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Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

Antibiotic resistance is one of the most important issues facing modern medicine. Some biocides have demonstrated the potential of selecting resistance to antibiotics in bacteria, but data are still very scarce and it is important to better identify the molecules concerned and the underlying mechanisms. This study aimed to assess the potential of polyhexamethylene biguanide (PHMB), a widely used biocide in a variety of sectors, to select antibiotic resistance in Escherichia coli grown in biofilms. Biofilms were grown on inox coupons and then exposed daily to sublethal concentrations of PHMB over 10 days. Antibiotic-resistant variants were then isolated and characterized phenotypically and genotypically to identify the mechanisms of resistance. Repeated exposure to PHMB led to the selection of an E. coli variant (Ec04m1) with stable resistance to gentamycin (8-fold increase in minimum inhibitory concentration (MIC) compared to the parental strain. This was also associated with a significant decrease in the growth rate in the variant. Sequencing and comparison of the parental strain and Ec04m1 whole genomes revealed a nonsense mutation in the aceE gene in the variant. This gene encodes the pyruvate dehydrogenase E1 component of the pyruvate dehydrogenase (PDH) complex, which catalyzes the conversion of pyruvate to acetyl-CoA and CO2. A growth experiment in the presence of acetate confirmed the role of this mutation in a decreased susceptibility to both PHMB and gentamicin (GEN) in the variant. This work highlights the potential of PHMB to select resistance to antibiotics in bacteria, and that enzymes of central metabolic pathways should be considered as a potential target in adaptation strategies, leading to cross-resistance toward biocides and antibiotics in bacteria.

In this contribution, the antimicrobial susceptibility toward 11 antibiotics and four biocides of a panel of 205 Listeria monocytogenes (Lm) strains isolated from different ecological niches (i.e., food, animals and natural environment) was evaluated. The impact of exposure to biocides on the antibiotic susceptibilities of Lm was also investigated. Lm strains isolated from food exhib-ited overall a lower susceptibility (higher minimal inhibitory concentrations, MIC) for ammonium quaternary compounds (QACs) and peracetic acid (PAC) than strains isolated from animals and natural environments. Conversely, the ecological origins of Lm strains did not significantly affect their susceptibilities towards antibiotics. Interestingly, repeated exposure to QACs recurrently led to a decrease in susceptibility toward ciprofloxacin (CIP), a fluoroquinolone antibiotic, largely used in human medicine. Moreover, these lower levels of susceptibility to CIP remained stable in most Lm strains even after subcultures without biocide selection pressure, suggesting an adaptation involving modifications at the genetic level. Results underlined the ability of Lm to adapt to biocides, especially QACs, and the potential link between this adaptation and the selection of resistance toward critical antibiotics such as ciprofloxacin. These data support a potential role of the extensive use of QACs from “farm to fork” in the selection of biocide and antibiotic resistance in pathogenic bacteria such as Lm.

International audience

International audience

International audience; This volume presents a compilation of various representative techniques and approaches currently used to study bacterial foodborne pathogens. Chapters guide the reader through bacterial pathogen detection and quantification in food, molecular, phenotypic, metabolic characterization of food pathogens, and ecology of foodborne bacterial pathogens. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.Authoritative and cutting-edge, Foodborne Bacterial Pathogens : Methods and Protocols aims to server as a guide both for researchers, students, and those in the food industry who want to have an overview of current approaches and protocols used to study bacterial foodborne pathogens.

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Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the xyl-doc cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics., This work was supported by a grant [R2DS 2010-08] from Conseil Regional d'Ile-de-France through DIM R2DS programs (http://www.r2ds-ile-de-france.com/). Irstea (www.irstea.fr/) contributed to the funding of a PhD grant for the first author. The funders provided support in the form of salaries for author [NB], funding for consumables and laboratory equipment, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Omics Services provided support in the form of salaries for authors [VS, MD], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors [NB, VS, MD] are articulated in the 'author contributions' section., info:eu-repo/semantics/publishedVersion

