Vitamin B12 analogues from gut microbes and diet differentially impact commensal propionate producers of the human gut.

To produce the health-associated metabolite propionate, gut microbes require vitamin B12 as a cofactor to convert succinate to propionate. B12 is sourced in the human gut from the unabsorbed dietary fraction and in situ microbial production. However, experimental data for B12 production by gut microbes is scarce, especially on their produced B12-analogues. Further, the promotion of propionate production by microbially-produced and dietary B12 is not yet fully understood. Here, we demonstrated B12 production in 6 out of 8 in silico predicted B12-producing bacteria from the human gut. Next, we showed in vitro that B12 produced by Blautia hydrogenotrophica, Marvinbryantia formatexigens, and Blautia producta promoted succinate to propionate conversion of two prevalent B12-auxotrophic gut bacteria, Akkermansia muciniphila and Bacteroides thetaiotaomicron. Finally, we examined the propiogenic effect of commercially available B12-analogues present in the human diet (cyano-B12, adenosyl-B12 and hydroxy-B12) at two doses. The low dose resulted in partial conversion of succinate to propionate for A. muciniphila when grown with adenosyl-B12 (14.6 ± 2.4 mM succinate and 18.7 ± 0.6 mM propionate) and hydroxy-B12 (13.0 ± 1.1 mM and 21.9 ± 1.2 mM), in comparison to cyano-B12 (0.7 ± 0.1 mM and 34.1 ± 0.1 mM). Higher doses of adenosyl-B12 and hydroxy-B12 resulted in significantly more conversion of succinate to propionate in both propionate-producing species, compared to the low dose. B12 analogues have different potential to impact the propionate metabolism of prevalent propionate producers in the gut. These results could contribute to strategies for managing gut disorders associated with decreased propionate production.

Glycerol and reuterin-producing Limosilactobacillus reuteri enhance butyrate production and inhibit Enterobacteriaceae in broiler chicken cecal microbiota PolyFermS model.

BACKGROUND: Administering probiotic strains of Limosilactobacillus reuteri to poultry has been shown to improve poultry performance and health. Some strains of L. reuteri taxa can produce reuterin, a broad-spectrum antimicrobial compound from glycerol conversion, with high inhibitory activity against enterobacteria. However, little is known about the metabolism of glycerol in the complex chicken cecal microbiota nor the effect of glycerol, either alone or combined with L. reuteri on the microbiota. In this study, we investigated the effect of L. reuteri PTA5_F13, a high-reuterin-producing chicken strain and glycerol, alone or combined, on broiler chicken cecal microbiota composition and activity using the continuous PolyFermS model recently developed to mimic chicken cecal fermentation. METHODS: Three independent PolyFermS chicken cecal microbiota models were inoculated with immobilized cecal microbiota from different animals and operated continuously. The effects of two additional levels of glycerol (50 and 100 mM) with or without daily supplementation of chicken-derived L. reuteri PTA5_F13 (107 CFU/mL final concentration) were tested in parallel second-stage reactors continuously inoculated with the same microbiota. We analyzed the complex chicken gut microbiota structure and dynamics upon treatment using 16S rRNA metabarcoding and qPCR. Microbiota metabolites, short-chain and branched-chain fatty acids, and glycerol and reuterin products were analyzed by HPLC in effluent samples from stabilized reactors. RESULTS: Supplementation with 100 mM glycerol alone and combined with L. reuteri PTA5_F13 resulted in a reproducible increase in butyrate production in the three modelled microbiota (increases of 18 to 25%). Glycerol alone resulted also in a reduction of Enterobacteriaceae in two of the three microbiota, but no effect was detected for L. reuteri alone. When both treatments were combined, all microbiota quantitatively inhibited Enterobacteriaceae, including in the last model that had very high initial concentrations of Enterobacteriaceae. Furthermore, a significant 1,3-PDO accumulation was measured in the effluent of the combined treatment, confirming the conversion of glycerol via the reuterin pathway. Glycerol supplementation, independent of L. reuteri addition, did not affect the microbial community diversity. CONCLUSIONS: Glycerol induced a stable and reproducible butyrogenic activity for all tested microbiota and induced an inhibitory effect against Enterobacteriaceae that was strengthened when reuterin-producing L. reuteri was spiked daily. Our in vitro study suggests that co-application of L. reuteri PTA5_F13 and glycerol could be a useful approach to promote chicken gut health by enhancing metabolism and protection against Enterobacteriaceae.

