Mice

For the conventional experiment, female C57BL/6J mice were purchased from Janvier (France) and used 1 week after delivery. All experiments were done with appropriate control groups (H 2 O), from the same batch of mice. Mice were kept 1 week in the same room prior starting treatments. Ten-week-old mice were fed ad libitum with Ssniff control diet V112x M-Z (Ssniff Spezialdiaten, Germany). All experiments were performed in accordance with the Comite d’Ethique en Experimentation Animale (COMETHEA C2EA – 45, Jouy en Josas, France). Every experiment was repeated two or three times.

Antibiotic treatment and induction of colitis with dextran sodium sulfate (DSS)

A broad-spectrum antibiotic cocktail was prepared by mixing ampicillin (2 g/L), neomycin (2 g/L), metronidazole (2 g/L), and vancomycin (1 g/L) in drinking water. To selectively deplete specific bacteria, we treated the mice until sacrifice with vancomycin (1 g/L in drinking water) or colistin (600 μg/gavage; Sigma). One week after starting the antibiotic treatment, mice were given 2% (wt/vol) DSS (molecular weight, 36,000–50,000; MP Biomedicals, Solon, OH) dissolved in sterile drinking water ad libitum for 7 days, followed by a recovery period (water only) of 5 days. Animals were monitored daily for weight loss and disease activity index (including three parameters: weight loss, stool consistency, and presence of blood in feces).

Gavage with fungi and E. coli strains

S. boulardii CNCM I-745 (syn. HANSEN CBS 5926, Biocodex Laboratories, Gentilly, France) and C. albicans SC5314 (ATCC, Molsheim, France) were used in this study. Both yeasts were grown on yeast extract peptone dextrose (YEPD) medium overnight at 37 °C. Aliquots containing a yeast suspension of 5 × 107 CFU/mL were prepared and stored at − 80 °C. A yeast suspension (107 CFU in 200 μL) or control medium was administered daily to 10-week-old mice by intragastric gavage.

The E. coli MCR1 strain, which is rendered resistant to colistin by the mobilized colistin resistance (MCR) gene, was used in this study. This strain is a veterinary isolate commensal [20] and was provided by Thomas Guillard (CHU Reims, Hôpital Robert Debré, Laboratoire de Bactériologie-Virologie-Hygiène, F-51092 Reims, France). The bacteria were grown in LB at 37 °C overnight and then aliquoted at 5 × 108 CFU/mL. A bacterial suspension (108 CFU in 200 μL) or control medium was administered daily to 10-week-old mice by intragastric gavage.

Histology

Colon samples for histological studies were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA), embedded in paraffin, and then stained with hematoxylin and eosin (Sigma-Aldrich, Saint Louis, USA) for histological scoring. Histological scoring was performed blinded according to the method previously described [21].

Gene expression analysis using quantitative reverse-transcription PCR (qRT-PCR)

Total RNA was isolated from colon samples using an RNeasy Mini Kit (Qiagen, Hilden, Germany), including a DNAse treatment step, according to the manufacturer’s instructions. Quantitative RT-PCR was performed using SuperScript II Reverse Transcriptase (Life Technologies, Saint Aubin, France) followed by a Takyon SYBR Green PCR kit (Eurogentec, Liège, Belgium) in a StepOnePlus apparatus (Applied Biosystems, Foster City, CA, USA) with specific mouse oligonucleotides. The oligonucleotides used were as follows: GAPDH—sense: 5′-AACTTTGGCATTGTGGAAGG-3′; antisense: 5′-ACACATTGGGGGTAGGAACA-3′, Reg3g—sense: 5′-TTCCTGTCCTCCATGATCAAAA-3′; antisense: 5′-CATCCACCTCTGTTGGGTTCA-3′. We used the 2−ΔΔCt quantification method with mouse GAPDH as an endogenous control and calibrated the assay to the wild type.

Cytokine quantification

The colonic explants were cultured (37 °C, 10% CO 2 ) overnight in 24-well tissue culture plates (Costar, Corning, Amsterdam, the Netherlands) in l mL of complete RPMI 1640 medium. The culture supernatants were collected and stored at − 80 °C until processing. ELISA was performed on the supernatants according to the manufacturer’s instructions in order to quantify IFN-γ (Mabtech, Nacka Strand, Sweden). For the colonic explants, cytokine concentrations were normalized according to the weight of each colonic explant.

