We investigated the effects of a 4-week oral synbiotic on ten IBS-D patients by comparing the following parameters pre- and post-treatment. Patients were examined by endoscopic evaluation of the upper and lower gastrointestinal tracts to obtain mucosal samples for FACS analysis and mucosal 16S rDNA analysis. Thereby, biopsies for FACS analysis were separately taken from the duodenum and ascending colon during the retraction of the endoscope and immediately processed. The colonic biopsies were obtained between the right-colonic flexure and the caecum. In addition, biopsies from the gastric corpus, duodenum, and ascending colon were obtained for mucosal microbiota analysis. Furthermore, analysis of fecal SCFAs, zonulin, and fecal 16S rDNA was performed.
Patients and controls
IBS-D patients (according to S3 guidelines) for this study were recruited consecutively from the outpatient clinics of the Department of Internal Medicine, Division of Gastroenterology and Hepatology, Medical University of Graz . The following inclusion criteria were applied: (1) symptomatic IBS patients according to current S3 guideline ; (2) age between 18 and 65 years; and (3) informed consent. Exclusion criteria were: (1) chronic inflammatory (IBD, celiac disease, and microscopic colitis were ruled out, and patients had the previous endoscopic evaluation and diagnostics), immune-, or neoplastic diseases; (2) recent application of immune-modifying medication; (3) pregnancy; and (4) alcohol or drug abuse. No patients received any new medications during the study period and no changes in medication dose were made during the study. No antibiotics were taken 4 weeks prior to study inclusion. All medications used by patients are systemically compiled in Table 1. All individuals included signed an informed consent prior to study inclusion. All protocols and informed consents were a priori waived by the local institutional review board (IRB number IRB00002556), vote 25-594 ex 12/13.
Study protocol and schedule
Patients were recruited from the outpatient department as mentioned. During a screening visit, inclusion and exclusion criteria were applied and physical status investigated. Patients fulfilling criteria signed an informed consent and were scheduled for the baseline study visit 1. At study visit 1, upper and lower GI tract endoscopy was performed and biopsies were taken, fecal samples and IBS-SSS were obtained, and synbiotic formulation was handed out. Patients recorded the oral administration of synbiotic mixture twice a day for 4 weeks. At study visit 2 (4 weeks later), all examinations were re-performed including endoscopy and obtaining of fecal samples and IBS-SSS.
The German version of the validated IBS-SSS questionnaire was obtained from the Zentrum für klinische Ernährung (ZKES, Wollgrasweg 49b, 70599 Stuttgart, Germany) and used as described . Symptoms were quantified prior and after 4 weeks of synbiotic therapy.
Patients were given a 4-week course (twice a day) commercially available synbiotic mixture (OMNi-BiOTiC® Stress Repair, Institut Allergosan, Graz) consisting of the following prebiotics corn starch, maltodextrin, inulin, fructooligosaccharides, potassium chloride, magnesium sulfate, mangan sulfate and enzymes as well as 7.5 × 109 of each of the following probiotic bacterial strains: Lactobacillus casei W56, Lactobacillus acidophilus W22, Lactobacillus paracasei W20, Lactobacillus salivarius W24, Lactobacillus plantarum W62, Lactococcus lactis W19, Bifidobacterium lactis W51 and W52, and Bifidobacterium bifidum W23.
Gastroduodeno- and ileocolonoscopy was performed with standard equipment (Olympus, Hamburg, Germany) in sedated subjects. Samples were obtained by forceps biopsy. Biopsies for FACS analysis were separately taken from the duodenum and ascending colon during the retraction of the endoscope and immediately processed. The colonic biopsies were obtained between the right-colonic flexure and the caecum. In addition, biopsies from the gastric corpus, duodenum, and ascending colon were obtained for mucosal microbiota analysis.
Isolation of lamina propria mononuclear cells
Mucosal biopsy specimens were obtained separately from the duodenum and ascending colon and immediately preserved in chilled RPMI medium (Sigma; supplemented with penicillin, streptomycin, and amphotericin). Biopsies were washed once with calcium- and magnesium-free HBSS (Life Technologies, Vienna, Austria) and then incubated in calcium- and magnesium-free HBSS containing 1 mM DTT and 5 mM EDTA at 37 °C for 20 min with gentle agitation to remove mucus and epithelial cells. Following a brief wash with calcium- and magnesium-free HBSS, tissue was digested with 1 mg/ml Collagenase A (Roche, Basel, Switzerland) and 5 units/ml DNase I (Roche, Basel, Switzerland) in HBSS at 37 °C for 60 min on a shaker and mechanically disrupted by gentle pipetting. Complete dissociation was verified by visual inspection. After passing through a 70 µm cell strainer, the released cells were washed twice with RPMI complete medium (containing 10% FCS and 1% penicillin/streptomycin) and finally re-suspended in RPMI complete medium. The cell suspension was kept on ice until further analysis.