Healthy adult gut microbiota sustains its own vitamin B12 requirement in an in vitro batch fermentation model.

Vitamin B12 (cobalamin) is present in the human lower gastrointestinal tract either coming from the unabsorbed dietary fraction or from in situ production of the gut microbiota. However, it is unclear whether the gut microbial communities need exogenous B12 for growth and metabolism, or whether B12 in low and high levels could affect gut community composition and metabolite production. Here, we investigated in vitro B12 production of human fecal microbiota and the effects of different levels of B12 (as cyanocobalamin) on composition and activity. Eight fecal communities from healthy human adults distributed over three enterotypes, dominated by Firmicutes (n = 5), Bacteroides (n = 1) or Prevotella (n = 2) were used to perform batch fermentations in Macfarlane medium supplemented with low B12 medium (Control, 5 ng/ml, within the tested fecal range), no B12 addition (NB12), and high B12 addition (ExtraB12, 2500 ng/ml). The microbiota community composition (qPCR, 16S rRNA metabarcoding), metabolic activity (HPLC-RI), and B12 levels (UHPLC-DAD) were measured after 24 h incubation at 37°C under strict anaerobic conditions. All fecal microbial communities produced B12 in the NB12 condition after 24 h, in the range from 152 ± 4 to 564 ± 25 ng/ml. None of the B12 treatments had an impact on total bacterial growth, community richness, diversity and total metabolite production, compared to the low B12 control. However, a significant increase of propionate was measured in ExtraB12 compared to NB12. Most taxonomic and metabolite changes compared to control incubations were donor-dependent, implying donor-microbiota-specific changes upon B12 treatments. Our in vitro data suggest that healthy human adult gut microbial communities have the capacity to produce B12 at levels fulfilling their own requirements, independently of the initial B12 content tested in the donor's feces. Further, supplementation of exogenous dietary B12 may have limited impact on the healthy human gut microbial community composition and function.

Pathogenic and Commensal Gut Bacteria Harboring Glycerol/Diol Dehydratase Metabolize Glycerol and Produce DNA-Reactive Acrolein.

Bacteria harboring glycerol/diol dehydratase (GDH) encoded by the genes pduCDE metabolize glycerol and release acrolein during growth. Acrolein has antimicrobial activity, and exposure of human cells to acrolein gives rise to toxic and mutagenic responses. These biological responses are related to acrolein's high reactivity as a chemical electrophile that can covalently bind to cellular nucleophiles including DNA and proteins. Various food microbes and gut commensals transform glycerol to acrolein, but there is no direct evidence available for bacterial glycerol metabolism giving rise to DNA adducts. Moreover, it is unknown whether pathogens, such as Salmonella Typhymurium, catalyze this transformation. We assessed, therefore, acrolein formation by four GDH-competent strains of S. Typhymurium grown under either aerobic or anaerobic conditions in the presence of 50 mM glycerol. On the basis of analytical derivatization with a heterocyclic amine, all wild-type strains were observed to produce acrolein, but to different extents, and acrolein production was not detected in fermentations of a pduC-deficient mutant strain. Furthermore, we found that, in the presence of calf thymus DNA, acrolein-DNA adducts were formed as a result of bacterial glycerol metabolism by two strains of Limosilactobacillus reuteri, but not a pduCDE mutant strain. The quantification of the resulting adducts with increasing levels of glycerol up to 600 mM led to the production of up to 1.5 mM acrolein and 3600 acrolein-DNA adducts per 108 nucleosides in a model system. These results suggest that GDH-competent food microbes, gut commensals, and pathogens alike have the capacity to produce acrolein from glycerol. Further, the acrolein production can lead to DNA adduct formation, but requires high glycerol concentrations that are not available in the human gut.

Stepwise establishment of functional microbial groups in the infant gut between 6 months and 2 years: A prospective cohort study.

