Revealing the dynamics and mechanisms of bacterial interactions in cheese production with metabolic modelling.

Cheese taste and flavour properties result from complex metabolic processes occurring in microbial communities. A deeper understanding of such mechanisms makes it possible to improve both industrial production processes and end-product quality through the design of microbial consortia. In this work, we caracterise the metabolism of a three-species community consisting of Lactococcus lactis, Lactobacillus plantarum and Propionibacterium freudenreichii during a seven-week cheese production process. Using genome-scale metabolic models and omics data integration, we modeled and calibrated individual dynamics using monoculture experiments, and coupled these models to capture the metabolism of the community. This model accurately predicts the dynamics of the community, enlightening the contribution of each microbial species to organoleptic compound production. Further metabolic exploration revealed additional possible interactions between the bacterial species. This work provides a methodological framework for the prediction of community-wide metabolism and highlights the added value of dynamic metabolic modeling for the comprehension of fermented food processes.

Deciphering the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice fermentation using phenotypic and transcriptional analysis.

In the context of sustainable diet, the development of soy-based yogurt fermented with lactic acid bacteria is an attractive alternative to dairy yogurts. To decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice (SJ) fermentation, the whole genome of the strain CIRM-BIA865 (Ld865) was sequenced and annotated. Then Ld865 was used to ferment SJ. Samples were analyzed throughout fermentation for their cell number, carbohydrate, organic acid, free amino acid, and volatile compound contents. Despite acidification, the number of Ld865 cells did not rise, and microscopic observations revealed the elongation of cells from 3.6 µm (inoculation) to 36.9 µm (end of fermentation). This elongation was observed in SJ but not in laboratory-rich medium MRS. Using transcriptomic analysis, we showed that the biosynthesis genes of peptidoglycan and membrane lipids were stably expressed, in line with the cell elongation observed, whereas no genes implicated in cell division were upregulated. Among the main sugars available in SJ (sucrose, raffinose, and stachyose), Ld865 only used sucrose. The transcriptomic analysis showed that Ld865 implemented the two transport systems that it contains to import sucrose: a PTS system and an ABC transporter. To fulfill its nitrogen needs, Ld865 probably first consumed the free amino acids of the SJ and then implemented different oligopeptide transporters and proteolytic/peptidase enzymes. In conclusion, this study showed that Ld865 enables fast acidification of SJ, despite the absence of cell division, leads to a product rich in free amino acids, and also leads to the production of aromatic compounds of interest. IMPORTANCE: To reduce the environmental and health concerns related to food, an alternative diet is recommended, containing 50% of plant-based proteins. Soy juice, which is protein rich, is a relevant alternative to animal milk, for the production of yogurt-like products. However, soy "beany" and "green" off-flavors limit the consumption of such products. The lactic acid bacteria (LAB) used for fermentation can help to improve the organoleptic properties of soy products. But metabolic data concerning LAB adapted to soy juice are lacking. The aim of this study was, thus, to decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during fermentation of a soy juice, based on a multidisciplinary approach. This result will contribute to give tracks for a relevant selection of starter. Indeed, the improvement of the organoleptic properties of these types of products could help to promote plant-based proteins in our diet.

Omnicrobe, an open-access database of microbial habitats and phenotypes using a comprehensive text mining and data fusion approach.

The dramatic increase in the number of microbe descriptions in databases, reports, and papers presents a two-fold challenge for accessing the information: integration of heterogeneous data in a standard ontology-based representation and normalization of the textual descriptions by semantic analysis. Recent text mining methods offer powerful ways to extract textual information and generate ontology-based representation. This paper describes the design of the Omnicrobe application that gathers comprehensive information on habitats, phenotypes, and usages of microbes from scientific sources of high interest to the microbiology community. The Omnicrobe database contains around 1 million descriptions of microbe properties. These descriptions are created by analyzing and combining six information sources of various kinds, i.e. biological resource catalogs, sequence databases and scientific literature. The microbe properties are indexed by the Ontobiotope ontology and their taxa are indexed by an extended version of the taxonomy maintained by the National Center for Biotechnology Information. The Omnicrobe application covers all domains of microbiology. With simple or rich ontology-based queries, it provides easy-to-use support in the resolution of scientific questions related to the habitats, phenotypes, and uses of microbes. We illustrate the potential of Omnicrobe with a use case from the food innovation domain.

