Growth medium.

YCFA medium consists of (per 100 ml) Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (0.1 g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g), (NH₄)₂SO₄ (0.09 g), MgSO₄ · 7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folic acid (5 μg), and pyridoxamine (15 μg). In addition, the following short-chain fatty acids (SCFA) are included (final concentrations): acetate (33 mM); propionate (9 mM); isobutyrate, isovalerate, and valerate (1 mM each). Cysteine is added to the medium following boiling and dispensed into Hungate tubes while the tubes are flushed with CO₂. After autoclaving, filter-sterilized solutions of thiamine and riboflavin are added to give final concentrations of 0.05 μg ml⁻¹ of each. For some experiments, the Casitone content was decreased to 0.2%; this modified medium is referred to as YcFA. Carbohydrate or other energy sources were added as indicated, and the final pH of the medium was adjusted to 6.8 ± 0.1.

DNA extraction, PCR amplification, and DGGE fingerprinting.

DNA was extracted and purified from 18-h-old cultures of F. prausnitzii strains grown on M2GSC medium by using the Wizard genomic purification kit (Promega Corporation, Madison, WI). 16S rRNA sequences were amplified using universal bacterial primers GC-357F (33) and 907R (34) to give an approximately 580-bp product flanking variable regions V3 to V5. PCR and denaturing gradient gel electrophoresis (DGGE) were carried out as previously reported (30).

16S rRNA gene amplification and sequencing.

16S rRNA genes were amplified using the universal bacterial primers 7F and 1510R (23) as described previously (12). PCR products were cleaned with the Wizard PCR product purification kit (Promega, Southampton, United Kingdom) and used to obtain bidirectional partial 16S rRNA gene sequences by using primers 7F, 519F, 519R, 916F, 916R, and 1510R (16, 23) on a Beckman capillary sequencer. All primers were obtained from Eurofins MWG.

16S rRNA gene sequence full-length construction and phylogenetic analysis.

Sequences from cultured isolates were manually inspected in order to assess quality. Sequence editing and assembling were carried out using the BioEdit sequence alignment editor, version 7.0.9.0 (17). Sequences were then aligned in Mothur (http://www.mothur.org) (46) using the SILVA bacterial database as a reference alignment, available at the same source. Alignment was then imported into the ARB software package (28) loaded with the SILVA 16S rRNA-ARB-compatible database (SSURef-100, August 2009, available through the SILVA rRNA database project at http://www.arb-silva.de/) (36). For the detection of chimeric sequences, each sequence was checked manually in the alignment, and phylogenetic trees were screened for sequences with unrealistically long branches or unique branching sites. Cultured representatives from the Ruminococcaceae were included as reference, and Eubacterium desmolans was used to root the tree. Phylogenetic analyses of the 16S rRNA gene sequences were conducted using the ARB software package, using the neighbor-joining (NJ) method (42) and the Jukes-Cantor (JC) algorithm for distance analysis. Tree topologies were evaluated using maximum parsimony and maximum likelihood methods. No filters or masks were used when constructing the trees. Bootstrapping analysis (1,000 replicates) was done to test the robustness of the NJ-JC tree using PHYLIP (13).

To assess which F. prausnitzii phylogroups were represented by the isolates, representative sequences of 16S rRNA genes directly amplified from fecal DNA were included (boldfaced in Fig. 2). These uncultured sequences were aligned and processed as described above and then added to the isolate-based tree using the Parsimony Quick Add Marked Tool already implemented in the ARB software package, thereby maintaining the overall tree topology.

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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.

RAPD-PCR.

Isolates were screened by random amplified polymorphic DNA PCR (RAPD-PCR) using the primer 1254, according to a previously described method (59). RAPD-PCR profiles were compared using the GelComparII software (Applied Maths, Belgium). The UPGMA (unweighted-pair group method using average linkages) method was used to build the dendrogram (see Fig. S1 in the supplemental material), and clusters were defined at a similarity score of >93.5%.

Carbohydrate utilization and assessment of bacterial growth.

Substrate utilization was determined by adding a final concentration of 0.5% (wt/vol) sugar to YCFA medium. Where possible, growth was measured spectrophotometrically as optical density at 650 nm (OD₆₅₀) for triplicate cultures at regular intervals up to stationary phase_. For insoluble xylan, however, fermentation was monitored by final pH measurement. To study competition for pectin, F. prausnitzii strains S3L/3 and A2-165 were inoculated individually and together with the known pectin-utilizing species B. thetaiotaomicron B5482 and E. eligens 3376 in cocultures and tricultures (see Table S2 in the supplemental material). These experiments used YcFA medium supplemented with 0.5% apple pectin (BDH Chemicals) that had been preadjusted to three different initial pH values (6.12, 6.45, and 6.79). Samples were collected at 0 h and 24 h to estimate bacterial numbers by fluorescent in situ hybridization (FISH), total sugar analysis, and SCFA concentrations. SCFA were analyzed by gas chromatography following conversion to t-butyldimethylsilyl derivatives (39). Total sugars were determined using the colorimetric phenol sulfuric assay (10).

