Investigation into In Vitro and In Vivo Caenorhabditis elegans Models to Select Cheese Yeasts as Probiotic Candidates for their Preventive Effects against Salmonella Typhimurium.

The design of multiscale strategies integrating in vitro and in vivo models is necessary for the selection of new probiotics. In this regard, we developed a screening assay based on the investigation of the potential of yeasts from cheese as probiotics against the pathogen Salmonella Typhimurium UPsm1 (ST). Two yeasts isolated from raw-milk cheese (Saccharomyces cerevisiae 16, Sc16; Debaryomyces hansenii 25, Dh25), as well as S. cerevisiae subspecies boulardii (CNCM I-1079, Sb1079), were tested against ST by applying in vitro and in vivo tests. Adherence measurements to Caco-2 and HT29-MTX intestinal cells indicated that the two tested cheese yeasts presented a better adhesion than the probiotic Sb1079 as the control strain. Further, the Dh25 was the cheese yeast most likely to survive in the gastrointestinal tract. What is more, the modulation of the TransEpithelial Electrical Resistance (TEER) of differentiated Caco-2 cell monolayers showed the ability of Dh25 to delay the deleterious effects of ST. The influence of microorganisms on the in vivo model Caenorhabditis elegans was evaluated by measuring the longevity of the worm. This in vivo approach revealed that this yeast increased the worm's lifespan and protected it against ST infection, confirming that this in vivo model can be useful for screening probiotic cheese yeasts.

Lactobacillus rhamnosus Lcr35 as an effective treatment for preventing Candida albicans infection in the invertebrate model Caenorhabditis elegans: First mechanistic insights.

The increased recurrence of Candida albicans infections is associated with greater resistance to antifungal drugs. This involves the establishment of alternative therapeutic protocols, such as probiotic microorganisms whose antifungal potential has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding the mechanisms of action of probiotic microorganisms has become a strategic need for the development of new therapeutics for humans. In this study, we investigated the prophylactic anti-C. albicans properties of Lactobacillus rhamnosus Lcr35® using the in vitro Caco-2 cell model and the in vivo Caenorhabditis elegans model. In Caco-2 cells, we showed that the strain Lcr35® significantly inhibited the growth (~2 log CFU.mL-1) and adhesion (150 to 6,300 times less) of the pathogen. Moreover, in addition to having a pro-longevity activity in the nematode (+42.9%, p = 3.56.10-6), Lcr35® protects the animal from the fungal infection (+267% of survival, p < 2.10-16) even if the yeast is still detectable in its intestine. At the mechanistic level, we noticed the repression of genes of the p38 MAPK signalling pathway and genes involved in the antifungal response induced by Lcr35®, suggesting that the pathogen no longer appears to be detected by the worm immune system. However, the DAF-16/FOXO transcription factor, implicated in the longevity and antipathogenic response of C. elegans, is activated by Lcr35®. These results suggest that the probiotic strain acts by stimulating its host via DAF-16 but also by suppressing the virulence of the pathogen.

Dialogue between Staphylococcus aureus SA15 and Lactococcus garvieae strains experiencing oxidative stress.

BACKGROUND: Staphylococcus aureus is an important foodborne pathogen. Lactococcus garvieae is a lactic acid bacterium found in dairy products; some of its strains are able to inhibit S. aureus growth by producing H2O2. Three strains of L. garvieae from different origins were tested for their ability to inhibit S. aureus SA15 growth. Two conditions were tested, one in which H2O2 was produced (high aeration) and another one in which it was not detected (low aeration). Several S. aureus genes related to stress, H2O2-response and virulence were examined in order to compare their level of expression depending on the inoculated L. garvieae strain. Simultaneous L. garvieae H2O2 metabolism gene expression was followed. RESULTS: The results showed that under high aeration condition, L. garvieae strains producing H2O2 (N201 and CL-1183) inhibited S. aureus SA15 growth and impaired its ability to deal with hydrogen peroxide by repressing H2O2-degrading genes. L. garvieae strains induced overexpression of S. aureus stress-response genes while cell division genes and virulence genes were repressed. A catalase treatment partially or completely restored the SA15 growth. In addition, the H2O2 non-producing L. garvieae strain (Lg2) did not cause any growth inhibition. The SA15 stress-response genes were down-regulated and cell division genes expression was not affected. Under low aeration condition, while none of the strains tested exhibited H2O2-production, the 3 L. garvieae strains inhibited S. aureus SA15 growth, but to a lesser extent than under high aeration condition. CONCLUSION: Taken together, these results suggest a L. garvieae strain-specific anti-staphylococcal mechanism and an H2O2 involvement in at least two of the tested L. garvieae strains.

