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Abstract Background Folate (vitamin B9) occurs naturally mainly as tetrahydrofolate (THF), methyl-tetrahydrofolate (M-THF), and formyl-tetrahydrofolate (F-THF), and as dietary synthetic form (folic acid). While folate auxotrophy and prototrophy are known for several gut microbes, the specific folate forms produced by gut prototrophs and their impact on gut auxotrophs and microbiota remain unexplored. Methods Here, we quantified by UHPLC-FL/UV folate produced by six predicted gut prototrophs (Marvinbryantia formatexigens DSM 14469, Blautia hydrogenotrophica 10507 T , Blautia producta DSM 14466, Bacteroides caccae DSM 19024, Bacteroides ovatus DSM 1896, and Bacteroides thetaiotaomicron DSM 2079 T) and investigated the impact of different folate forms and doses (50 and 200 µg/l) on the growth and metabolism of the gut auxotroph Roseburia intestinalis in pure cultures and during fecal anaerobic batch fermentations (48 h, 37 °C) of five healthy adults. Results Our results confirmed the production of folate by all six gut strains, in the range from 15.3 ng/ml to 205.4 ng/ml. Different folate forms were detected, with THF ranging from 12.8 to 41.4 ng/ml and 5-MTHF ranging from 0.2 to 113.3 ng/ml, and being detected in all strains. Natural folate forms, in contrast to folic acid, promoted the growth and metabolism of the auxotroph R. intestinalis L1-82, with dose-dependent effects. During fecal batch fermentations, folate forms at both levels had no detectable effect on total bacteria concentration, on gut community composition and metabolic activity and on Roseburia spp. abundance, compared to the control without folate addition. Conclusions Our study demonstrates for the first time in vitro the production of different natural folate forms by predicted gut prototrophs and the stimulation on the growth of the folate auxotrophic butyrate-producing R. intestinalis L1-82. Surprisingly, folate did not impact fecal fermentations. Our data suggest that the dietary folate forms at the tested levels may only have limited effects, if any, on the human gut microbiota in vivo.

Abstract 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.

Abstract Appropriate in vitro models to investigate the impact of novel nutritional strategies on the gut microbiota of infants living in rural Africa are scarce. Here, we aimed to develop such a continuous gut fermentation model based on the PolyFermS platform, which allows controlled and stable long-term cultivation of colon microbiota in conditions akin the host. Nine immobilized Kenyan infant fecal microbiota were used as inoculum for continuous PolyFermS colon models fed with medium mimicking the weaning infant diet. Fructo-oligosaccharides (FOS) supplementation (1, 4 and 8 g/L) and cultivation pH (5.8 and 6.3) were investigated stepwise. Conditions providing a close match between fecal and in vitro microbiota (pH 5.8 with 1 g/L FOS) were selected for investigating long-term stability of four Kenyan infant PolyFermS microbiota. The shared fraction of top bacterial genera between fecal and in vitro microbiota was high (74–89%) and stable during 107 days of continuous cultivation. Community diversity was maintained and two distinct fermentation metabolite profiles of infant fecal microbiota were observed. Three propiogenic and one butyrogenic metabolite profile of infant fecal microbiota established from day 8 onwards and stayed stable. We present here the first rationally designed continuous cultivation model of African infant gut microbiota. This model will be important to assess the effect of dietary or environmental factors on the gut microbiota of African infants with high enteropathogen exposure.

