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Pathogenic bacteria and their biofilms are involved in many human and animal diseases and are a major public health problem with, among other things, the development of antibiotic resistance. These biofilms are known to induce chronic infections for which classical treatments using antibiotic therapy are often ineffective. Sponges are sessile filter-feeding marine organisms known for their dynamic symbiotic partnerships with diverse microorganisms and their production of numerous metabolites of interest. In this study, we investigated the antibiofilm efficacy of different extracts from sponges, isolated in Wallis, without biocidal activity. Out of the 47 tested extracts, from 28 different genera, 11 showed a strong activity against Vibrio harveyi biofilm formation. Moreover, one of these extracts also inhibited two quorum-sensing pathways of V.harveyi.

The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota, and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands.

Lactobacillus paracasei FT700 was tested for its survival in simulated gastrointestinal conditions ( in vitro ) and for its sensitivity to antibiotics. The strain was used to produce fermented milk and the cell-free supernatant was freeze-dried and applied in cell cultures (monocytes and macrophages) to evaluate its cytotoxicity and immunomodulatory effects. After 180 min at pH 2.0 or in simulated gastric juice, L. paracasei FT700 concentrations were reduced by 4 and 3 log cfu/mL, respectively. Moreover, the strain was susceptible to the majority of antibiotics tested. Freeze dried cell-free fermented milk supernatant (FMS) was toxic to monocytes after 72 h of exposure at 10 mg/mL inducing their apoptosis. However, at concentrations below toxic level, FMS stimulated the differentiation of monocytes into macrophages, as observed by the increased expression of CD71 marker. This immune stimulation was not inflammatory since low production of TNF-α was observed. Overall, L. paracasei FT700 has potential to be used as probiotic in functional fermented milk.

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2017.02.013; The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensisK.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifyingtheir organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the actionmechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability,membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10 days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3 days incubation in control supernatant,while itwas prevented for up to 10 days by the active supernatant. Interestingly, between 66 and 99% of yeast cellswere under a viable but non-cultivable (VNC) state despite an absence ofmembrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products.

This study was carried out to screen for antimicrobial activities against common pathogenic and food spoilage bacteria, yeasts and molds, toxinogenic potential and sensitivity to antibiotics of 26 Bacillus strains (11 Bacillus subtilis, 5 Bacillus megaterium, 4 Bacillus amyloliquefaciens, 4 Bacillus pumilus and 2 Bacillus thuringiensis) isolated from different samples of Hibiscus sabdariffa fermented seeds (Mbuja). Antimicrobial assays were realised according to the cross-streak and the overlay methods. The main toxin and bacteriocin encoding genes were screened by PCR using specific primers and antibiotic resistance was assessed by the disc diffusion method. Bacillus species showed variable ability to inhibit bacterial and/or fungal species. The most antibacterial strains were B. amyloliquefaciens (S1 and S5) and B. subtilis (S12) whereas the most antifungal ones were B. megaterium S8 and S9. Subtilin and subtilosin A genes were detected in seven strains of B. subtilis but they were not associated with antimicrobial activities. All strains, except B. thuringiensis, did not contain toxin encoding genes and were sensitive to most tested antibiotics. This suggests that antimicrobial strains could be used in starter cultures for a controlled fermentation to produce Mbuja in order to better control the fermentation process of Mbuja and to increase consumer’s safety.Keywords: Bacillus, antimicrobial activities, toxins, antibiotic resistanceAfrican Journal of Biotechnology, Vol 13(31) 3617-3627

International audience; BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that significantly increases morbidity and mortality in nosocomial infections and cystic fibrosis patients. Its pathogenicity especially relies on the production of virulence factors or resistances to many antibiotics. Since multiplication of antibiotic resistance can lead to therapeutic impasses, it becomes necessary to develop new tools for fighting P. aeruginosa infections. The use of probiotics is one of the ways currently being explored. Probiotics are microorganisms that exert a positive effect on the host's health and some of them are known to possess antibacterial activities. Since most of their effects have been shown in the digestive tract, experimental data compatible with the respiratory environment are strongly needed. The main goal of this study was then to test the capacity of lactobacilli to inhibit major virulence factors (elastolytic activity and biofilm formation) associated with P. aeruginosa pathogenicity. RESULTS: Sixty-seven lactobacilli were isolated from the oral cavities of healthy volunteers. These isolates together with 20 lactobacilli isolated from raw milks, were tested for their capacity to decrease biofilm formation and activity of the elastase produced by P. aeruginosa PAO1. Ten isolates, particularly efficient, were accurately identified using a polyphasic approach (API 50 CHL, mass-spectrometry and 16S/rpoA/pheS genes sequencing) and typed by pulsed-field gel electrophoresis (PFGE). The 8 remaining strains belonging to the L. fermentum (6), L. zeae (1) and L. paracasei (1) species were sensitive to all antibiotics tested with the exception of the intrinsic resistance to vancomycin. The strains were all able to grow in artificial saliva. CONCLUSION: Eight strains belonging to L. fermentum, L. zeae and L. paracasei species harbouring anti-elastase and anti-biofilm properties are potential probiotics for fighting P. aeruginosa pulmonary infections. However, further studies are needed in order to test their innocuity and their capacity to behave such as an oropharyngeal barrier against Pseudomonas aeruginosa colonisation in vivo.