Genomic rearrangements in the aspA-dcuA locus of Propionibacterium freudenreichii are associated with aspartase activity.

Propionibacterium freudenreichii is crucial in Swiss-type cheese manufacture. Classic propionic acid fermentation yields the nutty flavor and the typical eyes. Co-metabolism of aspartate pronounces the flavor of the cheese; however, it also increases the size of the eyes, which can induce splitting and reduce the cheese quality. Aspartase (EC 4.3.1.1) catalyzes the deamination of aspartate, yielding fumarate and ammonia. The aspartase activity varies considerably among P. freudenreichii strains. Here, the correlation between aspartase activity and the locus of aspartase-encoding genes (aspA ) and dcuA encoding the C4-dicarboxylate transporter was investigated in 46 strains to facilitate strain selection for cheese culture. Low aspartase activity was correlated with a particular genomic rearrangement: low in vitro aspartase activity always occurred in strains with gene clusters aspA - dcuA where the dcuA was frameshifted, producing a stop codon or was disrupted by an ISL3-like element. The low aspartase activity could be due to the protein sequence of the aspartase or a dysfunctional DcuA. The highest values of aspartase activity were detected in strains with aspA1 - aspA2-dcuA with a DcuA sequence sharing 99.07 - 100% identity with the DcuA sequence of strain DSM 20271 T and an additional C4-dicarboxylate transporter belonging to the DcuAB family.

Deciphering the Microbiota and Volatile Profiles of Algerian Smen, a Traditional Fermented Butter.

In Algeria, Smen is a fermented butter produced in households using empirical methods. Smen fermentation is driven by autochthonous microorganisms; it improves butter shelf-life and yields highly fragrant products used as ingredients in traditional dishes as well as in traditional medicine. The present study is aimed at investigating microbial diversity and dynamics during Algerian Smen fermentation using both culture-dependent and culture-independent approaches, as well as by monitoring volatile organic compound production. To reach this goal, fifteen Smen samples (final products) produced in households from different regions in Algeria were collected and analyzed. In addition, microbial and volatile compound dynamics at the different stages of Smen manufacturing were investigated for one Smen preparation. The results showed that Smen is a microbiologically safe product, as all hygiene and safety criteria were respected. The dominant microorganisms identified by both techniques were LAB and yeasts. Lactococcus spp. and Streptococcus thermophilus were the main bacterial species involved in spontaneous raw milk fermentation preceding butter-making, while lactobacilli and enterococci were the only bacteria found to be viable during Smen maturation. Regarding fungal diversity, yeast species were only recovered from two mature Smen samples by culturing, while different species (e.g., Geotrichum candidum, Moniliella sp.) were identified in all samples by the culture-independent approach. Using microbial analysis of a single batch, many of these were found viable during manufacturing. Concerning the volatile profiles, they were highly diverse and characterized by a high prevalence of short chain fatty acids, methylketones, and esters. Correlation analysis between microbial diversity and volatile profiles showed that several yeast (Moniliella sp., K. marxianus) and LAB (e.g., Lactococcus spp., S. thermophilus) species were strongly correlated with one or more volatile organic compound families, including several ethyl esters and methyl ketones that can be linked to pleasant, sweetly floral, fruity, buttery, and creamy odors. This study clearly identified key microorganisms involved in Smen fermentation and maturation that could be used in the future for better fermentation control and improvement of quality attributes.

Brine salt concentration reduction and inoculation with autochthonous consortia: Impact on Protected Designation of Origin Nyons black table olive fermentations.