Abstract Background The combination of cultivation studies with molecular analysis approaches allows characterization of the complex human gut microbiota in depth. In vitro cultivation studies of infants living in rural sub-Saharan Africa are scarce. In this study, a batch cultivation protocol for Kenyan infant fecal microbiota was validated. Methods Fresh fecal samples were collected from 10 infants living in a rural area of Kenya. Samples were transported under protective conditions and subsequently prepared for inoculation within less than 30 h for batch cultivation. A diet-adapted cultivation medium was used that mimicked the daily intake of human milk and maize porridge in Kenyan infants during weaning. 16 S rRNA gene amplicon sequencing and HPLC analyses were performed to assess the composition and metabolic activity, respectively, of the fecal microbiota after 24 h of batch cultivation. Results High abundance of Bifidobacterium (53.4 ± 11.1%) and high proportions of acetate (56 ± 11% of total metabolites) and lactate (24 ± 22% of total metabolites) were detected in the Kenyan infant fecal microbiota. After cultivation started at an initial pH 7.6, the fraction of top bacterial genera (≥ 1% abundant) shared between fermentation and fecal samples was high at 97 ± 5%. However, Escherichia-Shigella, Clostridium sensu stricto 1, Bacteroides and Enterococcus were enriched concomitant with decreased Bifidobacterium abundance. Decreasing the initial pH to 6.9 lead to higher abundance of Bifidobacterium after incubation and increased the compositional similarity of fermentation and fecal samples. Despite similar total metabolite production of all fecal microbiota after cultivation, inter-individual differences in metabolite profiles were apparent. Conclusions Protected transport and batch cultivation in host and diet adapted conditions allowed regrowth of the top abundant genera and reproduction of the metabolic activity of fresh Kenyan infant fecal microbiota. The validated batch cultivation protocol can be used to study the composition and functional potential of Kenyan infant fecal microbiota in vitro.

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.

Abstract Several gut microbial species within the Faecalibacterium genus have emerged as promising next‐generation probiotics (NGP) due to their multifunctional protective effects against gastrointestinal and systemic disorders. To enable clinical studies and further applications, improved methods for cultivating Faecalibacterium must be developed in compliance with current Good Manufacturing Practice regulations, which is complicated by its oxygen sensitivity and complex nutritional requirements. Different yeast‐based nutrients (YBNs), including yeast extracts (YEs) and yeast peptones (YPs), are ubiquitously used when cultivating microbes to supply a broad range of macro‐ and micronutrients. In this study, we evaluated six experimental YBNs, namely three YEs, two YPs and a yeast cell wall product (YCW), and eight B‐vitamins in the cultivation of Faecalibacterium duncaniae A2‐165, former Faecalibacterium prausnitzii, using growth assays in microtitre plates, dose‐effect studies in Hungate tube fermentations and fully controlled bioreactor experiments. We demonstrated that YEs promote F. duncaniae A2‐165 growth in a nutritionally limited medium, while YPs and YCW lacked essential growth factors for enabling cell propagation. High cell density was obtained in controlled bioreactors using a medium containing 2–4% of a selected YE and 1% casein peptone (3.4 ± 1.7 × 109–5.1 ± 1.3 × 109 cells mL−1). Among all tested B‐vitamins, we identified B5 as a strong growth promoter. Replacing casein peptone with YP and supplementing with vitamin B5 further increased biomass by approximately 50% (6.8 ± 1.7 × 109 cells mL−1). Hence, empirical selection of YE, YP and B5 allowed formulation of a high‐yielding animal allergen‐free nutritive medium to produce F. duncaniae A2‐165. Selecting nutritionally suitable YBNs and combining these with other key nutrients are important steps for optimizing production of NGP with high yields and lower cost.

California poppy products are commonly used for the treatment of nervousness, anxiety and sleeping disorders. Pharmacologically relevant constituents include the main alkaloids californidine, escholtzine and protopine. However, only limited information is available about the alkaloid content in commercial preparations and their intestinal absorption. Moreover, a possible metabolization of these alkaloids by the gut microbiota, and their impact on microbial activity and viability have not been investigated. Californidine, escholtzine and protopine were quantified by UHPLC-MS/MS in eight commercial California poppy products. The intestinal permeability of alkaloids was studied in Caco-2 cell as a model for absorption in the small intestine. The gut microbial biotransformation was explored in artificial gut microbiota from the in vitro PolyFermS model. In addition, the impact of these alkaloids and a California poppy extract on the microbial production of short-chain fatty acids (SCFAs) and the viability of microbiota was investigated. Contents of californidine, escholtzine and protopine in California poppy products were in the ranges of 0.13–2.55, 0.05–0.63 and 0.008–0.200 mg/g, respectively. In the Caco-2 cell model, californidine was low-to-moderately permeable while escholtzine and protopine were highly permeable. An active transport process was potentially involved in the transfer of the three alkaloids. The three compounds were not metabolized by the artificial gut microbiota over 24 h. Neither the California poppy extract nor the alkaloids markedly impacted microbial SCFA production and bacterial viability.