A method for controlling the activity of an electrochemical device (11) comprising a bioanode (3) and a biocathode (6) immersed in an electrolyte (10A, 10C) containing microorganisms, the anode compartment (8) and cathode compartment (9) being separated by at least one membrane (14), optionally a reference electrode, a difference in potential being applied between the bioanode (3) and the biocathode (6), or between the bioanode and the reference electrode, characterised in that the operation of the device is governed by a dual control: - a priority control of the difference in potential between the bioanode and the biocathode, or between the bioanode and the reference electrode, between a minimum limit value allowing the development of an electroactive biofilm at the bioanode and a maximum limit value lower than the oxidation potential of said biofilm, and - a secondary control, when the first control is in place, optimising the Faradaic efficiency of the biocathode. A device allowing said method to be carried out., Procédé de régulation de l'activité d'un dispositif électrochimique (1 1 ) comportant une bioanode (3) et une biocathode (6) plongées dans un électrolyte (10A,10C) contenant des microorganismes, les compartiments anodique (8) et cathodique (9) étant séparés par au moins une membrane (14), éventuellement une électrode de référence, une différence de potentiel étant appliquée entre la bioanode (3) et la biocathode (6), ou entre la bioanode et l'électrode de référence, caractérisé en ce que le fonctionnement du dispositif est régi par une double régulation : - une régulation prioritaire de la différence de potentiel entre la bioanode et la biocathode, ou entre la bioanode et l'électrode de référence, entre une valeur limite minimale permettant le développement d'un biofilm électroactif à la bioanode et une valeur limite maximale inférieure au potentiel d'oxydation dudit biofilm, et - une régulation secondaire, lorsque la première régulation est mise en place, optimisant le rendement faradique de la biocathode. Dispositif permettant la réalisation dudit procédé.

Procédé de régulation de l'activité d'un dispositif électrochimique comportant une bioanode et une biocathode plongées dans un électrolyte (10A, 10C) contenant des microorganismes, les compartiments anodique et cathodique étant séparés par au moins une membrane, éventuellement une électrode de référence, une différence de potentiel étant appliquée entre la bioanode et la biocathode, ou entre la bioanode et l'électrode de référence, caractérisé en ce que le fonctionnement du dispositif est régi par une double régulation : -une régulation prioritaire de la différence de potentiel entre la bioanode et la biocathode, ou entre la bioanode et l'électrode de référence, entre une valeur limite minimale permettant le développement d'un biofilm électroactif à la bioanode et une valeur limite maximale inférieure au potentiel d'oxydation dudit biofilm, et -une régulation secondaire, lorsque la première régulation est mise en place, optimisant le rendement faradique de la biocathode. Dispositif permettant la réalisation dudit procédé.

International audience; Bioelectrochemical systems (BES) as microbial fuel cells take advantages of microorganisms to convert the chemical energy of organic waste into electricity. Recently, the discovery that BES can also be used for the synthesis of biocommoditiesvia microbial electrosynthesis (MES) has greatly expanded the horizons for their applications. Indeed, some microbes are able to use electrons and molecules such as CO2 to synthesize reduced products: volatile fatty acids, alcohols etc... By combining both these processes, it should thus theoretically be possible to use the electrons of organic waste to synthesize bio-based chemicals in a clean and controlled compartment. However, these technologies are only few years old and required scientific data before they can be practically applied. In this context, we developed a dual-chamber reactor with both biotic anode (carbon cloth) and cathode (stainless steel) separated by a cation-exchange membrane. Bioanode was inoculated using an anodic biofilm sample formed in biological wastes and biocathode by injecting a suspension of a homoacetogen-enriched culture. Acetate (600mg/l) was used as electron donor in the anodic compartment. Chronoamperometry experiments were carried out with a multi-channel potentiostat in order to monitor electroactivity of the microbial communities. Anode potential was poised at +0.158 volts versus saturated calomel electrode (SCE) for startup. Chemical analyses (volatile fatty acids (VFAs), cations/anions, chemical oxygen demand, and total organic carbon) were performed to evaluate the metabolic pathways. Microbial community diversity was investigated by 16S rDNA pyrosequencing (MiSeq sequencer, Illumina®). After the total consumption of acetate at anode (run 1), a second run (run 2) was launched by re-injecting 600 mg/L of acetate in anodic compartment and also 2-bromo-ethane sulfonate (2-BES) at the cathode to inhibit methanogenesis. Current density of 5 A/m² was reached after 24h of experiment. During run 1 and 2 78% and 89% of electrons from acetate were respectively transferred in the system revealing satisfying coulombic efficiency at the bioanode. During run 1, incoming electrons at biocathode were mainly used to produce methane (53% of total incoming electrons) and only traces of VFAs were detected. However, at the end of the second run, VFAs accumulated with a production rate of acetate reaching 11 g.m².d-1 and corresponding to 29% of the electrons coming from the anode. Using gas chromatography coupled with mass spectrometry, caprylate (C8H16O2) was also detected in reactors showing the ability of the MES system to produce molecules with an elongated carbon chain. Microbial diversity profiles showed a switch of archaeal and bacterial populations in cathodic compartment between run 1 and run 2 suggesting that VFAs production resulted from microbial adaptation to the addition of BES in the cathodic compartment. Overall this work constitutes a first step toward the utilization of MES systems for the conversion of organic wastes into biofuels and chemicals using coupled bioanode and biocathodes