Nyons table olives, named after the French city where they are processed, are naturally fermented black table olives. Their specificity relies on the use of the "Tanche" olive variety harvested at full maturity and their slow spontaneous fermentation in 10% salt brine driven by yeast populations. This study aimed at investigating the benefit of inoculating autochthonous consortia to produce Nyons table olives by fermentation in 10% salt brine and in reduced salt conditions (8%). Two strategies were evaluated: inoculation with a defined autochthonous consortium and inoculation by spent brine backslopping. To define the consortium, yeasts were selected among 48 autochthonous isolates and key features included high halotolerance, low pectinolytic and proteolytic activities, however none had ß-glucosidase activities. The consortium included eight yeast strains with distinct technological properties belonging to five dominant species, i.e. Citeromyces nyonsensis, Pichia membranifaciens, Wickerhamomyces anomalus, Zygotorulaspora mrakii and Candida atlantica. Fermentation trials were conducted over a year and compared by evaluating microbial community shifts (16S and ITS metagenetics) and volatile profiles (GC-MS). Regarding fermentations with the defined consortium, four out of five species implanted in early stages while one, Pichia membranifaciens, persisted and largely dominated by the end of the fermentation. Altogether, inoculation with the defined consortium did not disrupt microbial shifts compared to traditional fermentations although minor differences were observed in volatile profiles. The backslopping method yielded the highest impact on microbial populations and olive volatile profiles, with higher ester abundances at the end of fermentation. Finally, reduced salt in brine gave very promising results as no deleterious effects on microbial communities, volatile dynamics but also safety criteria of the olives were observed compared to traditional fermented olives.

Fine-Tuning of Process Parameters Modulates Specific Metabolic Bacterial Activities and Aroma Compound Production in Semi-Hard Cheese.

The formation of cheese flavor mainly results from the production of volatile compounds by microorganisms. We investigated how fine-tuning cheese-making process parameters changed the cheese volatilome in a semi-hard cheese inoculated with Lactococcus (L.) lactis, Lactiplantibacillus (L.) plantarum, and Propionibacterium (P.) freudenreichii. A standard (Std) cheese was compared with three variants of technological itineraries: a shorter salting time (7 h vs 10 h, Salt7h), a shorter stirring time (15 min vs 30 min, Stir15min), or a higher ripening temperature (16 °C vs 13 °C, Rip16°C). Bacterial counts were similar in the four cheese types, except for a 1.4 log10 reduction of L. lactis counts in Rip16°C cheeses after 7 weeks of ripening. Compared to Std, Stir15min and Rip16°C increased propionibacterial activity, causing higher concentrations of acetic, succinic, and propanoic acids and lower levels of lactic acid. Rip16°C accelerated secondary proteolysis and volatile production. We thus demonstrated that fine-tuning process parameters could modulate the cheese volatilome by influencing specific bacterial metabolisms.

Linking Pélardon artisanal goat cheese microbial communities to aroma compounds during cheese-making and ripening.

Pélardon is an artisanal French raw goat's milk cheese, produced using natural whey as a backslop. The aim of this study was to identify key microbial players involved in the acidification and aroma production of this Protected Designation of Origin cheese. Microbial diversity of samples, collected from the raw milk to 3-month cheese ripening, was determined by culture-dependent (MALDI-TOF analysis of 2877 isolates) and -independent (ITS2 and 16S metabarcoding) approaches and linked to changes in biochemical profiles (volatile compounds and acids). In parallel, potential dominant autochthonous microorganism reservoirs were also investigated by sampling the cheese-factory environment. Complex and increasing microbial diversity was observed by both approaches during ripening although major discrepancies were observed regarding Lactococcus lactis and Lacticaseibacillus paracasei fate. By correlating microbial shifts to biochemical changes, Lactococcus lactis was identified as the main acidifying bacterium, while L. mesenteroides and Geotrichum candidum were prevalent and associated with amino acids catabolism after the acidification step. The three species were dominant in the whey (backslop). In contrast, L. paracasei, Enterococcus faecalis, Penicillium commune and Scopulariopsis brevicaulis, which dominated during ripening, likely originated from the cheese-making environment. All these four species were positively correlated to major volatile compounds responsible for the goaty and earthy Pélardon cheese aroma. Overall, this work highlighted the power of MALDI-TOF and molecular techniques combined with volatilome analyses to dynamically follow and identify microbial communities during cheese-making and successively identify the key-players involved in aroma production and contributing to the typicity of Pélardon cheese.

Function-Driven Design of Lactic Acid Bacteria Co-cultures to Produce New Fermented Food Associating Milk and Lupin.

