Cultured representatives of two major phylogroups of human colonic Faecalibacterium prausnitzii can utilize pectin, uronic acids, and host-derived substrates for growth

Abstract

Faecalibacterium prausnitzii is one of the most abundant commensal bacteria in the healthy human large intestine, but information on genetic diversity and substrate utilization is limited. Here, we examine the phylogeny, phenotypic characteristics, and influence of gut environmental factors on growth of F. prausnitzii strains isolated from healthy subjects. Phylogenetic analysis based on the 16S rRNA sequences indicated that the cultured strains were representative of F. prausnitzii sequences detected by direct analysis of fecal DNA and separated the available isolates into two phylogroups. Most F. prausnitzii strains tested grew well under anaerobic conditions on apple pectin. Furthermore, F. prausnitzii strains competed successfully in coculture with two other abundant pectin-utilizing species, Bacteroides thetaiotaomicron and Eubacterium eligens, with apple pectin as substrate, suggesting that this species makes a contribution to pectin fermentation in the colon. Many F. prausnitzii isolates were able to utilize uronic acids for growth, an ability previously thought to be confined to Bacteroides spp. among human colonic anaerobes. Most strains grew on N-acetylglucosamine, demonstrating an ability to utilize host-derived substrates. All strains tested were bile sensitive, showing at least 80% growth inhibition in the presence of 0.5 μg/ml bile salts, while inhibition at mildly acidic pH was strain dependent. These attributes help to explain the abundance of F. prausnitzii in the colonic community but also suggest factors in the gut environment that may limit its distribution.

Fig 1

Phylogenetic relationship of F. prausnitzii isolates to other members of Clostridium cluster IV (Ruminococcaceae) based on 16S rRNA gene sequences. The tree was constructed using the ARB software package using the neighbor-joining method for distance analysis (Jukes-Cantor algorithm) with 1,533 informative positions considered (61 to 1,442 by E. coli 16S rRNA gene numbering). Bootstrap values above 80% (expressed as a percentage of 1,000 replications) are shown at branching points. Solid circles indicate branches that were consistent with calculations obtained by maximum-parsimony method. Empty circles represent those branches consistent with the maximum likelihood. The scale bar indicates the number of substitutions per site. F. prausnitzii isolates incorporated in this study are highlighted in bold. Sequence accession numbers are shown in parentheses. The database sequence for ATCC 27766 was included, but this strain was not studied here and it is not listed in Table 1.

Fig 2

Neighbor-joining phylogenetic tree showing the relationship between cultured F. prausnitzii strains and directly amplified partial 16S rRNA gene sequences from human fecal samples. 16S rRNA sequence accession numbers are given in parentheses. Squares indicate OTU representative sequences from two recent studies on gut microbiota of healthy subjects (shown in boldface): the Tap et al. (53) study (1,443 F. prausnitzii sequences out of 10,456 clones from 17 healthy adults of both sexes) and the Walker et al. (57) study (534 F. prausnitzii sequences out of 5,915 total sequences from six obese males). The percentage of all clones represented by each OTU in each of these studies is shown on the right.

Fig 3

PCR-DGGE fingerprints from F. prausnitzii isolates. Isolates are distributed in two separate bands that correlate with phylogroup designation (▵, phylogroup I; ○, phylogroup II). Asterisks indicate the ladder lanes (made by 16S rRNA gene fragments of Mucor sp., Pseudomonas fluorescens, and Micrococcus luteus, respectively, from the top to the bottom).

Fig 4

Tolerance of F. prausnitzii isolates to changes in initial medium pH values and bile salt concentrations. (A) Relative growth rates (h⁻¹) of F. prausnitzii strains on YCFAG medium at three initial pH values (6.7, 6.2, and 5.75) have been represented. For comparison, the growth rate determined for each strain at pH 6.7 is taken as 1.0. (B) Relative OD₆₅₀ after 24 h of F. prausnitzii isolates at four bile salt concentrations (0%, 0.1%, 0.25%, and 0.5%) on YCFAG medium. For comparison, the OD₆₅₀ after 24 h of incubation determined for each isolate in medium without bile salt has been taken as 1.0. Mean growth rates at pH 6.7 and mean OD₆₅₀s in the absence of bile salts for each strain (±standard deviation) were as follows: ●, ATCC 27768 (0.17 ± 0.02 and 0.33 ± 0.05, respectively); ▼, M21/2 (0.32 ± 0.07 and 1.39 ± 0.05, respectively); ■, S3L/3 (0.16 ± 0.02 and 0.52 ± 0.07, respectively); ♦, S4L/4 (0.20 ± 0.02 and 0.63 ± 0.06, respectively); ○, A2-165 (0.55 ± 0.04 and 0.77 ± 0.02, respectively); Δ, L2-6 (0.19 ± 0.01 and 0.47 ± 0.02, respectively); □, HTF-75H (0.15 ± 0.01 and 0.386 ± 0.046, respectively); ♢, HTF-F (0.18 ± 0.01 and 0.826 ± 0.089, respectively). Phylogroup I isolates have been represented in black while phylogroup II isolates are shown in white.

Fig 5

Competition for apple pectin. (A) Change in acidic product concentrations in the growth medium after 24-h fermentation of 0.5% apple pectin by monocultures and cocultures of isolated pectin-utilizing bacteria. F1, F. prausnitzii SL3/3; F2, F. prausnitzii A2-165; E, Eubacterium eligens 3376; B, B. thetaiotaomicron 5482 (monocultures). (B) F1+E+B and F2+E+B were tricultures of the three strains indicated. Negative values for acetate reflect the net consumption of acetate initially present in the medium by F. prausnitzii strains. Each strain or strain combination was inoculated into YcFA medium adjusted to three different initial pH values (6.12, 6.45, and 6.79). Final medium pH (measured in all cases and detailed in Table S2 in the supplemental material) had decreased after 24 h by up to 0.3 unit for F. prausnitzii monocultures, up to 0.7 unit for B. thetaiotaomicron, and up to 0.9 unit for E. eligens. The final pHs in the tricultures were 6.16 (F1+E+B) and 6.07 (F2+E+B) from initial pH 6.79, 5.79 (F1+E+B) and 5.64 (F2+E+B) from initial pH 6.45, and 5.47 (F1+E+B) and 5.33 (F2+E+B) from initial pH 6.12. Data for two-membered cocultures and on overall sugar utilization from the same experiment are given in Tables S1 and S2 in the supplemental material. (C) Numbers of F. prausnitzii cells detected by fluorescent in situ hybridization in cultures and cocultures. Counts/ml immediately after inoculation (t = 0) were as follows: S3L/3, 0.91 × 10⁷ ± 0.05 × 10⁷, and A2-165, 1.31 × 10⁷ ± 0.01 × 10⁷.