International audience; In natural and man-made environments, biofilms are known to be complex associations of microorganisms which development is controlled by a network of bacterial interactions. As biofilm is a dynamic environment, these interactions evolve over time, often illustrating the adaptation of the whole bacterial community to its environmental conditions on one hand and of the bacterial community members to each other on the other hand. From this process the structure and the functional properties of the whole biofilm finally emerges. Genetic diversification and selection of variants with increased fitness in the conditions experienced is one of the known mechanisms enabling the adaptation of species to the biofilm way of life. Therefore, a better understanding of the emergence of this genetic diversity and overall the dynamic mechanisms of adaptation of bacterial species in biofilm is thus a first step towards the managing of ecosystem functions and evolution. In this aim, we focused in this work on the adaptation of the model strain Pseudomonas putida KT2440 (GFP-tagged) in mixed biofilm with another strain from the same species (P. putida mCherry-tagged) more recently isolated from roots of plants. We first analysed the emergence of colony morphology variants of Pseudomonas putida KT2440 GFP in pure culture or in mixed biofilms with the P. putida mCherry strain. Three colonies variants of P. putida KT2440 GFP (2000 colonies screened) were obtained from the mixed biofilm of both P. putida strains after 168h of growth whereas no variant was identified from the axenic P. putida KT2440 biofilm on the basis of colony morphology. The three variants were then phenotypically characterized (growth rate, cell surface properties, swimming, swarming, macrocolonies) and their ability to form biofilms alone or with the P. putida mCherry strain was analysed using 3D confocal microscopy. Variants exhibited distinct phenotypes and produced biofilm with specific architecture in comparison with the ancestor. In the mixed biofilms, KT2440 variants also better competed with the P. putida mCherry strain compared to the ancestor. The mix of the three variants and ancestor produced biofilm with high biovolume and has a better ability to compete the P. putida PCL1480 mCherry strain illustrating the synergistical effect of strain association in biofilm production and thus in the adaptation and survival of the whole P. putida KT2440 GFP population. To sum up, this work provides evidence that an intra-specific competition can foster the emergence of phenotypic diversification which might increase the global fitness of the resulting population and highlights the importance of bacterial interactions in the evolution and adaptation of microbial communities.

International audience; Bioelectrochemical systems (BES) as microbial fuel cells take advantages of microorganisms to convert the chemical energy of organic waste including wastewaters and lignocellulosic biomass into electricity or hydrogen/chemical products. Recently, the discovering that BES can also be harness for product synthesis via microbial electrosynthesis (MES) has greatly expanded the horizon for these systems. MES is a promising strategy which consists in the direct powering of microbial activity with electricity to catalyze conversion of carbon dioxide to transportation fuels and other organic commodities. However, the MES concept is only few years old and scientific data about the functioning and the structure of microbial communities involved are required. In this context, we focused here on the study of communities developed in a BES system. For that, we constructed a dedicated system composed of dual-chamber reactor separated by an anion-exchange membrane and inoculated with biological sludges. Cathode potential was poised at -0.65 volts versus saturated calomel electrode (SCE). Chronoamperometry and cyclic voltammetry experiments were carried out with a multi-channel potentiostat in order to monitor electric activity of the microbial communities. Dynamics of population were achieved by ARISA during experiment and community diversity was investigated at specific time point of interest by sequencing 16S rDNA in bulk phases and in biofilm on the electrodes. Biofilm structures were also visualized using confocal microscopy. Chemical analyses were performed to highlight the metabolic pathways involved. Results showed that current reached intensity of 100 mA/ m². ARISA diversity profiles evolved during the experiment duration showing an adaptation of microbial communities. Pyrosequencing of the 16S rDNA genes revealed specific diversity profiles on the electrodes compared to the bulk. In particular, we observed that Dechloromonas sp. constitute de dominant species (~28% of the total diversity) at the anode whereas this species did not reach more than 1.5% in the bulk and at the cathode. The link between current production, chemical molecules and microbial species diversity is thus discussed. In conclusion, this contribution provides data on the structure and composition of electroactive communities which are crucial for the understanding, the development and the optimization of BES systems.