Designing bacterial co-cultures adapted to ferment mixes of vegetal and animal resources for food diversification and sustainability is becoming a challenge. Among bacteria used in food fermentation, lactic acid bacteria (LAB) are good candidates, as they are used as starter or adjunct in numerous fermented foods, where they allow preservation, enhanced digestibility, and improved flavor. We developed here a strategy to design LAB co-cultures able to ferment a new food made of bovine milk and lupin flour, consisting in: (i) in silico preselection of LAB species for targeted carbohydrate degradation; (ii) in vitro screening of 97 strains of the selected species for their ability to ferment carbohydrates and hydrolyze proteins from milk and lupin and clustering strains that displayed similar phenotypes; and (iii) assembling strains randomly sampled from clusters that showed complementary phenotypes. The designed co-cultures successfully expressed the targeted traits i.e., hydrolyzed proteins and degraded raffinose family oligosaccharides of lupin and lactose of milk in a large range of concentrations. They also reduced an off-flavor-generating volatile, hexanal, and produced various desirable flavor compounds. Most of the strains in co-cultures achieved higher cell counts than in monoculture, suggesting positive interactions. This work opens new avenues for the development of innovative fermented food products based on functionally complementary strains in the world-wide context of diet diversification.

Deciphering Microbial Community Dynamics and Biochemical Changes During Nyons Black Olive Natural Fermentations.

French PDO Nyons black table olives are produced according to a traditional slow spontaneous fermentation in brine. The manufacture and unique sensorial properties of these olives thus only rely on the autochthonous complex microbiota. This study aimed at unraveling the microbial communities and dynamics of Nyons olives during a 1.5-year-long spontaneous fermentation to determine the main microbial drivers and link microbial species to key metabolites. Fermentations were monitored at a local producer plant at regular time intervals for two harvests and two olive types (organically and conventionally grown) using culture-dependent and metabarcoding (ITS2 for fungi, V3-V4 region for bacteria) approaches. Olives and brines were also sampled for volatiles, organic acids and phenolic compounds. No major differences in microbiota composition were observed according to olive type or harvest period. Throughout the fermentation, yeasts were clearly the most dominant. ITS2 sequencing data revealed complex fungal diversity dominated by Citeromyces nyonsensis, Wickerhamomyces anomalus, Zygotorulaspora mrakii, Candida boidinii and Pichia membranifaciens species. Bacterial communities were dominated by the Celerinatantimonas genus, while lactic acid bacteria remained scarce. Clear shifts in microbial communities and biochemical profiles were observed during fermentation and, by correlating metabolites and microbiota changes, four different phases were distinguished. During the first 7 days, phase I, a fast decrease of filamentous fungal and bacterial populations was observed. Between days 21 and 120, phase II, W. anomalus and C. nyonsensis for fungi and Celerinatantimonas diazotrophica for bacteria dominated the fermentation and were linked to the pH decrease and citric acid production. Phase III, between 120 and 183 days, was characterized by an increase in acids and esters and correlated to increased abundances of Z. mrakii, P. membranifaciens and C. boidinii. During the last months of fermentation, phase IV, microbial communities were dominated by P. membranifaciens and C. boidinii. Both species were strongly correlated to an increase in fruity esters and alcohol abundances. Overall, this study provides an in-depth understanding about microbial species succession and how the microbiota shapes the final distinct olive characteristics. It also constitutes a first step to identify key drivers of this fermentation.

Diversity of the metabolic profiles of a broad range of lactic acid bacteria in soy juice fermentation.

This study explores the ability of lactic acid bacteria (LAB) to ferment soy juice. The ability of 276 LAB strains from 25 species to ferment the principal soy carbohydrates, sucrose, raffinose or stachyose was tested in synthetic media and a soy juice. Fermented soy juices (FSJs) were characterized for their odor. Selected FSJs were characterized by targeted metabolomics. All Streptococcus, 83% of Leuconostoc and Lactobacillus and 41% of Lactococcus strains were sucrose-positive, while only 36% of all the LAB strains tested were raffinose-positive and 6% stachyose-positive. Nearly all (97%) the sucrose-positive strains fermented soy juice, indicating that an ability to use sucrose is a good criterion to select strains for soy juice fermentation. Among the most efficient acidifying strains, 46 FSJs had an odor deemed to be acceptable. FSJ composition was dependent on both species and strains: 17/46 strains deglycosylated soy juice isoflavones, the 27 S. thermophilus strains converted a mean 4.4 ± 0.1 g/L of sucrose into 3.0 ± 0.1 g/L of lactic acid versus 5.2 ± 0.1 g/L into 2.2 ± 0.1 g/L for the 18 Lactobacillus and one Lactococcus strains. This study highlights the diversity of the metabolic profiles of LAB strains in soy juice fermentation.