Quantification of fecal lipocalin (LCN2) levels

Frozen fecal samples were weighed and suspended in cold PBS. Samples were then agitated on a FastPrep (MP Biomedicals, Santa Ana, USA) bead beating machine for 40 s on setting 6 using 4.5-mm glass beads to obtain a homogenous fecal suspension. Samples were then centrifuged for 5 min at 10,000g (4 °C), and clear supernatants were collected and stored at − 20 °C until analysis. LCN2 levels were estimated using a DuoSet murine LCN2 ELISA kit (R&D Systems, Minneapolis, USA) as per the manufacturer’s instructions and expressed as pg/mg of stool.

Quantification of fungi and bacteria in fresh stools

Fresh stools were collected over the course of the study to determine the quantities of yeast and bacteria remaining after intragastric gavage. Fresh stools were weighed and suspended in cold PBS (3 μL/mg of feces). Tenfold serial dilutions were performed until the desired concentrations were reached. For fungi quantification, diluted feces were plated on YEPD agar plates supplemented with ampicillin (100 mg/mL) and incubated at 30 °C. After 2 days of growth, fungi were counted, and the absolute quantities of yeast were determined according to the corresponding dilutions. For E. coli MCR1 (resistant to colistin) quantification, diluted feces were plated on LB agar plates supplemented with colistin (4 mg/L) and incubated at 37 °C. After 1 day of growth, bacteria were counted, and the absolute quantities of E. coli were determined according to the corresponding dilutions.

Fecal DNA extraction and fungal quantification via quantitative PCR (qPCR)

Fecal DNA was extracted from weighed stool samples as previously described [22]. More precisely, the fecal samples were weighed and then resuspended for 10 min at room temperature in 250 μL of 4 M guanidine thiocyanate in 0.1 M Tris (pH 7.5) (Sigma-Aldrich, Saint Louis, USA) and 40 μL of 10% N-lauroyl sarcosine (Sigma-Aldrich, Saint Louis, USA). After the addition of 500 μL of 5% N-lauroyl sarcosine in 0.1 M phosphate buffer (pH 8.0), the 2-mL tubes were incubated at 70 °C for 1 h. One volume (750 mL) of a mixture of 0.1- and 0.6-mm-diameter silica beads (Sigma-Aldrich, Saint Louis, USA) (previously sterilized by autoclaving) was added, and the tube was shaken at 6.5 m/s in three bouts of 30 s each in a FastPrep (MP Biomedicals, Santa Ana, USA) apparatus. Polyvinylpolypyrrolidone (15 mg) (Sigma-Aldrich, Saint Louis, USA) was added to the tube, which was then vortexed and centrifuged for 5 min at 20,000g. After recovery of the supernatant, the pellets were washed with 500 μL of TENP (50 mM Tris (pH 8), 20 mM EDTA (pH 8), 100 mM NaCl, 1% polyvinylpolypyrrolidone) and centrifuged for 5 min at 20,000g, and the new supernatant was added to the first supernatant. The washing step was repeated two times. The pooled supernatant (approximately 2 mL) was briefly centrifuged to remove particulate matter and then split into two 2-mL tubes. Nucleic acids were precipitated by the addition of 1 volume of isopropanol for 10 min at room temperature and centrifugation for 10 min at 20,000g. The pellets were resuspended and pooled in 450 μL of 100 mM phosphate buffer, pH 8, and 50 mL of 5 M potassium acetate. The tube was placed on ice overnight and centrifuged at 20,000g for 30 min. The supernatant was then transferred to a new tube containing 20 μL of RNase (1 mg/mL) and incubated at 37 °C for 30 min. Nucleic acids were precipitated by the addition of 50 μL of 3 M sodium acetate and 1 mL of absolute ethanol. The tube was incubated for 10 min at room temperature, and the nucleic acids were recovered by centrifugation at 20,000g for 15 min. The DNA pellet was finally washed with 70% ethanol, dried, and resuspended in 100 μL of Tris–EDTA (TE) buffer. The DNA suspensions were stored at − 20 °C for real-time qPCR analysis of the 16S rDNA or ITS2 sequences. DNA was then subjected to qPCR by using a Takyon SYBR Green PCR kit (Eurogentec, Maastricht, The Netherlands) for quantification of all fungal sequences or by using TaqMan Gene Expression Assays (Life Technologies) for quantification of all bacterial sequences. The probes and primers for the bacterial 16S DNA genes and primers for the fungal 18S DNA genes were as described previously [23]. Reference standard (with quantified C. albicans DNA) was used in the same qPCR, allowing the quantification of the CFU in each sample. The threshold cycle for each sample was determined for each gene and was normalized to the CT value of the all-bacteria 16S ribosomal RNA gene. Data were calculated using the 2−ΔΔCt method.

