Composition and metabolite profile of Kenyan infant donor fecal microbiota
Freshly collected fecal samples from 9 Kenyan infants were transported under anaerobic and cooled conditions from Kenya to Switzerland for processing, immobilization and reactor inoculation within 28 to 35 h after collection [12]. The dominant genus in all fecal microbiota was Bifidobacterium (60 ± 14%) except for fecal microbiota of infant 03, which was dominated by Bacteroides (26%), Veillonella (24%) and Bifidobacterium (23%) (Fig. 1a). Total fecal metabolites differed between the infants with concentrations ranging between 106 µmol/g feces (infant 3) and 421 µmol/g feces (infant 08) (Fig. 1b). Acetate was the most abundant fecal metabolite (41 to 73% of total metabolites). A high variability in detection and proportions of propionate (2 to 18%), butyrate (2 to 15%), formate (10 to 19%) and lactate (4 to 54%) was observed. The proportions of valerate and BCFA were low in the fecal samples of all infants when detected (ranging from 1 to 3% of total metabolites).
In summary, the Kenyan infant donor fecal microbiota was characterized by high proportions of Bifidobacterium and acetate, while the proportions and presence of other taxa and metabolites was variable. The accordance with results obtained by previous studies in the same infant population confirms the representativity of the fecal samples used in this study to assess the optimal conditions for the continuous cultivation in the PolyFermS model [5, 12].
Effect of different FOS doses on PolyFermS Kenyan infant fecal microbiota
Different concentrations of FOS were added to the cultivation medium, simulating the Kenyan infant diet and ileal chyme. The objective was to determine wether FOS could promote the in vitro growth of Bifidobacterium. This assessment was conducted using three independent PolyFermS models, where each bioreactor contained immobilized fecal microbiota of one Kenyan infant (Fig. 2). Composition and metabolite profile of the bioreactor effluent in vitro microbiota (IVM) were compared to the respective fecal inoculum.
Quantitative PCR enumeration of key bacterial taxa
IVM 01 and 02 were cultivated with 4 g/L and 8 g/L of FOS and IVM 03 was tested without supplementation and with 1 g/L and 4 g/L of FOS. Compared to the corresponding fecal inoculum, lower concentrations of Bifidobacterium in reactors effluent were detected for all FOS doses tested (Supplementary table 1). The concentrations of Bifidobacterium were lowest with 8 g/L of FOS and 0.99 and 0.60 log lower compared to 4 g/L of FOS for IVM 01 and 02, respectively. Concomitant, growth stimulation of Ruminococcaceae was observed with 8 g/L FOS compared to 4 g/L and to much higher concentrations than detected in the fecal microbiota. Therefore, in a next step, lower FOS doses of 1 g/L and 4 g/L were assessed for IVM 03. FOS supplementation did not impact Bifidobacterium concentrations in IVM 03, but higher concentrations of Ruminococcaceae were measured with 4 g/L FOS compared to the non-supplemented condition. FOS-induced infant-specific changes in key bacterial taxa were also detected. Concentrations of Lachnospiraceae, Enterobacteriaceae and Bacteroides were decreased in IVM 01 and Lachnospiraceae concentrations were lower in IVM 02 with 8 g/L compared to 4 g/L of FOS. Increased concentrations of Lachnospiraceae, Veillonella and Lactobacillus/Leuconostoc/Pediococcus (LLP) were detected in IVM 03 with 4 g/L of FOS compared to non-supplemented condition (Supplementary table 1).
In summary, FOS supplementation stimulated the growth of Ruminococcaceae in all IVM while Bifidobacterium decreased in IVM 01 and 02 supplemented with 8 g/L of FOS compared to 4 g/L.
Sequencing-based microbial community profile
Structural and compositional similarity of fecal and in vitro microbiota were evaluated with beta diversity metrics. The similarity between both was significantly lower for IVM 01 and 02 cultivated with 8 g/L of FOS compared to 4 g/L based on weighted Jaccard similarity index while no differences were observed for IVM 03 (Fig. 3a).
