Ethical compliance
All experimental protocols were approved by the Commissie Medische Ethiek, UZ KU Leuven. Study design complied with all relevant ethical regulations, aligning with the Declaration of Helsinki and in accordance with Belgian privacy laws. All participants gave their informed consent. Ethical approval of the study protocol was obtained (S59288).
Sample collection and preservation
An initial cohort of 51 healthy donors from the KU Leuven community provided the faecal samples used for this study [19]. No inclusion or exclusion criteria were imposed. A limited set of anthropometric metadata was compiled at enrolment, including gender, age, height, and weight. Participants were asked to collect a maximal amount of faeces (single defecation) in a plastic receptacle with a lid and to deposit the sample in a labelled non-transparent zip-lock bag at the research facility immediately after defecation. All faecal samples were processed within an hour of the reported time of egestion. Upon collection, fresh faecal samples were homogenized and divided in a series of aliquots for physicochemical analyses, 16S rRNA gene community profiling and comparative analyses of four different conditions. The latter comparison required two aliquots with specific weights. First, 1 g of fresh faecal sample was stored dry in a cryotube without the addition of a preservation medium or cryoprotectant (preservation condition P1). In parallel, 3.75 g of fresh sample was resuspended in 15 mL of sterile Cary-Blair transport medium with red phenol indicator (Remel, Lenexa, USA). The resulting suspension was distributed over three different cryotubes each containing four mL of faecal suspension. The first tube was preserved without a cryoprotective agent (preservation condition P2), the second was supplemented with 1 mL sterile glycerol (Alfa Aesar, Thermofisher, Germany) to a final concentration of 20% v/v (preservation condition P3) and the third with 0.2 mL DMSO (Sigma Aldrich, USA) to a final concentration of 5% v/v (preservation condition P4; Fig. 1).
Faecal samples selection
Out of the initial 51 faecal sample donors, 11 were selected along a gradient of physicochemical (i.e. faecal pH, water activity, and moisture content) and microbiological (i.e. microbial load) parameters (Supplementary Table S1). Water activity was measured in duplicate at 37°C using a resistive electrolytic hygrometer (Labmaster, Novasina, Lachen, Switzerland) after manual homogenization of the fresh faecal sample. Faecal pH of fresh samples was measured in triplicate after mechanical homogenization (5 min, 150 r.p.m; Stomacher 3500 [Seward Ltd., Worthing, UK]) using a FG2 pH meter coupled to an InLab Solids electrode (Mettler Toledo, Greifensee, Switzerland). Stool moisture content was determined in duplicate on 0.2 g of frozen faecal material (-80°C) as the percentage of mass loss after lyophilization. Microbial load of frozen cells was determined in triplicate using a C6 Accuri flow cytometer (BD Biosciences, New Jersey, USA) as described previously [19].
Cultivation, colony counts and isolation
All post-preservation sample dilution, culturing and isolation steps were performed at 37°C under anaerobic conditions (10% H2, 10% CO2, 80% N2) in a Don Whitley A35 Anaerobic Workstation with HEPA filter (Don Whitley Scientific, Shipley, UK). All dilution and culture media were anaerobically preincubated overnight.
Per post-preservation sample, a dilution series (10−1 to 10−7) for each preservation condition was prepared in PBS and dilutions 10−3 to 10−7 were plated in triplicate on modified Gifu Anaerobic Medium (mGAM; Hyserve) agar and incubated for 5 days. Plates corresponding to countable dilutions (i.e. plate dilutions producing CFU in the range of 20–200, n=203) were first subjected to CFU counting and subsequently used for isolation. Colonies were selected (i.e. 48 colonies per countable dilution when possible, up to 95 per preservation condition) for isolation based on morphological differences (i.e. diameter, edge, shape and colour) and were transferred to a 96-well microplate in mGAM broth. Following incubation for 48h, 15% (v/v) glycerol was added and homogenized into each well after which the microwell plates were stored at − 80°C to use for identification by partial 16S rRNA gene sequencing. For all dilutions that yielded >100 colonies, one plate was kept aside for harvesting its entire biomass (from here on referred to as cultured fraction) which was resuspended in PBS. Following centrifugation (18,000×g, 2 min) and removal of supernatant, these fractions were stored at − 80°C for further 16S rRNA gene MiSeq community profiling. The entire experimental design is visualized in Fig. 1.