Identification of proteins involved in the anti-inflammatory properties of Propionibacterium freudenreichii by means of a multi-strain study.

Propionibacterium freudenreichii, a dairy starter, can reach a population of almost 109 propionibacteria per gram in Swiss-type cheese at the time of consumption. Also consumed as a probiotic, it displays strain-dependent anti-inflammatory properties mediated by surface proteins that induce IL-10 in leukocytes. We selected 23 strains with varied anti-inflammatory potentials in order to identify the protein(s) involved. After comparative genomic analysis, 12 of these strains were further analysed by surface proteomics, eight of them being further submitted to transcriptomics. The omics data were then correlated to the anti-inflammatory potential evaluated by IL-10 induction. This comparative omics strategy highlighted candidate genes that were further subjected to gene-inactivation validation. This validation confirmed the contribution of surface proteins, including SlpB and SlpE, two proteins with SLH domains known to mediate non-covalent anchorage to the cell-wall. Interestingly, HsdM3, predicted as cytoplasmic and involved in DNA modification, was shown to contribute to anti-inflammatory activity. Finally, we demonstrated that a single protein cannot explain the anti-inflammatory properties of a strain. These properties therefore result from different combinations of surface and cytoplasmic proteins, depending on the strain. Our enhanced understanding of the molecular bases for immunomodulation will enable the relevant screening for bacterial resources with anti-inflammatory properties.

Adaptation of Propionibacterium freudenreichii to long-term survival under gradual nutritional shortage.

BACKGROUND: Propionibacterium freudenreichii is an Actinobacterium widely used in the dairy industry as a ripening culture for Swiss-type cheeses, for vitamin B12 production and some strains display probiotic properties. It is reportedly a hardy bacterium, able to survive the cheese-making process and digestive stresses. RESULTS: During this study, P. freudenreichii CIRM-BIA 138 (alias ITG P9), which has a generation time of five hours in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, was incubated for 11 days (9 days after entry into stationary phase) in a culture medium, without any adjunct during the incubation. The carbon and free amino acids sources available in the medium, and the organic acids produced by the strain, were monitored throughout growth and survival. Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the strain sustained a high population level of 9.3 log10 CFU/mL. Its physiological adaptation was investigated by RNA-seq analysis and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase. CONCLUSIONS: P. freudenreichii adapts its metabolism during entry into stationary phase by down-regulating oxidative phosphorylation, glycolysis, and the Wood-Werkman cycle by exploiting new nitrogen (glutamate, glycine, alanine) sources, by down-regulating the transcription, translation and secretion of protein. Utilization of polyphosphates was suggested.

Bovine Teat Microbiome Analysis Revealed Reduced Alpha Diversity and Significant Changes in Taxonomic Profiles in Quarters with a History of Mastitis.

Mastitis is a mammary gland inflammatory disease often due to bacterial infections. Like many other infections, it used to be considered as a host-pathogen interaction driven by host and bacterial determinants. Until now, the involvement of the bovine mammary gland microbiota in the host-pathogen interaction has been poorly investigated, and mainly during the infectious episode. In this study, the bovine teat microbiome was investigated in 31 quarters corresponding to 27 animals, which were all free of inflammation at sampling time but which had different histories regarding mastitis: from no episode of mastitis on all the previous lactations (Healthy quarter, Hq) to one or several clinical mastitis events (Mastitic quarter, Mq). Several quarters whose status was unclear (possible history of subclinical mastitis) were classified as NDq. Total bacterial DNA was extracted from foremilk samples and swab samples of the teat canal. Taxonomic profiles were determined by pyrosequencing on 16s amplicons of the V3-4 region. Hq quarters showed a higher diversity compared to Mq ones (Shannon index: ~8 and 6, respectively). Clustering of the quarters based on their bacterial composition made it possible to separate Mq and Hq quarters into two separate clusters (C1 and C2, respectively). Discriminant analysis of taxonomic profiles between these clusters revealed several differences and allowed the identification of taxonomic markers in relation to mastitis history. C2 quarters were associated with a higher proportion of the Clostridia class (including genera such as Ruminococcus, Oscillospira, Roseburia, Dorea, etc.), the Bacteroidetes phylum (Prevotella, Bacteroides, Paludibacter, etc.), and the Bifidobacteriales order (Bifidobacterium), whereas C1 quarters showed a higher proportion of the Bacilli class (Staphylococcus) and Chlamydiia class. These results indicate that microbiota is altered in udders which have already developed mastitis, even far from the infectious episode. Microbiome alteration may have resulted from the infection itself and or the associated antibiotic treatment. Alternatively, differences in microbiome composition in udders with a history of mastitis may have occurred prior to the infection and even contributed to infection development. Further investigations on the dynamics of mammary gland microbiota will help to elucidate the contribution of this endogenous microbiota to the mammary gland health.