In line with quantitative PCR (qPCR) analysis, the relative abundance of Bifidobacterium was lower in vitro compared to the fecal inoculum microbiota in all three IVM (Fig. 3b). In IVM 01 and 02, supplementation with 8 g/L compared to 4 g/L of FOS, promoted Faecalibacterium (35.5% and 35.0% relative abundance versus 0 and < 0.1%, respectively) at the expense of Bacteroides (13.6% and 14.6% versus 27% and 25.6%, respectively) and Bifidobacterium (2.1% and 6.5% versus 13.7% and 21.2%, respectively). In IVM 03, the relative abundance of Bifidobacterium was low (1.6 to 1.9%) independent of FOS treatment, while the relative abundance of Faecalibacterium was also increased with FOS supplementation (13.2% with 4 g/L, 7.1% with 1 g/L and 8.7% without FOS). Several bacterial genera harbouring potential pathogens were detected in IVM effluents but at a low er relative abundance compared to the respective fecal inoculum. For example, the relative abundance of Escherichia-Shigella was below 1% (0 to 0.6%) in all IVM, while it ranged from 4.0–4.5% in the fecal microbiota. Also, Klebsiella was detected at a relative abundance of 8.4% in fecal microbiota of infant 03 but only at 0.1–0.4% in IVM 03.
Differential abundance analysis confirmed the promotion of Faecalibacterium with increasing FOS doses in vitro (Supplementary Fig. 1). Faecalibacterium (ASV 037 assigned to F. prausnitzii) showed an increase in relative abundance with 8 g/L versus 4 g/L of FOS in IVM 01 (log2-fold increase 16.9) and IVM 02 (log2-fold increase 13.9), and with 1 g/L versus no FOS and 4 g/L versus 1 g/L of FOS in IVM 03 (log2-fold increase 1). Concomitant, a decreased relative abundance of Bifidobacterium was detected with 8 g/L versus 4 g/L of FOS in IVM 01 (log2-fold decrease − 2.3) and IVM 02 (log2-fold decrease − 1.2).
Increasing FOS doses also impacted alpha diversity in vitro. The community richness of IVM 01 was lower at 8 g/L compared to 4 g/L of FOS and compared to the corresponding fecal microbiota (Supplementary Fig. 2). Furthermore, the community evenness of IVM 01 and 02 was higher compared to the corresponding fecal microbiota and increasing FOS doses reduced the evenness in all tested IVM, which might be explained by the strong promotion of Faecalibacterium.
Fermentation metabolite profile
Compared to the fecal metabolite profile, the ratio of intermediate to total metabolites decreased, and that of propionate and butyrate increased in IVM 01 and 02 (Fig. 3c). Butyrate production was largely enhanced in IVM 01 and 02 with 8 g/L of FOS compared to 4 g/L, representing 41% and 42% versus 11% and 16% of total metabolites, respectively, and at the expense of acetate. The metabolite profile of IVM 03 was very similar compared to the fecal metabolite profile, independent of the FOS treatment, except for lactate which was only detected in the feces.
Overall, Bifidobacterium and genera harbouring potential pathogens were maintained in vitro but at lower abundance compared to the fecal microbiota. With the high FOS doses of 8 g/L and 4 g/L the relative abundance of Bifidobacterium decreased concomitant with increased Faecalibacterium and largely enhanced butyrate production, compared to the fecal microbiota. Therefore, FOS at 1 g/L, was selected for reproducing a representative profile of the Kenyan infant fecal microbiota in the in vitro PolyFermS fermentation model.
Effect of pH on in vitro establishment of Kenyan infant fecal microbiota
The effect of an increased cultivation pH from 5.8 to 6.3 was tested in four continuous fermentation models inoculated with fecal microbiota from infant 04 to 07 to assess if a higher cultivation pH supports the in vitro establishment of potential enteropathogens present in Kenyan infant feces.