Phytomedicines such as valerian and St. John’s wort are widely used for the treatment of sleeping disorders, anxiety and mild depression. They are perceived as safe alternatives to synthetic drugs, but limited information is available on the intestinal absorption and interaction with human intestinal microbiota of pharmacologically relevant constituents valerenic acid in valerian, and hyperforin and hypericin in St. John’s wort. The intestinal permeability of these compounds and the antidepressant and anxiolytic drugs citalopram and diazepam was investigated in the Caco-2 cell model with bidirectional transport experiments. In addition, interaction of compounds and herbal extracts with intestinal microbiota was evaluated in artificial human gut microbiota. Microbiota-mediated metabolisation of compounds was assessed, and bacterial viability and short-chain fatty acids (SCFA) production were measured in the presence of compounds or herbal extracts. Valerenic acid and hyperforin were highly permeable in Caco-2 cell monolayers. Hypericin showed low-to-moderate permeability. An active transport process was potentially involved in the transfer of valerenic acid. Hyperforin and hypericin were mainly transported through passive transcellular diffusion. All compounds were not metabolized over 24 h in the artificial gut microbiota. Microbial SCFA production and bacterial viability was not substantially impaired nor promoted by exposure to the compounds or herbal extracts.

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.

Abstract Background Auto-aggregation is a desired property for probiotic strains because it is suggested to promote colonization of the human intestine, to prevent pathogen infections and to modulate the colonic mucosa. We recently reported the generation of adapted mutants of Lactiplantibacillus plantarum NZ3400, a derivative of the model strain WCFS1, for colonization under adult colonic conditions of PolyFermS continuous intestinal fermentation models. Here we describe and characterize the emerge of an auto-aggregating phenotype in L. plantarum NZ3400 derivatives recovered from the modelled gut microbiota. Results L. plantarum isolates were recovered from reactor effluent of four different adult microbiota and from spontaneously formed reactor biofilms. Auto-aggregation was observed in L. plantarum recovered from all microbiota and at higher percentage when recovered from biofilm than from effluent. Further, auto-aggregation percentage increased over time of cultivation in the microbiota. Starvation of the gut microbiota by interrupting the inflow of nutritive medium enhanced auto-aggregation, suggesting a link to nutrient availability. Auto-aggregation was lost under standard cultivation conditions for lactobacilli in MRS medium. However, it was reestablished during growth on sucrose and maltose and in a medium that simulates the abiotic gut environment. Remarkably, none of these conditions resulted in an auto-aggregation phenotype in the wild type strain NZ3400 nor other non-aggregating L. plantarum, indicating that auto-aggregation depends on the strain history. Whole genome sequencing analysis did not reveal any mutation responsible for the auto-aggregation phenotype. Transcriptome analysis showed highly significant upregulation of LP_RS05225 (msa) at 4.1–4.4 log2-fold-change and LP_RS05230 (marR) at 4.5–5.4 log2-fold-change in all auto-aggregating strains compared to non-aggregating. These co-expressed genes encode a mannose-specific adhesin protein and transcriptional regulator, respectively. Mapping of the RNA-sequence reads to the promoter region of the msa-marR operon reveled a DNA inversion in this region that is predominant in auto-aggregating but not in non-aggregating strains. This strongly suggests a role of this inversion in the auto-aggregation phenotype. Conclusions L. plantarum NZ3400 adapts to the in vitro colonic environment by developing an auto-aggregation phenotype. Similar aggregation phenotypes may promote gut colonization and efficacy of other probiotics and should be further investigated by using validated continuous models of gut fermentation such as PolyFermS.

Humans ingest many microorganisms, which may colonize and interact with the resident gut microbiota. However, extensive knowledge about host-independent microbe-microbe interactions is lacking. Here, we investigated such colonization process using a derivative of the model probiotic Lactiplantibacillus plantarum WCFS1 into continuously cultivated gut microbiota in the intestinal PolyFermS fermentation model inoculated with five independently immobilized human adult fecal microbiota. L. plantarum successfully colonized and organized itself spatially in the planktonic, that is, the reactor effluent, and sessile, that is, reactor biofilm, fractions of distinct human adult microbiota. The microbiota carrying capacity for L. plantarum was independent of L. plantarum introduction dose and second supplementation. Adult microbiota (n = 3) dominated by Prevotella and Ruminoccocus exhibited a higher carrying capacity than microbiota (n = 2) dominated by Bacteroides with 105 and 103 CFU/ml of L. plantarum, respectively. Cultivation of human adult microbiota over 3 months resulted in decreased carrying capacity and correlated positively with richness and evenness, suggesting enhanced resistance toward colonizers. Our analyses ultimately allowed us to identify the fermentation metabolite valerate as a modulator to increase the carrying capacity in a microbiota-independent manner. In conclusion, by uncoupling microbe-microbe interactions from host factors, we showed that L. plantarum colonizes the in vitro colonic community in a microbiota-dependent manner. We were further able to demonstrate that L. plantarum colonization levels were not susceptible to the introduction parameters dose and repeated administration but to microbiota features. Such knowledge is relevant in gaining a deeper ecological understanding of colonizer-microbiota interactions and developing robust probiotic strategies.