The early intestinal colonization of functional microbial groups plays an essential role in infant gut health, with most studies targeting the initial colonization period from birth to 6 months of age. In a previous report, we demonstrated the metabolic cross-feeding of lactate and identified keystone species specified for lactate utilization in fecal samples of 40 healthy infants. We present here the extension of our longitudinal study for the period from 6 months to 2 years, with a focus on the colonization of functional groups involved in lactate metabolism and butyrate production. We captured the dynamic changes of the gut microbiota and reported a switch in the predominant lactate-producing and lactate-utilizing bacteria, from Veillonella producing propionate in the first year to Anaerobutyrycum hallii producing butyrate in the second year of life. The significant increase in butyrate producers and fecal butyrate concentration was also pinpointed to the weaning period between 6 and 10 months. Correlation analyses further suggested, for the first time, the metabolic cross-feeding of hydrogen in infants. In conclusion, our longitudinal study of 40 Swiss infants provides important insights into the colonization of functional groups involved in lactate metabolism and butyrate production in the first 2 years of life.

Impact of microcin J25 on the porcine microbiome in a continuous culture model.

The increased prevalence of Salmonella spp. resistance in swine spurs the search for alternatives to antibiotics. Microcin J25 (MccJ25), a bacteriocin produced by Escherichia coli, is a potent inhibitor of several pathogenic bacteria including Salmonella enterica. In this study, we aimed to evaluate in vitro the impact of MccJ25 on the composition and the metabolic activity of the swine colonic microbiota. The PolyFermS in vitro continuous fermentation model was used here with modified Macfarlane medium to simulate the porcine proximal colon. During 35 days of fermentation, a first-stage reactor containing immobilized swine fecal microbiota fed two second-stage control and test reactors operated in parallel and used to test the effects of MccJ25 on the composition and the metabolic activity of the microbiota. Reuterin, a broad-spectrum antimicrobial compound produced by Limosilactobacillus reuteri, a lactic acid bacterium naturally present in the gastro-intestinal tract of human and animals, and the antibiotic rifampicin were tested for comparison. Sequencing of 16S rRNA was performed using the Illumina MiSeq technology to evaluate microbial diversity, and liquid chromatography coupled to mass spectrometry (LC-MS) followed by multivariate analysis was used to assess the bacteriocin/antibiotic degradation products and to monitor changes in the swine colonic microbiota metabolome. The results show that MccJ25 or reuterin treatments only induce subtle changes of both the microbial diversity and the metabolome of the swine colon microbiota, while rifampicin induces significant modification in amino acid levels. Although these findings need being validated in vivo, this study affords a first proof of concept for considering MccJ25 as a possible alternative to antibiotics for veterinary and farming applications, taking into account its pathogen-selective and potent inhibitory activity.

Impact of manipulation of glycerol/diol dehydratase activity on intestinal microbiota ecology and metabolism.

Glycerol/diol dehydratases (GDH) are enzymes that catalyse the production of propionate from 1,2-propanediol, and acrolein from glycerol. Acrolein reacts with dietary carcinogenic heterocyclic amines (HCA), reducing HCA mutagenicity, but is itself also an antimicrobial agent and toxicant. Gut microbial GDH activity has been suggested as an endogenous acrolein source; however, there is limited information on the potential of the intestinal microbiota to have GDH activity, and what impact it can have on the intestinal ecosystem and host health. We hypothesized that GDH activity of gut microbiota is determined by the abundance and distribution of GDH-active taxa and can be enhanced by supplementation of the GDH active Anaerobutyricum hallii, and tested this hypothesis combining quantitative profiling of gdh, model batch fermentations, microbiota manipulation, and kinetic modelling of acrolein formation. Our results suggest that GDH activity is a common trait of intestinal microbiota shared by a few taxa, which was dependent on overall gdh abundance. Anaerobutyricum hallii was identified as a key taxon in GDH metabolism, and its supplementation increased the rate of GDH activity and acrolein release, which enhanced the transformation of HCA and reduced fermentation activity. The findings of this first systematic study on acrolein release by intestinal microbiota indicate that dietary and microbial modulation might impact GDH activity, which may influence host health.