International audience; Few antifungal protective cultures adapted to fermented dairy products are commercially available because of the numerous constraints linked to their market implementation. Consumer's demand for naturally preserved food products is growing and the utilization of lactic acid bacteria is a promising way to achieve this goal. In this study, using a 2(5-1) factorial fractional design, we first evaluated the effects of fermentation time, of initial sucrose concentration and of the initial contamination amount of a spoilage yeast, on antifungal activities of single and mixed cultures of Lactobacillus rhamnosus K.C8.3.1I and Lactobacillus harbinensis K.V9.3.1Np in yogurt. L. harbinensis K.V9.3.1Np, the most relevant strain with regard to antifungal activity was then studied to determine its minimal inhibitory inoculation rate, its antifungal stability during storage and its impact on yogurt organoleptic properties. We showed that L. harbinensis K.V9.3.1Np maintained a stable antifungal activity over time, which was not affected by initial sucrose, nor by a reduction of the fermentation time. This inhibitory activity was an all-or-nothing phenomenon. Once L. harbinensis K.V9.3.1Np reached a population of ∼2.5 × 10(6) cfu/g of yogurt at the time of contamination, total inhibition of the yeast was achieved. We also showed that an inoculation rate of 5 × 10(6) cfu/ml in milk had no detrimental effect on yogurt organoleptic properties. In conclusion, L. harbinensis K.V9.3.1Np is a promising antifungal bioprotective strain for yogurt preservation.

British Journal of Nutrition, 103 (3), ISSN:0007-1145, ISSN:1475-2662

Background Reuterin produced from glycerol by Lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. It has been postulated that reuterin could play a role in the probiotic effects of Lb. reuteri. Reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. Moreover reuterin's mode of action has not yet been elucidated. Glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin. The aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols (LMW-SH) such as glutathione, hydrogen peroxide and/or reuterin sensitivity. Reuterin was produced by Lb. reuteri SD2112 in pure glycerol solution, purified and used to test the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). Hydrogen peroxide sensitivity and intracellular LMW-SH concentration were also analysed. Results Our data showed that most tested intestinal bacteria showed MIC below that for a sensitive indicator Escherichia coli (7.5–15 mM). Lactobacilli and Clostridium clostridioforme were more resistant with MIC ranging from 15 to 50 mM. No correlation between bacterial intracellular concentrations of LMW-SH, including glutathione, and reuterin or hydrogen peroxide sensitivities were found. Conclusion Our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of LMW-SH was not directly linked to their capacity to resist reuterin or hydrogen peroxide. This suggests that detoxification by LMW-SH such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance to reuterin and hydrogene peroxide., BMC Microbiology, 7, ISSN:1471-2180

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri , E. biforme , E. contortum , E. cylindroides (two probes), E. dolichum , E. hadrum , E. lentum , E. limosum , E. moniliforme , and E. ventriosum . The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme , E. cylindroides , E. hadrum , E. lentum , and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10 7 cells g (dry weight) of feces −1 . The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.

Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia and which is involved in themortality of mechanically-ventilated or cystic fibrosis patients.These infections are difficult to treat because of the existence of many antibiotic resistances in P. aeruginosa and therapeutic alternatives are needed. Our hypothesis was that the use of probiotics could be an alternative to antibiotic therapy in order to reduce P. aeruginosa infections and its injurious and pro-inflammatory effects in lungs.The main goal of this work was to evaluate the effects of lactobacilli in a murine model of P. aeruginosa pneumonia.The first step of this work was to screen lactobacilli isolated from oral cavities of healthy volunteers against biofilmformation and elastolytic activity of P. aeruginosa PAO1. The effects of selected lactobacilli were then evaluated in amodel of infection of lung epithelial cells by P. aeruginosa PAO1 and in a murine model of P. aeruginosa PAO1pneumonia. Eighty-seven lactobacilli were tested in vitro, leading to the selection of 3 and 5 strains respectively active against biofilm formation and elastolytic activity. The most active strains (L. fermentum K.C6.3.1E, L. paracasei ES.D.88and L. zeae Od.76) toward biofilm formation and elastolytic activity were chosen to be tested in vitro, in a cell model of P. aeruginosa PAO1 infection. This mix showed cytoprotective effect against P. aeruginosa PAO1. Finally, the prophylactic intratracheal administration of the mix of lactobacilli in mice allowed to reduce the pulmonary loads in P.aeruginosa PAO1. In the same time, the pro-inflammatory effects(IL-6 and TNF- α) of the infection were reduced. These promising results suggest the possibility of new therapeutic applications for probiotics., Pseudomonas aeruginosa est un pathogène opportuniste responsable de pneumonies. Il est particulièrement impliqué dans la mortalité des patients sous ventilation mécanique et des patients atteints de la mucoviscidose. Ces infections sont difficiles à traiter en raison de l’existence de nombreuses résistances aux antibiotiques chez cette bactérie et des alternatives thérapeutiques s’avèrent donc nécessaires. Nous avons ainsi émis l’hypothèse qu’une application oropharyngée de lactobacilles pourrait permettre de limiter les infections à P. aeruginosa et leurs effets chez les patients concernés. L’objectif principal de ce travail était d’évaluer les effets d’un mélange de lactobacilles dans un modèle murin de pneumonie à P. aeruginosa. Les effets de lactobacilles isolés dans les cavités orales de volontaires sains sur la formation de biofilm et l’activité élastolytique de P. aeruginosa PAO1 ont été mesurés in vitro. Les effets des lactobacilles sélectionnés ont ensuite été évalués dans un modèle d’infection de cellules épithéliales respiratoires(A549) par P. aeruginosa PAO1 puis dans un modèle murin de pneumonie à P. aeruginosa PAO1. Les effets de 87 lactobacilles sur la formation de biofilm et l’activité élastolytique de P. aeruginosa PAO1 ont été déterminés in vitro,aboutissant à la sélection de 3 et 5 souches ayant respectivement inhibé la formation de biofilm et l’activité élastolytique de P. aeruginosa PAO1. Parmi ces souches, L. fermentum K.C6.3.1E, L. paracasei ES.D.88 et L. zeae Od.76,qui étaient les souches les plus actives contre la formation de biofilm ou l’activité élastolytique et les plus acidifiantes lors de croissances dans une salive artificielle, ont été associées dans un mélange testé dans un modèle cellulaire d’infection à P. aeruginosa PAO1. Ce mélange n’a pas eu d’effet cytotoxique et a démontré un effet cytoprotecteur vis-à-vis de l’infection à P. aeruginosa PAO1. In vivo, l’administration intratrachéale de ces mêmes bactéries de façon prophylactique a permis d’une part de réduire les charges pulmonaires en P. aeruginosa PAO1 et d’autre part de réduire ses effets pro-inflammatoires au niveau pulmonaire (IL-6, TNF-α). Ces résultats prometteurs laissent entrevoir la possibilité de nouvelles applications thérapeutiques pour les probiotiques.