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.

Dietary fibers such as arabinoxylan (AX) are promising food constituents to prevent particular diet-related chronic diseases because of their prebiotic properties. Arabinoxylan fermentation by the gut microbiota depends on the structural architecture of AX, which can be modified during food processing and consequently affect its prebiotic potential, but it is little investigated. Therefore, the aim of this study was to evaluate the effects of naturally occurring and processing-induced structural alterations of the soluble AX of wheat bran and rye flour on the in vitro human colon fermentation. It was found that fermentation behavior is strongly linked to the AX fine structure and their processing-induced modifications. The short-chain fatty acid (SCFA) metabolism, acidification kinetics, bacterial growth, and bacterial composition revealed that wheat bran AX (WBAX) was fermented faster than rye flour AX. Increased levels of bound phenolic acids resulting from processing were identified as the inhibiting factor for AX fermentation kinetics. Bacterial genera promoted by AX varied between AX source and processing type, but also between microbiota. Extruded WBAX promoted butyrate production and growth of butyrate-producing Faecalibacterium in the butyrogenic microbiota while it did not enhance fermentation and inhibited the growth of Prevotella in the propiogenic microbiota. We anticipate that the findings of this study are a starting point for further investigation on the impact of processing-induced changes on the prebiotic potential of dietary fibers prior to human studies.

Abstract Background Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia coli (EHEC) infection, whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities. Results We utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites—4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid—that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity. Conclusion Organ-on-chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection.

ABSTRACT Research and marketing of probiotics demand holistic strain improvement considering both the biotic and abiotic gut environment. Here, we aim to establish the continuous in vitro colonic fermentation model PolyFermS as a tool for adaptive evolutionary engineering. Immobilized fecal microbiota from adult donors were steadily cultivated up to 72 days in PolyFermS reactors, providing a long-term compositional and functional stable ecosystem akin to the donor’s gut. Inoculation of the gut microbiota with immobilized or planktonic Lactiplantibacillus plantarum NZ3400, a derivative of the probiotic model strain WCFS1, led to successful colonization. Whole-genome sequencing of 45 recovered strains revealed mutations in 16 genes involved in signaling, metabolism, transport, and cell surface. Remarkably, mutations in LP_RS14990, LP_RS15205, and intergenic region LP_RS05100

ABSTRACT 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.

ABSTRACT The metabolism of lactate impacts infant gut health and may lead to acute accumulation of lactate and/or H2 associated with pain and crying of colicky infants. Because gut microbiota studies are limited due to ethical and safety concerns, in vitro fermentation models were developed as powerful tools to assess effects of environmental conditions on the gut microbiota. In this study, we established a continuous colonic fermentation model (PolyFermS), inoculated with immobilized fecal microbiota and mimicking the proximal colon of 2-month-old infants. We investigated the effects of pH and retention time (RT) on lactate metabolism and of lactate-utilizing bacteria (LUB) exhibiting little or no H2 production. We observed that a drop in pH from 6.0 to 5.0 increased the number of lactate-producing bacteria (LPB) and decreased LUB concomitantly with lactate accumulation. Increasing RT from 5 to 10 h at pH 5.0 resulted in complete lactate consumption associated with increased LUB. Supplementation with dl-lactate (60 mM) to mimic lactate accumulation promoted propionate and butyrate production with no effect on acetate production. We further demonstrated that lactate-utilizing Propionibacterium avidum was able to colonize the reactors 4 days after spiking, suggesting its ability to compete with other lactate-utilizing bacteria producing H2. In conclusion, we showed that PolyFermS is a suitable model for mimicking young infant colonic microbiota. We report for the first time pH and RT as strong drivers for composition and metabolic activity of infant gut microbiota, especially for the metabolism of lactate, which is a key intermediate product for ecology and infant health. IMPORTANCE The metabolism of lactate is important for infant gut health and may lead to acute lactate and/or H2 accumulation, pain, and crying as observed in colicky infants. Functional human studies often faced ethical challenges due to invasive medical procedures; thus, in this study, we implemented PolyFermS fermentation models to mimic the infant proximal colon, which were inoculated with immobilized fecal microbiota of two 2-month-old infants. We investigated the impact of pH, retention time, and accumulation of dl-lactate on microbiota composition and metabolic activity. We found that a drop in pH from 6.0 to 5.0 led to increased LPB and decreased LUB concomitantly with lactate accumulation. Increasing the RT resulted in complete lactate consumption associated with increased LUB. Our data highlight for the first time the impact of key abiotic factors on the metabolism of lactate, which is an important intermediate product for ecology and infant health.