In vitro Modeling of Chicken Cecal Microbiota Ecology and Metabolism Using the PolyFermS Platform.

Continuous in vitro fermentation models provide a useful tool for a fast, reproducible, and direct assessment of treatment-related changes in microbiota metabolism and composition independent of the host. In this study, we used the PolyFermS model to mimic the conditions of the chicken cecum and evaluated three nutritive media for in vitro modeling of the chicken cecal microbiota ecology and metabolism. We observed that our model inoculated with immobilized cecal microbiota and fed with a modified Viande Levure medium (mVL-3) reached a high bacterial cell density of up to approximately 10.5 log cells per mL and stable microbiota composition, akin to the host, during 82 days of continuous operation. Relevant bacterial functional groups containing primary fibrolytic (Bacteroides, Bifidobacteriaceae, Ruminococcaceae), glycolytic (Enterococcus), mucolytic (Bacteroides), proteolytic (Bacteroides), and secondary acetate-utilizing butyrate-producing and propionate-producing (Lachnospiraceae) taxa were preserved in vitro. Besides, conserved metabolic and functional Kyoto Encyclopedia of Genes and Genomes pathways were observed between in vitro microbiota and cecal inoculum microbiota as predicted by functional metagenomics analysis. Furthermore, we demonstrated that the continuous inoculation provided by the inoculum reactor generated reproducible metabolic profiles in second-stage reactors comparable to the chicken cecum, allowing for the simultaneous investigation and direct comparison of different treatments with a control. In conclusion, we showed that PolyFermS is a suitable model for mimicking chicken cecal microbiota fermentation allowing ethical and ex vivo screening of environmental factors, such as dietary additives, on chicken cecal fermentation. We report here for the first time a fermentation medium (mVL-3) that closely mimics the substrate conditions in the chicken cecum and supports the growth and metabolic activity of the cecal bacterial akin to the host. Our PolyFermS chicken cecum model is a useful tool to study microbiota functionality and structure ex vivo.

Intestinal inflammation alters mucosal carbohydrate foraging and monosaccharide incorporation into microbial glycans.

Endogenous carbohydrates released from the intestinal mucus represent a constant source of nutrients to the intestinal microbiota. Mucus-derived carbohydrates can also be used as building blocks in the biosynthesis of bacterial cell wall components, thereby influencing host mucosal immunity. To assess the uptake of endogenous carbohydrates by gut microbes in healthy mice and during intestinal inflammation, we applied azido-monosaccharides that can be tracked on bacterial cell walls after conjugation with fluorophores. In interleukin-10 deficient mice, changes in the gut microbiota were accompanied by decreased carbohydrate hydrolase activities and increased lumenal concentrations of host glycan-derived monosaccharides. Tracking of the monosaccharide N-azidoacetylglucosamine (GlcNAz) in caecum bacteria revealed a preferential incorporation of this carbohydrate by Xanthomonadaceae in healthy mice and by Bacteroidaceae in interleukin-10 deficient mice. These GlcNAz-positive Bacteroidaceae fractions mainly belonged to the species B. acidifaciens and B. vulgatus. Growth of Bacteroides species in the presence of specific monosaccharides changed their stimulatory activity toward CD11c+ dendritic cells. Expression of activation markers and cytokine production was highest after stimulation of dendritic cells with B. vulgatus. The variable incorporation of monosaccharides by related Bacteroides species underline the necessity to investigate intestinal bacteria down to the species level when addressing microbiota-host interactions.

Isolation and Comparative Genomic Analysis of Reuterin-Producing Lactobacillus reuteri From the Chicken Gastrointestinal Tract.