International audience; Biofilms are structured communities of microorganisms and are now recognized as the prevailing mode of life of bacteria in natural and man-made environments. Bacterial biofims participate therefore to a large number of beneficial ecological processes but can also have a considerable negative impact on human well-being throughout their involvement in biodeterioration or biofouling processes and even by causing serious infectious diseases. The functional roles of biofilms leading to these positive or negative impacts are intimately related to the spatial architecture and organization of the community. Since biofilms are recognized as complex associations of numerous different species in our environments, a better deciphering of the nature of the interactions between bacterial populations become an important ecological parameter required to better understand, predict and control biofilm functions. Individuals of two different bacterial species can interact in biofilms in different ways. They may compete for ressources and space, just as conspecific individuals do. They may also interact in more complex ways, in which intra-specific interactions differ from inter-specific ones. For instance, different species may share different common goods or have complementary feeding needs. We are here interested in these last types of interactions, which modify the dynamics of each species in the presence of the other. These interactions make the coupled dynamics of the two species different from the juxtaposition of the two monospecific dynamics, so that it should be possible to detect these by the careful analysis of biofilm growth patterns. In this aim, mono-species and bi-species biofilms were grown under flow in three-channel flow-cells. 3D biofilm structure was then scanned every hour during biofilm development using time-lapse confocal laser scanning microscopy combined to genetically modified bacterial strains expressing constitutively green or red fluorescent protein (GFP or mCherry). We built a simple individual-based model of biofilm development, where intra-specific interactions did not differ from inter-specific ones. We developed a statistical method to parameterize the different processes represented in the biofilm model (growth, mobility, attachment and detachment) using our temporal series of biofilm images obtained by confocal microscopy. By setting the model directly with these experimental data, we limit the number of hypothesis that we formulated and therefore, we limit the bias caused by these hypothesis in the model. This parameterization relied on the comparison between the structural patterns observed during biofilm development in vitro and in silico. Namely, biofilm structural patterns were quantified thanks to a number of summary statistics (number of clusters and the first four moments of the cluster size distribution) at the different time points both in the simulations and in the experimental images. And model parameter values with which model simulations best fit empirical observations were retained. We demonstrate in this contribution that our simple model of biofilm growth can be parameterized with temporal series of biofilm images, and that it satisfactorily represents the biofilm growth dynamics observed in our experiments. We finally demonstrate how to use this simple model of biofilm growth as a null model to detect non-trivial interactions between pairs of bacterial species.

International audience; In natural and man-made environments, biofilms are known to be complex associations of microorganisms which development is controlled by a network of bacterial interactions. As biofilm is a dynamic environment, these interactions evolve over time, often illustrating the adaptation of the whole bacterial community to its environmental conditions on one hand and of the bacterial community members to each other on the other hand. From this process the structure and the functional properties of the whole biofilm finally emerges. Genetic diversification and selection of variants with increased fitness in the conditions experienced is one of the known mechanisms enabling the adaptation of species to the biofilm way of life. Therefore, a better understanding of the emergence of this genetic diversity and overall the dynamic mechanisms of adaptation of bacterial species in biofilm is thus a first step towards the managing of ecosystem functions and evolution. In this aim, we focused in this work on the adaptation of the model strain Pseudomonas putida KT2440 (GFP-tagged) in mixed biofilm with another strain from the same species (P. putida mCherry-tagged) more recently isolated from roots of plants. We first analysed the emergence of colony morphology variants of Pseudomonas putida KT2440 GFP in pure culture or in mixed biofilms with the P. putida mCherry strain. Three colonies variants of P. putida KT2440 GFP (2000 colonies screened) were obtained from the mixed biofilm of both P. putida strains after 168h of growth whereas no variant was identified from the axenic P. putida KT2440 biofilm on the basis of colony morphology. The three variants were then phenotypically characterized (growth rate, cell surface properties, swimming, swarming, macrocolonies) and their ability to form biofilms alone or with the P. putida mCherry strain was analysed using 3D confocal microscopy. Variants exhibited distinct phenotypes and produced biofilm with specific architecture in comparison with the ancestor. In the mixed biofilms, KT2440 variants also better competed with the P. putida mCherry strain compared to the ancestor. Mixed biofilms of 2 or the 3 variants and ancestor with the P. putida mCherry strain are currently under evaluation to highlight potential synergistic effect resulting from the strain association. To sum up, this work provides evidence that an intra-specific competition can foster the emergence of phenotypic diversification which might increase the global fitness of the resulting population and highlights the importance of bacterial interactions in the evolution and adaptation of microbial communities.