Permanent draft genome sequence of the probiotic strain Propionibacterium freudenreichii CIRM-BIA 129 (ITG P20).

Propionibacterium freudenreichii belongs to the class Actinobacteria (Gram positive with a high GC content). This "Generally Recognized As Safe" (GRAS) species is traditionally used as (i) a starter for Swiss-type cheeses where it is responsible for holes and aroma production, (ii) a vitamin B12 and propionic acid producer in white biotechnologies, and (iii) a probiotic for use in humans and animals because of its bifidogenic and anti-inflammatory properties. Until now, only strain CIRM-BIA1T had been sequenced, annotated and become publicly available. Strain CIRM-BIA129 (commercially available as ITG P20) has considerable anti-inflammatory potential. Its gene content was compared to that of CIRM-BIA1 T. This strain contains 2384 genes including 1 ribosomal operon, 45 tRNA and 30 pseudogenes.

Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti-inflammatory cheese.

SCOPE: Inflammatory bowel disease (IBD) constitutes a growing public health concern in western countries. Bacteria with anti-inflammatory properties are lacking in the dysbiosis accompanying IBD. Selected strains of probiotic bacteria with anti-inflammatory properties accordingly alleviate symptoms and enhance treatment of ulcerative colitis in clinical trials. Such properties are also found in selected strains of dairy starters such as Propionibacterium freudenreichii and Lactobacillus delbrueckii (Ld). We thus investigated the possibility to develop a fermented dairy product, combining both starter and probiotic abilities of both lactic acid and propionic acid bacteria, designed to extend remissions in IBD patients. METHODS AND RESULTS: We developed a single-strain Ld-fermented milk and a two-strain P. freudenreichii and Ld-fermented experimental pressed cheese using strains previously selected for their anti-inflammatory properties. Consumption of these experimental fermented dairy products protected mice against trinitrobenzenesulfonic acid induced colitis, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. As a control, the corresponding sterile dairy matrix failed to afford such protection. CONCLUSION: This work reveals the probiotic potential of this bacterial mixture, in the context of fermented dairy products. It opens new perspectives for the reverse engineering development of anti-inflammatory fermented foods designed for target populations with IBD, and has provided evidences leading to an ongoing pilot clinical study in ulcerative colitis patients.

New insights about phenotypic heterogeneity within Propionibacterium freudenreichii argue against its division into subspecies.

Propionibacterium freudenreichii is widely used in Swiss-type cheese manufacture, where it contributes to flavour and eye development. It is currently divided into two subspecies, according to the phenotype for lactose fermentation and nitrate reduction (lac+/nit- and lac-/nit+ for P. freudenreichii subsp. shermanii and subsp. freudenreichii, respectively). However, the existence of unclassifiable strains (lac+/nit+ and lac-/nit-) has also been reported. The aim of this study was to revisit the relevance of the subdivision of P. freudenreichii into subspecies, by confirming the existence of unclassifiable strains. Relevant conditions to test the ability of P. freudenreichii for lactose fermentation and nitrate reduction were first determined, by using 10 sequenced strains, in which the presence or absence of the lactose and nitrate genomic islands were known. We also determined whether the subdivision based on lac/nit phenotype was related to other phenotypic properties of interest in cheese manufacture, in this case, the production of aroma compounds, analysed by gas chromatography-mass spectrometry, for a total of 28 strains. The results showed that a too short incubation time can lead to false negative for lactose fermentation and nitrate reduction. They confirmed the existence of four lac/nit phenotypes instead of the two expected, thus leading to 13 unclassifiable strains out of the 28 characterized (7 lac+/nit+ and 6 lac-/nit-). The production of the 15 aroma compounds detected in all cultures varied more within a lac/nit phenotype (up to 20 times) than between them. Taken together, these results demonstrate that the division of P. freudenreichii into two subspecies does not appear to be relevant.