Lactobacillus reuteri is a natural inhabitant of selected animal and human gastrointestinal tract (GIT). Certain strains have the capacity to transform glycerol to 3-hydroxypropionaldehyde (3-HPA), further excreted to form reuterin, a potent antimicrobial system. Reuterin-producing strains may be applied as a natural antimicrobial in feed to prevent pathogen colonization of animals, such as in chicken, and replace added antimicrobials. To date, only seven L. reuteri strains isolated from chicken have been characterized which limits phylogenetic studies and host-microbes interactions characterization. This study aimed to isolate L. reuteri strains from chicken GIT and to characterize their reuterin production and antimicrobial resistance (AMR) profiles using phenotypic and genetic methods. Seventy strains were isolated from faces, crops and ceca of six chicken from poultry farms and samples from slaughterhouse. Twenty-five strains were selected for further characterization. Draft genomes were generated for the new 25 isolates and integrated into a phylogenetic tree of 40 strains from different hosts. Phylogenetic analysis based on gene content as well as on core genomes showed grouping of the selected 25 L. reuteri chicken isolates within the poultry/human lineage VI. Strains harboring pdu-cob-cbi-hem genes (23/25) produced between 156 mM ± 11 and 330 mM ± 14 3-HPA, from 600 mM of glycerol, in the conditions of the test. All 25 chicken strains were sensitive to cefotaxime (MIC between 0.016 and 1 µg/mL) and penicillin (MIC between 0.02 and 4 µg/mL). Akin to the reference strains DSM20016 and SD2112, the novel isolates were resistant to penicillin, possibly associated with identified point mutations in ponA, pbpX, pbpF and pbpB. All strains resistant to erythromycin (4/27) carried the ermB gene, and it was only present in chicken strains. All strains resistant to tetracycline (5/27) harbored tetW gene. This study confirms the evolutionary history of poultry/human lineage VI and identifies pdu-cob-cbi-hem as a frequent trait but not always present in this lineage. L. reuteri chicken strains producing high 3-HPA yield may have potential to prevent enteropathogen colonization of chicken.

Reuterin Demonstrates Potent Antimicrobial Activity Against a Broad Panel of Human and Poultry Meat Campylobacter spp. Isolates.

Reuterin is a broad-spectrum antimicrobial system produced by specific strains of Lactobacillus reuteri during anaerobic metabolism of glycerol. Acrolein is the main component responsible for its antimicrobial activity. Here, the sensitivity of Campylobacter jejuni (n = 51) and Campylobacter coli (n = 20) isolates from chicken meat and human stool samples to reuterin was investigated. The minimum inhibitory concentration (MIC) of C. jejuni and C. coli strains was measured between 1.5 and 3.0 µM of acrolein, below the MIC of the sensitive indicator strain Escherichia coli K12 (16.5 µM acrolein). The interaction of C. jejuni N16-1419 and the reuterin-producing L. reuteri PTA5_F13 was studied during 24 h co-cultures with or without glycerol. A high C. jejuni growth was observed in cultures without glycerol. In contrast, C. jejuni growth decreased from 7.3 ± 0.1 log CFU/mL to below detection limit (1 log CFU/mL) during co-cultures added with 28 mM glycerol. This bactericidal effect could be attributed to in situ reuterin production. The low MIC observed and the high sensitivity towards in situ produced reuterin suggests L. reuteri combined with glycerol, as a possible intervention option to reduce Campylobacter in the food chain.

Planktonic and Sessile Artificial Colonic Microbiota Harbor Distinct Composition and Reestablish Differently upon Frozen and Freeze-Dried Long-Term Storage.