Influence of initial pH and bile salts on bacterial growth.

Each strain was inoculated into YCFA medium supplemented with 10 mM glucose (YCFAG) that had been adjusted to the three different initial pH values (6.7, 6.2, and 5.75) as described previously (12). Growth was followed for 24 h by measuring absorbance at 650 nm for triplicate cultures, and specific growth rates (h⁻¹) were calculated in exponential phase. The influence of bile salts (Sigma B8631) was assessed by inoculating culture into YCFAG medium containing 0% (control), 0.1%, 0.25%, or 0.5% bile salts (all percentages in wt/vol), in triplicate. Growth was measured spectrophotometrically up to 24 h using absorbance at the 650-nm wavelength. The pH of the medium was also monitored at the beginning and at the end of each experiment.

Enumeration of F. prausnitzii bacteria by FISH analysis.

Cultures were prepared for analysis as described previously (19). Cell suspensions were applied to gelatin-coated slides. Dried slides were hybridized with 10 μl of the Fprau645 oligonucleotide probe (52) (50-ng/μl stock solution) and washed. Between 25 and 30 fields were counted per well using an epifluorescence microscope (Olympus) and image analysis software (Olympus Cell F digital imaging software) or manual counting for numbers of less than 10 fluorescent cells per field.

Statistical analysis.

Quantitative parameters, such as growth rates and relative OD₆₅₀, were compared by one-way analysis of variance (ANOVA). The Bonferroni post hoc test was applied for multicomparisons of those variables with more than two subgroups of samples. Previously, data normality was assessed by the Shapiro-Wilks test and the Leven test was conducted to assess for homoscedasticity. The Kruskal-Wallis nonparametric test was performed when required. All statistical analyses were conducted via SPSS 15.0 (SPSS Inc., Chicago, IL).

Substrate utilization by Faecalibacterium prausnitzii isolates.

Growth on carbohydrates of dietary and host origin by four phylogroup I and six phylogroup II isolates is shown in Table 2. The basal YCFA medium (described in Materials and Methods) contained 33 mM acetate, which is known to stimulate the growth of F. prausnitzii strains (11). Growth was assessed where possible by the change in OD₆₅₀, but for insoluble substrates such as xylan, it was necessary to rely on change in medium pH as an indicator of substrate fermentation. The ability of F. prausnitzii to utilize dietary polysaccharides was somewhat limited with no growth on arabinogalactan, no fermentation of xylan, and little or no growth on soluble starch. While two strains grew well on inulin, the remainder grew poorly. Stimulation of F. prausnitzii 16S rRNA sequences by inulin has been reported in vivo in healthy human volunteers (38), but it appears likely from the present work that this stimulation may favor certain strains. Interestingly, most isolates grew on apple pectin, although not on citrus pectin. Salyers et al. (43, 44) noted that the utilization of uronic acids was unusual in genera from the human colon other than Bacteroides species. In the present study, several F. prausnitzii strains were able to utilize galacturonic acid, which is an important constituent of pectin.

Growth was also detected for most F. prausnitzii strains on the host-derived sugar N-acetylglucosamine and for some strains on d-glucosamine and d-glucuronic acid, while β-glucuronidase activity has been reported previously in some F. prausnitzii isolates (8). This suggests that F. prausnitzii has the ability to switch between diet- and host-derived substrates, in common with several other dominant human colonic species (48). None of the carbohydrates tested allowed differentiation between the two phylogroups.

Very little growth was observed when carbohydrates were omitted from the medium, although the basal YCFA medium contains 1% Casitone. This indicates that F. prausnitzii strains have little or no ability to grow with peptides as their sole energy source. No evidence was found for fermentation of porcine gastric mucin.

Tolerance of Faecalibacterium prausnitzii isolates to the gut environment.