Mutations and genomic islands can explain the strain dependency of sugar utilization in 21 strains of Propionibacterium freudenreichii.

BACKGROUND: Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF is also extremely adaptable to several ecological niches and can grow on a variety of carbon and nitrogen sources. The aim of this work was to discover the genetic basis for strain-dependent traits related to its ability to use specific carbon sources. High-throughput sequencing technologies were ideal for this purpose as they have the potential to decipher genomic diversity at a moderate cost. RESULTS: 21 strains of PF were sequenced and the genomes were assembled de novo. Scaffolds were ordered by comparison with the complete reference genome CIRM-BIA1, obtained previously using traditional Sanger sequencing. Automatic functional annotation and manual curation were performed. Each gene was attributed to either the core genome or an accessory genome. The ability of the 21 strains to degrade 50 different sugars was evaluated. Thirty-three sugars were degraded by none of the sequenced strains whereas eight sugars were degraded by all of them. The corresponding genes were present in the core genome. Lactose, melibiose and xylitol were only used by some strains. In this case, the presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes, with the exception of xylitol. Furthermore, the simultaneous presence of these genes was in line the metabolic pathways described previously in other species. We also considered the genetic origin (transduction, rearrangement) of the corresponding genomic islands. Ribose and gluconate were degraded to a greater or lesser extent (quantitative phenotype) by some strains. For these sugars, the phenotypes could not be explained by the presence/absence of a gene but correlated with the premature appearance of a stop codon interrupting protein synthesis and preventing the catabolism of corresponding carbon sources. CONCLUSION: These results illustrate (i) the power of correlation studies to discover the genetic basis of binary strain-dependent traits, and (ii) the plasticity of PF chromosomes, probably resulting from horizontal transfers, duplications, transpositions and an accumulation of mutations. Knowledge of the genetic basis of nitrogen and sugar degradation opens up new strategies for the screening of PF strain collections to enable optimum cheese starter, probiotic and white biotechnology applications.

Surface proteins of Propionibacterium freudenreichii are involved in its anti-inflammatory properties.

Propionibacterium freudenreichii is a beneficial bacterium used in the food industry as a vitamin producer, as a bio-preservative, as a cheese ripening starter and as a probiotic. It is known to adhere to intestinal epithelial cells and mucus and to modulate important functions of the gut mucosa, including cell proliferation and immune response. Adhesion of probiotics and cross-talk with the host rely on the presence of key surface proteins, still poorly identified. Identification of the determinants of adhesion and of immunomodulation by P. freudenreichii remains a bottleneck in the elucidation of its probiotic properties. In this report, three complementary proteomic methods are used to identify surface-exposed proteins in a strain, previously selected for its probiotic properties. The role of these proteins in the reported immunomodulatory properties of P. freudenreichii is evidenced. This work constitutes a basis for further studies aimed at the elucidation of mechanisms responsible for its probiotic effects, in a post-genomic context. BIOLOGICAL SIGNIFICANCE: Dairy propionibacteria, mainly the species Propionibacterium freudenreichii, are consumed in high amounts within Swiss type cheeses. These peculiar bacteria are considered both as dairy starters and as probiotics. Their consumption modulates the gut microbiota, which makes them both probiotic and prebiotic. Promising immunomodulatory properties have been identified in these bacteria, in vitro, in animals and in humans. However, the mechanisms responsible for such anti-inflammatory properties are still unknown. In this work, we identify surface proteins involved in adhesion and immunostimulation by P. freudenreichii. This opens new perspectives for its utilization in new functional fermented food products, in clinical trials, and in understanding modulation of gut inflammation by products containing propionibacteria.

Fine-tuned characterization of Staphylococcus aureus Newbould 305, a strain associated with mild and chronic mastitis in bovines.

S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity.