comparisons and interpretations are often difficult due to an absence of standardised expression of the results. The objective of this work was to propose a complete procedure for the use of combined and half-cell platinum (Pt) sensors to obtain stable, repeatable and reproducible E h measurements and to standardise data. Three measurement systems, each made up of a sensor, cable and interface, were compared in tap water. A control chart was used to calculate a confidence interval (CI = 20 mV) to verify whether the process was under control. This complete procedure was then applied to the measurement of E h in milk samples to characterise both pure strains of lactic acid bacteria and complex mixed starters, to study the impact of a process parameter (oxygen) on starter activity, to follow E h during cheese making and to measure E h gradients in cheeses. This manuscript presents a procedure for the measurement of E h as well as the feasibility and interest of this procedure in the dairy chain