Biofilm-associated, sessile communities represent the major bacterial lifestyle, whereas planktonic cells mainly appear during initial colonization of new surfaces. Previous research, mainly performed with pathogens, demonstrated increased environmental stress tolerance of biofilm-growing compared to planktonic bacteria. The lifestyle-specific stress response of colonic microbiota, both natural and fermentation produced, has not been addressed before. Planktonic and sessile "artificial" colonic microbiota delivered by PolyFermS continuous fermentation models can provide a controllable and reproducible alternative to fecal transplantation in treating gastrointestinal disorders. We therefore characterized planktonic and sessile microbiota produced in two PolyFermS models inoculated with immobilized fecal microbiota and comparatively tested their levels of tolerance of frozen storage (-80°C) and freeze-dried storage (4°C) for 9 months to mimic preservation strategies for therapeutic applications. Sessile microbiota harbored next to shared taxa a unique community distinguishable from planktonic microbiota. Synergistetes and Proteobacteria were highly represented in sessile microbiota, while Firmicutes were more abundant in planktonic microbiota. The community structure and metabolic activity of both microbiota, monitored during standardized reactivation batch fermentations, were better preserved after frozen storage than dried storage, indicated by higher Bray-Curtis similarity and enhanced recovery of metabolite production. For both lifestyles, reestablishment of Bacteroidaceae was impaired after frozen and dried storage along with reduced propionate formation. In contrast, butyrate production was maintained after reactivation despite compositional rearrangements within the butyrate-producing community. Unexpectedly, the rate of recovery of metabolite production was lower after preservation of sessile than planktonic microbiota. We speculate that higher functional dependencies between microbes might have led to the lower stress tolerance of sessile than planktonic microbiota.IMPORTANCE Fecal microbiota transplantation has been successfully applied in the treatment of recurrent Clostridium difficile infection and has been suggested as an alternative therapy for other intestinal disorders such as inflammatory bowel disease or metabolic syndrome. "Artificial" colonic microbiota delivered by PolyFermS continuous fermentation models can provide a controllable and reproducible alternative to fecal transplantation, but effective preservation strategies must be developed. In this study, we systematically investigated the response of sessile and planktonic artificial colonic microbiota to cryopreservation and lyophilization. We suggest that functional redundancy is an important factor in providing functional stability with respect to exposure to stress during processing and storage. Functional redundancy in compositionally reduced microbial systems may be considered when designing microbial products for therapy.

Decontamination of Minimally-Processed Fresh Lettuce Using Reuterin Produced by Lactobacillus reuteri.

Over the last years the demand for pre-washed, fresh-cut, and minimally-processed (MP) produce has increased. MP fresh vegetable are rapidly spoiled, whereas there is consumers' concern about chemical disinfection treatments such as with chlorine. A promising antimicrobial is reuterin, a broad-spectrum-antimicrobial compound produced by food-grade Lactobacillus reuteri from glycerol. In aqueous solution, reuterin is a dynamic system consisting of 3-hydroxypropionaldehyde (3-HPA), its hydrate, its dimer as well as acrolein, which was recently identified as the main antimicrobial component of the system. Here, we tested the use of reuterin containing similar 3-HPA levels but different acrolein concentrations for decontaminating and preserving fresh-cut lettuce. Crude reuterin (CR) was produced by biotransformation of 600 mM glycerol using L. reuteri DSM 20016T. CR preparations were further incubated for 16 h at 50°C to produce enhanced reuterin (ER) with raised concentration of acrolein. Fresh-cut iceberg lettuce (Lactuca sativa) was washed using CR (1.5-1.9 mM acrolein) and ER (7.2-21.9 mM acrolein) solutions at 4°C, or sodium hypochloride (250 mg/L) and tap water, and compared with unwashed lettuce. Washed lettuce samples were packed under modified atmosphere (2% O2, 5% CO2, and 93% N2) and stored for 13 days at 4°C. Application of ER containing 12.1, 20.9, or 21.9 mM acrolein reduced the initial viable plate counts of Enterobacteriaceae (by 2.1-2.8 log CFU/g), and yeasts and molds (by 1.3-2.0 log CFU/g) when compared with unwashed samples. In contrast, reuterin solutions containing 7.2 mM acrolein, sodium hypochlorite and tap water only showed very limited and transient, or no effects on the cell loads of lettuce after washing and during storage. Visual assessment of leaves washed with ER showed acrolein concentration-dependent discoloration noticeable already after 3 days of storage for the highest acrolein concentrations. Discoloration became severe for all ER treatments after 7 days, while the other treatments preserved the aspect of washed lettuce. Our data show the predominant role of acrolein as the main antimicrobial component of the reuterin system for food biopreservation. Reuterin preparations with enhanced acrolein concentration of 12.1 mM and higher were effective to reduce plate counts of Enterobacteriaceae and yeasts and molds washed lettuce until day 7 but induced pronounced discoloration of lettuce.

Understanding the prebiotic potential of different dietary fibers using an in vitro continuous adult fermentation model (PolyFermS).