Previous studies have reported that F. prausnitzii growth is inhibited by slightly acidic pH (12). The eight isolates tested showed growth rates at pH 5.75 ranging between 20% (for A2-165) and 80% (for HTF-F) of those at pH 6.7 (Fig. 4A). On average, there was a 14% decrease at pH 6.2, but a 60% decrease at pH 5.75, compared with pH 6.7. Tolerance of bile salts, whose concentrations have been reported to increase in certain gut disorders (24, 35), is also considered to be an important factor for survival in the intestine. Bile salt tolerance differed among isolates, particularly at the lowest concentration tested (0.1%), but all the strains tested were bile salt sensitive, showing on average 76%, 95%, and 97% inhibition at 0.1%, 0.25%, and 0.5% bile salts, respectively (Fig. 4B). In contrast, other species of intestinal bacteria such as Bacteroides spp. and Enterococcus faecium have been reported to be resistant to up to 20% and 40% bile salt concentrations, respectively (5). Bile acids are synthesized in the liver and released into the small intestine, where it is estimated that 90 to 95% of secreted bile is absorbed. The concentration of bile in the healthy large intestine is approximately 0.05 to 0.3%. The sensitivity of all the F. prausnitzii isolates tested to bile salts suggests that this is a factor that may restrict populations of this species in regions of high bile concentration, e.g., within the small intestine.

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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.

While these differences in sensitivity to bile salts and pH seem likely to influence the distribution of individual strains, there was no statistically significant evidence for consistent differences between phylogroups.

Potential role of Faecalibacterium prausnitzii in the fermentation of pectin in the colon.

Pectin is extensively fermented in the human colon (7, 55), but the ability to utilize pectin for growth has been reported for relatively few groups of human colonic bacteria. Salyers et al. (43, 44) showed that pectin utilization was relatively common among Bacteroides spp., occurring in 47% of 188 isolates surveyed and prompting subsequent studies on B. thetaiotaomicron (9, 54). In contrast, of the 154 strains of Gram-positive anaerobes tested, which included five strains reported as Fusobacterium prausnitzii, only Eubacterium eligens was previously found to utilize pectin or polygalacturonic acid (43) (Table 3). The present data, however, indicate that F. prausnitzii could have a major role in pectin utilization (Table 3).

In order to test this hypothesis further, we examined the ability of two F. prausnitzii strains (S3L/3 and A2-165) to compete for apple pectin with representatives of the two other known groups of pectin-utilizing bacteria, B. thetaiotaomicron and E. eligens. As previous studies have shown that pH plays a critical role in determining the outcome of competition between Bacteroides spp. and Firmicutes (12, 58), incubations were performed at three initial pH values typical of the range seen in the distal colon (Fig. 5; see also Tables S1 and S2 in the supplemental material). In pure cultures, the major fermentation products produced from pectin were butyrate for F. prausnitzii, acetate and succinate for B. thetaiotaomicron, and formate and acetate for E. eligens (Fig. 5A). As previously observed for growth on starch and glucose (12), the lowest pH (6.12) curtailed fermentation of pectin by B. thetaiotaomicron. As expected (Fig. 4A), both F. prausnitzii strains grew well at the lowest pH (Fig. 5). Tricultures including all three species showed large amounts of butyrate at all three pH values, thus confirming the ability of F. prausnitzii to compete for this substrate with the other two pectin-utilizing species (Fig. 5B). Counts estimated by FISH for F. prausnitzii after 24 h of incubation indicated greater numbers in the triculture at the lowest pH than at the highest pH (Fig. 5C). Butyrate concentration was less affected by pH, indicating continued fermentative activity by F. prausnitzii in spite of decreased cell growth at the highest pH. Data for two-membered cocultures from this experiment are shown in Tables S1 and S2 in the supplemental material. Pectin utilization (measured by decrease in total sugar) was highest for cultures including B. thetaiotaomicron at pH 6.79 (see Table S2 in the supplemental material).

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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.

We acknowledge the Scottish Government Rural Environment Research and Analysis Directorate for support. Mireia Lopez-Siles was awarded a fellowship from the Spanish Ministerio de Educación to support a research stay at the RINH and is also the recipient of an FI grant from the Generalitat de Catalunya (2010FI_B2 00135), which receives support from the European Union Commissionate. This work was partially funded by the Spanish Ministry of Education and Science through project SAF2006-0041).

We thank Isabelle Laugaudin for contributing to growth tests, Donna Henderson for SCFA analysis, and Pauline Young for bacterial 16S rRNA sequencing. We also thank Anna Plasencia, Corran Musk, and Carles Borrego for their useful suggestions and technical assistance with phylogenetic analysis and Julien Tap, Petra Louis, and Alan Walker for help in identifying representative sequences for F. prausnitzii OTUs from published studies (53, 57) examining 16S rRNA clone libraries.