In natural, industrial or medical environments, microorganisms are present mainly in being associated with surfaces in highly organized communities called biofilms. These biological structures cosntitute a surprisingly effective survival strategy showing a large ability to withstand environmental stresses such as cleaning and disinfection treatments. Therefore, biofilms have a considerable impact on public health because they allow the persistence and transmission of pathogens. In this context, this work aimed to better understand the phenomena limiting the effectiveness of disinfectants in biofilms noticeably by using innovative imaging fluorescence non-invasive techniques. The ultimate goal was to provide data which can help to optimize disinfection treatments. In the first part, a high-throughput structural method based on confocal microscopy was developed and used to study the architectural diversity of bacterial biofilms formed by a wide range of strains. This study allowed us to identify strains of interest in terms of biofilm structure for the second part of the work. In particular, we demonstrated the ability of B. subtilis to form protruding structures with a specific architecture in a submerged system. In the second part, the spatiotemporal dynamic of the action of disinfectants were visualized in the biofilms of P. aeruginosa or B. subtilis strains by a time-lapse fluorescence confocal microscopy method. Using this technique, we showed that benzalkonium chloride encountered problems of penetration in the biofilms formed by P. aeruginosa strains. The correlation of kinetic inactivation parameters and data obtained by the characterization biochemical matrix suggested a key role of extracellular substances in the penetration limitations of the disinfectant. We also observed a pronounced resistance of the biofilm formed by a strain of B. subtilis isolated from a medical device to peracetic acid at the in-use concentration and time of biocide in medical areas. In addition, three-dimensional structures formed by this strains afforded protection to the pathogen Staphylococcus aureus in mixed biofilm against the same treatment This point highlights the importance of multi-species interactions in bacterial resistance to disinfectants and in the persistence of pathogens in our environments.; Dans les environnements naturels, industriels ou médicaux, les microorganismes sont majoritairement présents en étant associés aux surfaces dans des communautés hautement organisées appelées biofilms. Ces édifices biologiques constituent une stratégie de survie étonnement efficace témoignant d’une grande capacité de résistance à différent stress environnementaux tels que les traitements de nettoyage et de désinfection. L’impact des biofilms d’un point de vue sanitaire est donc considérable du fait qu’ils permettent la persistance et la transmission de germes pathogènes dans l’environnement. Dans ce contexte, ce travail de thèse avait pour objectif une meilleure compréhension des phénomènes limitant l’efficacité de désinfectants au sein des biofilms en s’appuyant notamment sur des techniques innovantes d’imagerie de fluorescence non-invasive. Le but final étant d’apporter des éléments utiles à l’optimisation des traitements de désinfection. Dans une première partie, une méthode d’investigation structurale à haut-débit par microscopie confocale a été développée et utilisée pour étudier la diversité architecturale des biofilms bactériens formés par un large panel de souches. Cette étude nous a permis d’identifier des souches d’intérêt en termes de structures de biofilms formés pour la suite du travail. Nous avons notamment pu mettre en évidence la capacité de B. subtilis à former des structures importantes et avec une architecture spécifique dans un système immergé. Dans une deuxième partie, les dynamiques d’action spatiotemporelles de désinfectants ont été visualisées dans les biofilms de souches de P. aeruginosa ou B. subtilis par des approches de microscopie confocale de fluorescence en temps réel. L’utilisation de cette technique nous a permis de mettre en évidence les difficultés de pénétration du chlorure de benzalkonium au sein des structures formées par différentes souches de P. aeruginosa. La corrélation des paramètres cinétiques d’inactivation et des données obtenues par la caractérisation biochimique de la matrice suggère un rôle majeur des substances extracellulaires dans la limitation de pénétraton du désinfectant. Nous avons également pu montrer une résistance marquée du biofilm formé par une souche de B. subtilis isolée d’un dispositif médical à l’acide péracétique, à la concentration et au temps d’utilisation du biocide dans le milieu médical. De plus, les structures tridimensionnelles formées par cette souche étaient capables de protéger le pathogène Staphylococcus aureus dans un biofilm mixte vis-à-vis du même traitement soulignant l’importance des interactions multi-espèces dans la résistance des bactéries aux désinfectants et la persistance de pathogènes dans nos environnements.