Bioprotective Effect of Lactococcus piscium CNCM I-4031 Against Listeria monocytogenes Growth and Virulence.

Listeria monocytogenes is a Gram-positive pathogen occurring in many refrigerated ready-to-eat foods. It is responsible for foodborne listeriosis, a rare but severe disease with a high mortality rate (20-30%). Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp and in a chemically defined medium using a cell-to-cell contact-dependent mechanism. To characterize this inhibition further, the effect of L. piscium was tested on a collection of 42 L. monocytogenes strains. All strains were inhibited but had different sensitivities. The effect of the initial concentration of the protective and the target bacteria revealed that the inhibition always occurred when L. piscium had reached its maximum population density, whatever the initial concentration of the protective bacteria. Viewed by scanning electron microscopy, L. monocytogenes cell shape and surface appeared modified in co-culture with L. piscium CNCM I-4031. Lastly, L. monocytogenes virulence, evaluated by a plaque-forming assay on the HT-29 cell line, was reduced after cell pre-treatment by the protective bacteria. In conclusion, the bioprotective effect of L. piscium toward L. monocytogenes growth and virulence was demonstrated, and a hypothesis for the inhibition mechanism is put forward.

Complete Genome Sequence of Lactococcus piscium CNCM I-4031, a Bioprotective Strain for Seafood Products.

Lactococcus piscium CNCM I-4031 is a psychotrophic foodborne lactic acid bacterium showing potential interest for the biopreservation of seafood products due to its inhibition properties toward pathogenic and spoilage bacteria. The analysis of its genome will provide a better understanding of the mechanisms of interaction between these bacteria.

Improving simultaneously the quality and safety of cooked and peeled shrimp using a cocktail of bioprotective lactic acid bacteria.

Tropical shrimp is of considerable economic importance in the world but is highly perishable due to microbial and chemical degradation. Biopreservation is a food preservation technology based on the addition of "positive" bacteria able to kill or prevent the growth of undesirable microorganisms. Two strains of lactic acid bacteria (LAB) have previously been selected for a biopreservation strategy: Lactococcus piscium CNCM I-4031, for its ability to prevent the sensory deterioration of seafood and Carnobacterium divergens V41, which inhibits growth of Listeria monocytogenes. The objective was to test the association of the two strains to improve both the quality and safety of shrimp. In a first trial, the two LAB were inoculated alone or in a cocktail in cooked and peeled shrimp (CPS) Penaeus vannamei at 5×105CFU/g. Chemical, sensory and microbiological analyses by culture-dependent and -independent methods were performed during storage under modified atmosphere packaging (MAP) at 8°C. The results were compared to a non-inoculated batch. In a second trial, the same experiments were repeated in the presence of 102CFU/g of L. monocytogenes RF191. The microbiota of CPS was composed of LAB, Shewanella spp. and Enterobacteriaceae. Brochothrix thermosphacta was not detected. L. piscium and C. divergens reached 108 and 109CFU/g, respectively, in 7days and did not inhibit each other when co-inoculated. L. piscium reduced L. monocytogenes by 1Log (CFU/g) for 28days. C. divergens had an immediate listericidal effect lasting 7days. A regrowth of L. monocytogenes was then observed but the count was always 2 to 5Log (CFU/g) lower than in the control. No additional or synergic effect between protective strains was observed and the cocktail had the same inhibitory effect as C. divergens alone. C. divergens was very effective at preventing the sensory deterioration of CPS. This may be related to the inhibition of Shewanella and Enterobacteriaceae. However, the panelists could detect the presence of C. divergens during the first 10days of storage, with slight unpleasant odors and flavors. L. piscium improved the sensory quality of CPS for 14days only. In co-culture, L. piscium eliminated the off-odors and flavors released by C. divergens in the early stage of storage and the co-culture allowed maintaining a good quality of CPS throughout the storage. Therefore, the use of a cocktail of L. piscium CNCM I-4031 and C. divergens V41 is recommended in a strategy of biopreservation of shrimp.

Inhibition mechanism of Listeria monocytogenes by a bioprotective bacteria Lactococcus piscium CNCM I-4031.