qPCR enumeration of key bacterial taxa
Cultivation pH had a donor-dependent impact on the growth of Enterobacteriaceae, a bacterial family that harbours many potential pathogenic genera including Escherichia, Shigella and Salmonella. Enterobacteriaceae concentrations were 1.6 log higher at pH 6.3 compared to pH 5.8 in IVM 04, while they were 0.5 and 0.8 log lower in IVM 06 and 07, respectively (Supplementary table 2). A pH of 6.3 promoted the growth of Bacteroides in IVM 04 (+ 0.5 log) and IVM 06 (+ 2.6 log), and of Lachnospiraceae in IVM 04 (+ 0.8 log) and IVM 05 (+ 0.5 log) compared to pH 5.8. Moreover, higher cultivation pH 6.3 decreased the concentrations of infant-characteristic Lactobacillus/Leuconostoc/Pediococcus in IVM 04 to 07 (-0.5 to -2.2 log) and Bifidobacterium in IVM 04 to 06 (-0.1 to -1.2 log) compared to pH 5.8. Veillonella concentrations were also 1.7 log lower at pH 6.3 compared to 5.8 in IVM 06.
Sequencing-based microbial community profile
The community distance of IVM 05 and IVM 06 to the corresponding fecal microbiota was higher after cultivation at pH 6.3 compared to pH 5.8 (Fig. 4a). Infant-specific and common compositional shifts in relative genera abundance were detected after cultivation (Fig. 4b). The relative abundance of Escherichia-Shigella was low in all IVM (0.02–6.32%) compared to the fecal microbiota (3.3–29.1%) and an enrichment of Bacteroides (12.6–38.5% in IVM versus 0.2–2.0% in feces) was observed in IVM 04, 05 and 07. Increased cultivation pH resulted in several infant-specific shifts in relative genus abundance. For example, in IVM 04 at pH 6.3 compared to 5.8, the relative abundance of Enterococcus was increased (18.9% at pH 6.3 versus 0.2% at pH 5.8, respectively) at the expense of Lactobacillus (0.2% versus 37.7%, respectively). In IVM 05, the relative abundance of Faecalibacterium (11% versus 0%, respectively) and Blautia (9% versus 0%, respectively) was increased while the relative abundance of Prevotella (2% versus 15%, respectively) was decreased at pH 6.3 compared to 5.8. In IVM 06, the strongest increase in relative abundance at pH 6.3 compared to 5.8 was observed for Clostridium sensu stricto 1 (23% versus 4%, respectively), Dialister (16% versus 1%, respectively) and Actinomyces (26% versus 0%, respectively). Furthermore, at pH 6.3, the relative abundance of Bifidobacterium was strongly decreased to 0.2% and 3.9% in IVM 04 and 06, respectively, compared to pH 5.8 where it was among the three most abundant genera (27% and 37%, respectively). In contrast, independent of cultivation pH, Bifidobacterium relative abundance was low in IVM 05 and 07 (0.2–2.1%).
Differential abundance analysis was performed to detect pH-induced significant differences in relative genus abundance. Higher cultivation pH 6.3 promoted genera harbouring potential pathogenic gut members (Supplementary Fig. 3). The relative abundance of Escherichia-Shigella increased at pH 6.3 compared to 5.8 for IVM 04, 05 and 06 with a log2-fold increase from 3.0 to 3.9. Clostridium sensu stricto 1 was also promoted at pH 6.3 as indicated by a log2-fold increase of 7.3 and 2.9 compared to pH 5.8 in IVM 05 and 06, respectively. Moreover, at pH 6.3 compared to pH 5.8, a log2-fold increase of 10.9 and 3.2 was detected for Campylobacter and Clostridioides in IVM 06 and 07, respectively. On the other hand, and in line with qPCR results, cultivation pH 6.3 decreased the relative abundance of infant-characteristic beneficial gut taxa such as Bifidobacterium in IVM 04, 06 and 07 and Lactobacillus in IVM 04 and 06, respectively, at pH 6.3 compared to 5.8.
Alpha diversity was also affected by cultivation pH, with a much higher number of observed ASVs at pH 6.3 (85 ± 4 ASVs) compared to 5.8 (49 ± 6 ASVs) in IVM 05 and compared to its fecal inoculum (38 ASVs) (Supplementary Fig. 4). The community evenness of all IVM was higher compared to the corresponding fecal microbiota and cultivation at pH 6.3 compared to 5.8 further increased the evenness in IVM 04 and 06.