16S DNA gene and ITS2 sequencing

DNA was isolated from the feces of mice before and after DSS treatment using the protocol described above. Bacterial diversity was determined for each sample by targeting a portion of the ribosomal genes. A 16S DNA gene fragment comprising the V3 and V4 hypervariable regions (16S (sense) 5′-TACGGRAGGCAGCAG-3′ and (antisense) 5′-CTACCNGGGTATCTAAT-3′) was amplified using an optimized and standardized 16S-amplicon-library preparation protocol (Metabiote, GenoScreen, Lille, France). Briefly, 16S DNA gene PCR was performed using 5 ng of genomic DNA according to the manufacturer’s protocol (Metabiote) using 192 bar-coded primers (Metabiote MiSeq Primers) at final concentrations of 0.2 μM and an annealing temperature of 50 °C for 30 cycles. The PCR products were purified using an Agencourt AMPure XP-PCR Purification system (Beckman Coulter, Brea, CA, USA), quantified according to the manufacturer’s protocol, and multiplexed at equal concentrations. Sequencing was performed using a 300-bp paired-end sequencing protocol on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) at GenoScreen, Lille, France. Raw paired-end reads were subjected to the following processes: (1) quality-filtering using the PRINSEQ-lite PERL script [24] by truncating from the 3′ end those bases that did not exhibit a quality < 30 based on the Phred algorithm; (2) paired-end read assembly using FLASH [25] (fast length adjustment of short reads to improve genome assemblies) with a minimum overlap of 30 bases and a 97% overlap identity; and (3) searching for and removing both forward and reverse primer sequences using CutAdapt, with no mismatches allowed in the primer sequences. Assembled sequences for which perfect forward and reverse primers were not found were eliminated. A similar approach was used for fungi microbiota using the primers ITS2 (sense) 5′-GTGARTCATCGAATCTTT-3′ and (antisense) 5′-GATATGCTTAAGTTCAGCGGGT-3′ and the optimized and standardized ITS2-amplicon-library preparation protocol (Metabiote, GenoScreen).

16S and ITS2 sequence analysis

The sequences were demultiplexed and quality filtered using the QIIME version 1.8.0 software package [26]. The sequences were then assigned to OTUs using the UCLUST algorithm [27] with a 97% pairwise identity threshold and classified taxonomically using the Greengenes reference database (version 13.5) for bacteria and the UNITE ITS database (alpha version 12_11) for fungi [28]. Rarefaction was performed (20,000 and 250 sequences per sample for 16S and ITS2, respectively) and used to compare the relative abundance of OTUs across samples. Alpha diversity was estimated using the Shannon diversity index or the number of observed species. Beta diversity was measured by a Bray–Curtis distance matrix and was used to build principal coordinates analysis (PCoA) plots. The linear discriminant analysis (LDA) effect size (LEfSe) algorithm was used to identify taxa that were specific to diet and/or treatment. Deposition of the raw sequence data in the European Nucleotide Archive is in process; the accession number is pending.

Statistics

GraphPad Prism version 6.0 (San Diego, CA, USA) was used for all analyses and preparation of graphs. For all data displayed in graphs, the results are expressed as the mean ± s.e.m. (n = 5 to 12 per group). For comparisons between two groups, a two-tailed Student’s t test for unpaired data or a nonparametric Mann–Whitney test was used. For comparisons among more than two groups, one-way analysis of variance (ANOVA) and a post hoc Tukey test or a nonparametric Kruskal–Wallis test followed by a post hoc Dunn’s test was used. For all statistical tests, differences with a P value less than 0.05 were considered significant.

Correlation between bacterial and fungal taxon relative abundance was measured by distance correlation [29] and as described previously [7]. Only taxa present in at least 50% of the samples of each group were included. In addition to the distance correlation, the sign of Spearman’s correlation was computed to describe heuristically the direction of association between microbial taxa. The distance correlation was computed in R-3.2.3 using the package energy v1.6.2. The P values were corrected using the Benjamini–Hochberg procedure to control the false discovery rate (P < 0.25).