Listeria monocytogenes is a pathogenic Gram positive bacterium and the etiologic agent of listeriosis, a severe food-borne disease. Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp. To investigate the inhibititory mechanism, a chemically defined medium (MSMA) based on shrimp composition and reproducing the inhibition observed in shrimp was developed. In co-culture at 26 °C, L. monocytogenes was reduced by 3-4 log CFU g(-1) after 24 h. We have demonstrated that the inhibition was not due to secretion of extracellular antimicrobial compounds as bacteriocins, organic acids and hydrogen peroxide. Global metabolomic fingerprints of these strains in pure culture were assessed by liquid chromatography coupled with high resolution mass spectrometry. Consumption of glucose, amino-acids, vitamins, nitrogen bases, iron and magnesium was measured and competition for some molecules could be hypothesized. However, after 24 h of co-culture, when inhibition of L. monocytogenes occurred, supplementation of the medium with these compounds did not restore its growth. The inhibition was observed in co-culture but not in diffusion chamber when species were separated by a filter membrane. Taken together, these data indicate that the inhibition mechanism of L. monocytogenes by L. piscium is cell-to-cell contact-dependent.

Lactic Acid Bacteria Isolated from Bovine Mammary Microbiota: Potential Allies against Bovine Mastitis.

Bovine mastitis is a costly disease in dairy cattle worldwide. As of yet, the control of bovine mastitis is mostly based on prevention by thorough hygienic procedures during milking. Additional strategies include vaccination and utilization of antibiotics. Despite these measures, mastitis is not fully under control, thus prompting the need for alternative strategies. The goal of this study was to isolate autochthonous lactic acid bacteria (LAB) from bovine mammary microbiota that exhibit beneficial properties that could be used for mastitis prevention and/or treatment. Sampling of the teat canal led to the isolation of 165 isolates, among which a selection of ten non-redundant LAB strains belonging to the genera Lactobacillus and Lactococcus were further characterized with regard to several properties: surface properties (hydrophobicity, autoaggregation); inhibition potential of three main mastitis pathogens, Staphylococcus aureus, Escherichia coli and Streptococcus uberis; colonization capacities of bovine mammary epithelial cells (bMEC); and immunomodulation properties. Three strains, Lactobacillus brevis 1595 and 1597 and Lactobacillus plantarum 1610, showed high colonization capacities and a medium surface hydrophobicity. These strains are good candidates to compete with pathogens for mammary gland colonization. Moreover, nine strains exhibited anti-inflammatory properties, as illustrated by the lower IL-8 secretion by E. coli-stimulated bMEC in the presence of these LAB. Full genome sequencing of five candidate strains allowed to check for undesirable genetic elements such as antibiotic resistance genes and to identify potential bacterial determinants involved in the beneficial properties. This large screening of beneficial properties while checking for undesirable genetic markers allowed the selection of promising candidate LAB strains from bovine mammary microbiota for the prevention and/or treatment of bovine mastitis.

A unique in vivo experimental approach reveals metabolic adaptation of the probiotic Propionibacterium freudenreichii to the colon environment.

BACKGROUND: Propionibacterium freudenreichii is a food grade bacterium consumed both in cheeses and in probiotic preparations. Its promising probiotic potential, relying largely on the active release of beneficial metabolites within the gut as well as the expression of key surface proteins involved in immunomodulation, deserves to be explored more deeply. Adaptation to the colon environment is requisite for the active release of propionibacterial beneficial metabolites and constitutes a bottleneck for metabolic activity in vivo. Mechanisms allowing P. freudenreichii to adapt to digestive stresses have been only studied in vitro so far. Our aim was therefore to study P. freudenreichii metabolic adaptation to intra-colonic conditions in situ. RESULTS: We maintained a pure culture of the type strain P. freudenreichii CIRM BIA 1, contained in a dialysis bag, within the colon of vigilant piglets during 24 hours. A transcriptomic analysis compared gene expression to identify the metabolic pathways induced by this environment, versus control cultures maintained in spent culture medium.We observed drastic changes in the catabolism of sugars and amino-acids. Glycolysis, the Wood-Werkman cycle and the oxidative phosphorylation pathways were down-regulated but induction of specific carbohydrate catabolisms and alternative pathways were induced to produce NADH, NADPH, ATP and precursors (utilizing of propanediol, gluconate, lactate, purine and pyrimidine and amino-acids). Genes involved in stress response were down-regulated and genes specifically expressed during cell division were induced, suggesting that P. freudenreichii adapted its metabolism to the conditions encountered in the colon. CONCLUSIONS: This study constitutes the first molecular demonstration of P. freudenreichii activity and physiological adaptation in vivo within the colon. Our data are likely specific to our pig microbiota composition but opens an avenue towards understanding probiotic action within the gut in further studies comparing bacterial adaptation to different microbiota.