Fermented dairy products such as yogurt, fermented milks and fresh cheeses are of great importance in the French economy. The stability of these products and export opportunities are however limited by frequent fungal spoilages. In addition, the increase of fungal resistances to Chemical preservatives and the strong consumer demand for products deprived of Chemical additives are pushing manufacturers to reduce the quantities of added Chemical preservatives in these products. In this context, it is necessary to develop alternatives to classical food preservation methods, such as biopreservation. Lactic acid bacteria (LAB), used for millennia for diverse food fermentations, and generally recognized as safe for human health, can potentially be used for biopreservation. In order to select 1 or 2 LAB strains with antifungal activity capable to compete in fermented dairy products such as yogurt, a collection of antifungal LAB was first created. This was done by screening colonies isolated from cow, goat and ewe raw milk samples over a one-year period. This screening step, targeted against 4 fungi found in contaminated dairy products, resulted in the isolation of 1235 antifungal colonies. The biodiversity of the isolated antifungal LAB was then determined along with that of the fungi found in the different raw milks, in order to observe a possible correlation between the presence of fungi and the expression of antifungal activities. The influence of milk origin, sampling period, isolation medium and targeted fungus on the percentage of isolated active colonies was highlighted and clearly showed that both cow and goat milks were reservoirs of antifungal LAB. Among the targeted fungi, P. expansum was more easily inhibited, and the majority of colonies were isolated during the third sampling period, mainly on MRS-based media. The majority of identified antifungal LAB belonged to the genus Lactobacillus, mainly to the Lb. casei group. Eleven isolates, including 10 belonging to the Lb. casei group, were selected from these lactobacilli, according to their activity level and action spectrum in MRS medium. Some of their technological properties were characterized for their potential use as protective cultures in fermented dairy products and their antifungal activity was tested in milk and yogurt. To do this, 6 fungal contaminants commonly encountered in yogurt spoilage were used. A particular strain, Lb. harbinensis K.V9.3.1Np, showed a strong antifungal activity in yogurt. Additional experiments showed that the variation of technological parameters (presence or absence of sugar, fermentation time) had no influence on the antifungal activity of this strain. This is the first time that an antifungal potential has been observed for Lb. harbinensis, a species isolated for the first time in 2005 from a fermented vegetable product. Because of its effectiveness in yogurt, Lb. harbinensis K.V9.3.1Np is a promising strain for biopreservation of fermented dairy product(s)., Les produits laitiers fermentés tels que les yaourts, les laits fermentés et les fromages frais, occupent une place importante dans l’économie française. La stabilité de ces produits et leurs chances d’exportation sont cependant limitées par de fréquentes altérations fongiques. De plus, l’augmentation des résistances de certains champignons aux conservateurs chimiques ainsi que la forte demande des consommateurs pour des produits dépourvus d’additifs poussent les industriels à en réduire l’ajout. Dans ce contexte, il est nécessaire de développer des alternatives innovantes, telle que la bio-conservation. Les bactéries lactiques, utilisées depuis des millénaires dans la fermentation de nombreux aliments, et généralement reconnues comme inoffensives pour la santé, sont potentiellement de bonnes candidates pour la bio-conservation. Dans le but d’obtenir une ou deux souches de bactéries lactiques antifongiques, capables d’être compétitives au sein de produits laitiers fermentés tels que le yaourt, une collection de bactéries lactiques antifongiques a d’abord été constituée. Pour cela, un criblage de colonies isolées de laits crus de vache, de chèvre et de brebis a été effectué sur une période d’une année. Ce criblage, ciblé contre 4 champignons communément retrouvés dans les produits laitiers contaminés, a abouti à l’isolement de 1235 colonies de bactéries lactiques antifongiques. Ceci a permis d’évaluer la biodiversité des bactéries lactiques antifongiques d’une part, et celle des champignons, d’autre part, dans ces échantillons de lait, afin d’observer une éventuelle corrélation entre la présence de champignons et l’expression des activités antifongiques. L’influence de l’origine du lait, de la période d’échantillonnage, du milieu d’isolement et du champignon ciblé, sur le pourcentage de colonies actives isolées a été mise en évidence. Parmi les champignons ciblés, Pénicillium expansum était le plus facilement inhibé, et la majorité des colonies ont été isolées durant la troisième période d’échantillonnage, majoritairement sur les milieux à base de MRS. Les laits de vache et de chèvre se sont révélés être des réservoirs de bactéries lactiques antifongiques, contrairement aux laits de brebis. La majorité des bactéries lactiques antifongiques isolées et identifiées appartenait au genre Lactobacillus, majoritairement du groupe Lb. casei. Onze isolats, dont 10 appartenant au groupe Lb. casei, ont été sélectionnés selon l’intensité de leur activité antifongique et leur spectre d’action sur milieu MRS. Certaines de leurs propriétés technologiques ont été caractérisées, en vue de leur utilisation comme cultures protectrices dans des produits laitiers fermentés. Leur activité antifongique a également été testée dans le lait et dans le yaourt, contre 6 contaminants fongiques communément responsables d’altérations dans les yaourts. Une souche, Lb. harbinensis K.V9.3.1Np, a révélé de fortes activités antifongiques dans le yaourt contre 6 cibles fongiques. Les études complémentaires effectuées ont permis de montrer que la variation de certains paramètres technologiques (présence ou absence de saccharose, temps de fermentation) n’avait pas d’influence sur l’activité antifongique de cette souche. La découverte du potentiel antifongique de Lb. harbinensis K.V9.3.1Np est innovante. Cette espèce, isolée pour la première fois en 2005 d’un produit fermenté végétal, n’avait encore jamais été décrite comme présentant des activités antimicrobiennes. Sa forte activité antifongique dans le yaourt fait de Lb. harbinensis K.V9.3.1Np un bon candidat pour la bioconservation de produit(s) laitier(s) fermenté(s).