Milk oligosaccharides exert a prebiotic action that contributes to the development of the infant gut microbiota during lactation. Given that milk oligosaccharides remain intact after passage through stomach and small intestine, they can potentially influence the composition of the gut microbiota when ingested as dietary supplements after weaning. To address the regulatory effects of specific oligosaccharides in colitis linked to the microbiota composition, we have supplemented interleukin-10 null (Il10-/-) mice with four fucosylated and sialylated oligosaccharides. We found that oral supplementation with 2-fucosyllactose significantly decreased the severity of colitis as displayed by reduced inflammatory marker expression, histological and diarrhea scores, an increased epithelial integrity and less pronounced colon shortening. Oral supplementation with 2-fucosyllactose led to a marked expansion of the commensal Ruminococcus gnavus, which was accompanied by an enhanced cecal concentration of propionate. Decreased activation of immune cells by R. gnavus was confirmed by reconstitution of antibiotic-treated Il10-/- mice and by stimulation of dendritic cells in vitro. This study demonstrates that post-weaning administration of specific oligosaccharides can shift the composition of the gut microbiota to lessen chronic inflammation as observed in Il10-/- mice. The expansion of R. gnavus sets a positive microbial environment at the cost of pro-inflammatory Gram-negative bacteria, thereby lowering intestinal inflammation.

Murine models are valuable tools to study the role of gut microbiota in health or disease. However, murine and human microbiota differ in species composition, so further investigation of the murine gut microbiota is important to gain a better mechanistic understanding. Continuous in vitro fermentation models are powerful tools to investigate microbe-microbe interactions while circumventing animal testing and host confounding factors, but are lacking for murine gut microbiota. We therefore developed a novel continuous fermentation model based on the PolyFermS platform adapted to the murine caecum and inoculated with immobilized caecal microbiota. We followed a stepwise model development approach by adjusting parameters [pH, retention time (RT), growth medium] to reach fermentation metabolite profiles and marker bacterial levels similar to the inoculum. The final model had a stable and inoculum-alike fermentation profile during continuous operation. A lower pH during startup and continuous operation stimulated bacterial fermentation (115 mM short-chain fatty acids at pH 7 to 159 mM at pH 6.5). Adjustments to nutritive medium, a decreased pH and increased RT helped control the in vitro Enterobacteriaceae levels, which often bloom in fermentation models, to 6.6 log gene copies/mL in final model. In parallel, the Lactobacillus, Lachnospiraceae, and Ruminococcaceae levels were better maintained in vitro with concentrations of 8.5 log gene copies/mL, 8.8 log gene copies/mL and 7.5 log gene copies/mL, respectively, in the final model. An independent repetition with final model parameters showed reproducible results in maintaining the inoculum fermentation metabolite profile and its marker bacterial levels. Microbiota community analysis of the final model showed a decreased bacterial diversity and compositional differences compared to caecal inoculum microbiota. Most of the caecal bacterial families were represented in vitro, but taxa of the Muribaculaceae family were not maintained. Functional metagenomics prediction showed conserved metabolic and functional KEGG pathways between in vitro and caecal inoculum microbiota. To conclude, we showed that a rational and stepwise approach allowed us to model in vitro the murine caecal microbiota and functions. Our model is a first step to develop murine microbiota model systems and offers the potential to study microbiota functionality and structure ex vivo.

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at -80 °C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.