Consumption of fermentable dietary fibers (DFs), which can induce growth and/or activity of specific beneficial populations, is suggested a promising strategy to modulate the gut microbiota and restore health in microbiota-linked diseases. Until today, inulin and fructo-oligosaccharides (FOS) are the best studied DFs, while little is known about the gut microbiota-modulating effects of ß-glucan, α-galactooligosaccharide (α-GOS) and xylo-oligosaccharide (XOS). Here, we used three continuous in vitro fermentation PolyFermS model to study the modulating effect of these DFs on two distinct human adult proximal colon microbiota, independently from the host. Supplementation of DFs, equivalent to a 9 g daily intake, induced a consistent metabolic response depending on the donor microbiota. Irrespective to the DF supplemented, the Bacteroidaceae-Ruminococcaceae dominated microbiota produced more butyrate (up to 96%), while the Prevotellaceae-Ruminococcaceae dominated microbiota produced more propionate (up to 40%). Changes in abundance of specific bacterial taxa upon DF supplementation explained the observed changes in short-chain fatty acid profiles. Our data suggest that the metabolic profile of SCFA profile may be the most suitable and robust read-out to characterize microbiota-modulating effects of a DF and highlights importance to understand the inter-individual response to a prebiotic treatment for mechanistic understanding and human application.

Genomic assessment in Lactobacillus plantarum links the butyrogenic pathway with glutamine metabolism.

The butyrogenic capability of Lactobacillus (L.) plantarum is highly dependent on the substrate type and so far not assigned to any specific metabolic pathway. Accordingly, we compared three genomes of L. plantarum that showed a strain-specific capability to produce butyric acid in human cells growth media. Based on the genomic analysis, butyric acid production was attributed to the complementary activities of a medium-chain thioesterase and the fatty acid synthase of type two (FASII). However, the genomic islands of discrepancy observed between butyrogenic L. plantarum strains (S2T10D, S11T3E) and the non-butyrogenic strain O2T60C do not encompass genes of FASII, but several cassettes of genes related to sugar metabolism, bacteriocins, prophages and surface proteins. Interestingly, single amino acid substitutions predicted from SNPs analysis have highlighted deleterious mutations in key genes of glutamine metabolism in L. plantarum O2T60C, which corroborated well with the metabolic deficiency suffered by O2T60C in high-glutamine growth media and its consequent incapability to produce butyrate. In parallel, the increase of glutamine content induced the production of butyric acid by L. plantarum S2T10D. The present study reveals a previously undescribed metabolic route for butyric acid production in L. plantarum, and a potential involvement of the glutamine uptake in its regulation.

Potential probiotic Pichia kudriavzevii strains and their ability to enhance folate content of traditional cereal-based African fermented food.

With the aim of selecting starter cultures with interesting probiotic potential and with the ability to produce folate in a food matrix, yeast strains isolated from fermented cereal-based African foods were investigated. A total of 93 yeast strains were screened for their tolerance to pH 2 and 0.3% of bile salts. Pichia kudriavzevii isolates gave the best results. Selected P. kudriavzevii strains were tested for survival to the simulated human digestion and for adhesion to Caco-2 cells. Moreover, presence of folate biosynthesis genes was verified and production of extra and intra-cellular folate determined during growth in culture medium. 31% of yeast strains could tolerate pH 2, while 99% bile salts. Survival rate after simulated digestion ranged between 11 and 45%, while adhesion rate between 12 and 40%. Folate production was mainly intracellular, maximum after 24 h of growth. To be closer to traditional cereal-based fermentations, a P. kudriavzevii strain with good probiotic potential was co-inoculated with Lactobacillus fermentum strains in a pearl millet gruel. This resulted in in situ folate production that peaked after 4 h. The use of strains with both probiotic and nutritional enrichment properties may have a greater impact for the consumers.

Impact of Nisin-Activated Packaging on Microbiota of Beef Burgers during Storage.