Fermentation metabolite profile
Total metabolite concentrations were lower in all IVM compared to the fecal microbiota (Fig. 4c). Compared to the fecal metabolite profile, proportions of intermediate metabolites were decreased concomitant with increased proportions of acetate, propionate and butyrate in all IVM except for IVM 05 (due to formate production in vitro). Cultivation pH 6.3 stimulated propionate production while pH 5.8 promoted butyrate and valerate production (except in IVM 06 where butyrate was not detected at pH 5.8). Large increase of propionate proportion (33 ± 1% at pH 6.3 versus 12 ± 2% at pH 5.8), concomitant with decreased acetate proportions were observed for IVM 06.
In summary, higher cultivation pH 6.3 stimulated propionate production and genera harbouring potential pathogenic gut members compared to pH 5.8. However, infant-characteristic Bifidobacterium and Lactobacillus were better maintained in vitro at pH 5.8 resulting in higher similarity between fecal and IVM compared to pH 6.3. Therefore, pH 5.8 was chosen for long-term continuous cultivation.
Model stability during long-term continuous cultivation of four Kenyan infant fecal microbiota
Four Kenyan infant fecal microbiota were continuously cultivated for up to 107 days to assess the community stability at the selected conditions (pH 5.8 and 1g/L FOS).
Sequencing-based microbial community profile
Principal coordinate analysis (PCoA) was conducted to assess whether the infant-specific fecal microbiota profile was maintained in vitro during cultivation at pH 5.8 with 1 g/L of FOS (Fig. 5a). PCoA of binary Jaccard showed clustering of in vitro microbial communities by infant. However, in the PCoA of weighted Jaccard, all the fecal microbial communities clustered together, which is likely due to similar high relative abundance of Bifidobacterium in contrast to the IVM. The percentage of shared genera between feces and IVM was stable over 107 days of continuous cultivation at 52 ± 5%, 61 ± 4%, 56 ± 3% and 47 ± 3% for IVM 06, 07, 08 and 09, respectively (Fig. 5b). When only the most abundant genera (≥ 1%) were considered, the percentage of shared genera between feces and IVM was higher at 89 ± 5%, 78 ± 5%, 86 ± 4% and 74 ± 4% for IVM 06, 07, 08 and 09, respectively.
Compared to the fecal microbiota, Lactococcus, Streptococcus, Bacteroides and Collinsella were enriched in vitro (+ 8% to + 40% compared to feces) concomitant with a decrease in Bifidobacterium abundance (-28% to -65% compared to feces) (Fig. 6). However, the abundant genera of all IVM were maintained over time, except for Olsenella outcompeting Streptococcus in IVM 07. Further, the abundance of Streptococcus was high (28–35%) in IVM 06 from day 20 onwards compared to below 1% at day 9–11. The community richness decreased while the evenness increased in all IVM compared to the fecal inoculum (Supplementary Fig. 5). Alpha diversity was stable over the course of fermentation and similar between all IVM (observed ASVs: 46–55, Pielou’s evenness index: 0.60–0.66). Only IVM 06 showed a significant but small increase of evenness (0.07, p = 0.02) from day 9–11 to day 98–100.
Fermentation metabolite profile
The main metabolites detected in all IVM were acetate, followed by propionate, formate and butyrate (Fig. 7). After the initial 2 days of continuous fermentation, the intermediate metabolite lactate decreased to very low or non-detectable levels in all IVM. IVM 06, 08 and 09 established a propiogenic metabolite profile characterized by main SCFA ratio (acetate:propionate:butyrate) of 76:19:5, 66:26:8 and 74:18:8, respectively, in agreement with the propiogenic fecal metabolite profile (Fig. 3). In contrast, IVM 07 developed a butyrogenic profile with a main SCFA ratio of 57:15:28, while the fecal metabolite profile was propiogenic (83:13:4). The metabolite profiles were maintained over the complete cultivation period in all IVM, except for IVM 09 where a decrease in propionate between day 23 and 33 was observed followed by a recovery. Total metabolic activity over time was assessed considering total carbon concentrations to account for different carbon content of fermentation metabolites. The coefficient of variation over the complete fermentation time was comparable between IVM with 19%, 13%, 11% and 14% for IVM 06, 07, 08 and 09, respectively.
In summary, the Kenyan infant PolyFermS model maintained the most abundant genera, community diversity and specific metabolite profiles in IVM effluents over up to 107 days continuous fermentation.