Community profiling of faecal samples and cultured fractions
Before processing for 16S rRNA gene-based community profiling, cultured fraction pellets were resuspended in 4.5 mL PBS (Sigma Aldrich, USA) and split in 3 aliquots of 1.5 mL. Samples were centrifuged and supernatant was removed prior to storage at − 80°C in cryovials. Frozen faecal aliquots (between 150 and 200 mg) and cultured fractions were subjected to DNA extraction using the PowerMicrobiome DNA/RNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, USA) according to the manufacturer’s instructions as described previously [20]. The V4 hypervariable region of the 16S rRNA gene was amplified using forward primer 515F (5′ GTGYCAGCMGCCGCGGTAA 3′) and the reverse primer 806R (5′ GGACTACNVGGGTWTCTAAT 3′), modified with adapters and barcodes [20]. Sequencing was then performed using the Illumina MiSeq platform (MiSeq Reagent Kit v2, 500 cycles, 20% PhiX) according to the manufacturer’s specifications to generate paired-end reads of 250 bases in length in each direction. After demultiplexing with sdm as part of the LotuS [21] pipeline without allowing for mismatches, fastq sequences were pre-processed using DADA2 pipeline v1.14.1 [22]. The taxonomy was assigned using GTDB release 95 [23, 24]. For the comparison with FGFP [20] samples, taxonomy classification was also performed using the RDP classifier [25]. For relative microbiome analyses, each sample depth was rarefied to 10,000 reads.
Identification of isolated colonies
From individual bacterial colony suspensions stored in 96-well microplates, 20 μL was transferred into a new microplate and washed with PBS to remove the excess medium. Plates were centrifuged for 10 min at 3,000 x g, after which the supernatant was removed. Following a second washing step, pellets were resuspended in 20 μL alkaline lysis solution containing 2.5 mL 10% SDS (Invitrogen, USA), 5.0 mL 1N NaOH (Merck, New Jersey, USA) and 92.5 mL sterile MilliQ per 100 mL. Samples were heated at 95°C for 15 min and immediately put on ice for 10 min. Finally, 180 μL of sterile MilliQ water was added and after gentle mixing, samples were centrifuged for 20 min at 3,000 x g and stored at − 20°C until further analysis. For identification based on partial 16S rRNA gene sequencing, 2 μL of each extract was used as a template for PCR with forward primer 27F (5′AGAGTTTGATCCTGGCTCAG 3′) and reverse primer 1492R (5′ ACGGCTACCTTGTTACGACTT 3′). Following agarose electrophoresis check, amplicons were sent for 16S rRNA gene Sanger sequencing to Eurofins (Germany). Sequences were taxonomically assigned by GTDB release 95 [23, 24].
Statistical analyses
All statistical analyses and graphical representations were performed in R using the packages phyloseq [26] vegan [27], CoDaSeq [28] and ggplot2 [29]. Observed richness was calculated with the R package phyloseq. Microbiome variation between individuals was visualized by PCoA using Bray-Curtis distances, only samples with more than 10,000 reads and amplicon sequence variants (ASV) with a relative abundance higher than 0.001 across the dataset were included in the 16S rRNA data analysis. Correlations between continuous variables were analysed using non-parametric Spearman tests.
The contribution of metadata variables to the cultured fraction community variation was determined by distance-based redundancy analysis (dbRDA) on genus-level Aitchison distance (Bray-Curtis distances between samples after rarefication) with the capscale function in the vegan R package [27]. Correction for multiple testing (Benjamin–Hochberg procedure, FDR) was applied and significance was defined at FDR < 0.1. The cumulative contribution of metadata variables was determined by forward model selection on dbRDA with the ordiR2step function in vegan [27], with variables that showed a significant contribution to cultured fraction community variation in the previous step.
Alpha-diversity, number of observed genera, species and ASV (richness) of both the cultured fraction and faecal samples were determined using the vegan R package [27]. Associations between the richness and diversity of the cultured fractions and preservation conditions were assessed by fitting generalized linear models (GLMs). The model used multivariate analysis assessing the effect of preservation conditions, dilution factors and initial richness (RichnessF) from the faecal samples. The significance of was assessed by performing log-likelihood (χ2) tests.
$$\mathrm{Nullmodel}\ \mathrm{richness}=\mathrm{specnumber}\sim \mathrm{Dilution}+\mathrm{Preservation}+\mathrm{RichnessF}$$
with specnumber as the number of species found in the sample (richness).