Beef burgers were stored at 4°C in a vacuum in nisin-activated antimicrobial packaging. Microbial ecology analyses were performed on samples collected between days 0 and 21 of storage to discover the population diversity. Two batches were analyzed using RNA-based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The active packaging retarded the growth of the total viable bacteria and lactic acid bacteria. Culture-independent analysis by pyrosequencing of RNA extracted directly from meat showed that Photobacterium phosphoreum, Lactococcus piscium, Lactobacillus sakei, and Leuconostoc carnosum were the major operational taxonomic units (OTUs) shared between control and treated samples. Beta diversity analysis of the 16S rRNA sequence data and RNA-DGGE showed a clear separation between two batches based on the microbiota. Control samples from batch B showed a significant high abundance of some taxa sensitive to nisin, such as Kocuria rhizophila, Staphylococcus xylosus, Leuconostoc carnosum, and Carnobacterium divergens, compared to control samples from batch A. However, only from batch B was it possible to find a significant difference between controls and treated samples during storage due to the active packaging. Predicted metagenomes confirmed differences between the two batches and indicated that the use of nisin-based antimicrobial packaging can determine a reduction in the abundance of specific metabolic pathways related to spoilage. The present study aimed to assess the viable bacterial communities in beef burgers stored in nisin-based antimicrobial packaging, and it highlights the efficacy of this strategy to prolong beef burger shelf life.

Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains.

Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces marxianus, Millerozyma farinosa, Candida glabrata, Wickerhamomyces anomalus, Hanseniaspora guilliermondii and Debaryomyces nepalensis) were screened for phytase production on solid and liquid media. 95% were able to grow in the presence of phytate as sole phosphate source, P. kudriavzevii being the best growing species. A phytase coding gene of P. kudriavzevii (PHYPk) was identified and its expression was studied during growth by RT-qPCR. The expression level of PHYPk was significantly higher in phytate-medium, compared to phosphate-medium. In phytate-medium expression was seen in the lag phase. Significant differences in gene expression were detected among the strains as well as between the media. A correlation was found between the PHYPk expression and phytase extracellular activity.

Monitoring of the microbiota of fermented sausages by culture independent rRNA-based approaches.

In Italy, fermented sausages (called "salami") are consumed in large quantities. Salami samples from a local meat factory in the area of Torino were analyzed at 0, 3, 7, 30 and 45 days of ripening. Swab samples from the production environment were also collected at the beginning of the experiment. The diversity of metabolically active microbiota occurring during the natural fermentation of salami was evaluated by using RT-PCR-DGGE coupled with RNA-based pyrosequencing of the 16S rRNA gene. A culture-dependent approach was also applied to identify and characterize isolated Staphylococcaceae and LAB populations. Staphylococcus succinus, Staphylococcus xylosus and Lactobacillus sakei were the species most frequently isolated during the maturation time. Rep-PCR analysis showed that S. succinus and S. xylosus isolated from swabs and salami samples clustered together, suggesting possible contamination during the production process. RT-PCR-DGGE and rRNA-based pyrosequencing showed that the metabolically active populations were dominated by S. succinus, Lb. sakei and Leuconostoc carnosum. In this specific case study, only a few species belonging to Staphylococcaceae, Lactobacillaceae and Leuconostocaceae may be metabolically active and contribute to determine the final characteristics of the products.

Gene transcription patterns of pH- and salt-stressed Listeria monocytogenes cells in simulated gastric and pancreatic conditions.

A Listeria monocytogenes subgenomic array, targeting 54 genes involved in the adhesion, adaptation, intracellular life cycle, invasion, and regulation of the infection cycle was used to investigate the gene expression patterns of acid- and salt-stressed Listeria cells after exposure to conditions similar to those in gastric and pancreatic fluids. Three L. monocytogenes strains, one laboratory reference strain (EGDe) and two food isolates (wild strain 12 isolated from milk and wild strain 3 isolated from fermented sausage), were used during the studies. Differences in the expressed genes were observed between the gastric and pancreatic treatments and also between the serotypes. Increased transcripts were observed of the genes belonging to the adaptation and regulation group for serotype 4b (strain 12) and to the invasion and regulation group for serotype 1/2a (strain EGDe). Interestingly, no significantly differentially expressed genes were found for serotype 3c (strain 3) in most cases. The genes related to adaptation (serotype 1/2a) and to intracellular life cycle and invasion (serotype 4b) were down-regulated in order to cope with the hostile environment of the gastric and pancreatic fluids. These findings may provide experimental evidence for the dominance of serotypes 1/2a and 4b in clinical cases of listeriosis and for the sporadic occurrence of serotype 3c.