FACS analysis was performed as previously described [27,28,29]. Briefly, the cell suspension was washed once with staining buffer (PBS containing 3% FCS and 2 mM EDTA) and the cells were stained in 100 µl staining buffer for 20 min at room temperature in the dark. For enumeration of lamina propria dendritic cells, directly labeled monoclonal antibodies for the following markers were used: lin (lineage) 1-FITC (CD3, CD14, CD16, CD19, CD20, CD56, and CD34), HLA-DR-PerCP-Cy5.5, CD11c-APC, and CD103-PE. LPDCs were identified as lin1-/HLA-DR+ cells. For determination of Tregs, anti-CD3-APC-Cy7, anti-CD4-V450, anti-CD8-FITC, anti-CD25-PE, and anti-CD127-Alexa Fluor 647 antibodies were used. With the exception of CD103-PE (eBioscience, San Diego, USA), all antibodies were purchased from BD Bioscience (San Jose, USA). FMO (fluorescence-minus-one) controls were employed to set the boundaries for gating of positively stained cells. After the staining reaction, the cells were washed once with staining buffer and re-suspended in 100 µl staining buffer. For the exclusion of dead cells, propidium iodide (PI) was added to the samples immediately prior to acquisition on an LSR II (BD Bioscience, San Jose, USA) flow cytometer. The data files were analyzed using FlowJo (FlowJo, LLC) software.
Isolation of total genomics DNA, 16S library preparation, and Illumina sequencing
Stool and mucosal samples were stored at − 80 °C and used for total DNA isolation combining mechanical and enzymatic lysis with the MagnaPure LC DNA Isolation Kit III (Bacteria, Fungi) (Roche, Mannheim, Germany) according to manufacturer’s instructions as described . Modifications were made for stool and mucosal specimens. Briefly, stool samples were homogenized in 500 µl PBS and 250 µl of the suspension was mixed with 250 µl of bacterial lysis buffer and further transferred to Magna Lyser green bead tubes (Roche, Mannheim, Germany). Mechanical lysis was two times performed at 6500 rpm in a MagNA Lyser Instrument (Roche, Mannheim, Germany). Mucosal specimens were prepared with bead beating for four times at 6500 rpm in 500 µl lysis buffer and enzymatic lysis samples were mixed with 25 µl lysozyme (100 mg/ml) and incubated at 37 °C for 30 min. Afterwards, samples were mixed with 30 µl Proteinase K and stool samples were incubated at 65 °C for 1 h. Mucosal specimens were incubated overnight at 65 °C. Enzymes were heat inactivated at 95 °C for 10 min and further steps were performed according to Magna Pure DNA isolation kit III (Bacteria, Fungi) manufacturer’s instruction. 250 µl of mucosal specimens and 100 µl of stool samples were taken for DNA purification that was eluted in 100 µl. For target specific PCR amplification, the primers 27f (AGAGTTTGATCCTGGCTCAG) and 357r (CTGCTGCCTYCCGTA) were taken as described by Baker et al.  and synthesized at Eurofins (MWG, Ebersberg, Germany). Then, 5 µl of total DNA from mucosal sample and 2 µl from stool sample extracts were taken for a 25 µl PCR reactions as described . Triplicates were pooled and amplification was verified using a 1% agarose gel. The sequencing library was amplified, quantified, and sequenced on a MiSeqII desktop sequencer (Illumina, Eindhoven, The Netherlands) as described previously . Version 3.600 cycles chemistry (Illumina, Eindhoven, The Netherlands) was taken according to manufacturer‘s instructions to run the 6 pM library with 20% PhiX (Illumina, Eindhoven, The Netherlands). FASTQ files were then further taken to perform data analysis.
Microbiota analysis and statistical methods
Quality filtering and analysis of raw 16S rRNA gene sequence data (hypervariable region V1–V2) was performed with mothur (version 1.22.0) according to the recommended standard operating procedure of mothur for Illumina MiSeq data (https://www.mothur.org/wiki/MiSeq_SOP, accessed June 2016) with additional removal of singletons (default settings and parameters were used, if not specified otherwise) [32, 33]. Briefly: paired reads were merged using mothur’s make.contigs command, whereby reads less than 200 bps were filtered out of the data set. In addition, sequences containing ambiguous bases or more than eight homopolymeres were removed together with chimeric sequences or sequences outside of the core alignment with the SILVA reverence database (version 119). Furthermore, noisy sequences were identified using pre.cluster and finally deleted from the data set. Remaining pre-processed and filtered sequences were clustered by mothur’s de novo OTU-picking strategy into OTUs at a distance of 0.03. Finally, taxonomic classification was assigned using the RDP Bayesian classifier (version 2.10.1, trainingsset 10/29.10.2014) with default settings and a classification confidence cutoff of 80% . Subsequent OTU-based microbiota analyses were performed in QIIME (version 1.8.0), including core.diversity analysis with rarefaction to a sampling depth of 9.538 reads per sample for all four locations (COR = corpus, COL = colon, FEC = feces, and DUO = duodenum) . Unweighted UniFrac distance metrics as measure of between-sample (beta) diversity was calculated and applied for principal coordinates analysis (PCoA) to visualize patterns of diversity . Within-samples (alpha) diversity was calculated using four different measures (1) observed species, (2) ChaoI Index, (3) Shannon Index, and (4) Faith’s phylogenetic diversity [37, 38]. Statistical significant differences between sample (alpha) diversity were assessed by a nonparametric two-sample t test (p values were determined by Monte Carlo permutations. Calculations are based on the greatest rarefaction depth. Bonferroni correction was used to account for multiple comparisons). Differences in taxonomic microbiota compositions (differentially abundant features/genera) within the four locations and between treatments were determined using linear discriminant effect size analysis (LEfSe) on the filtered data sets at species level. If not otherwise specified p values below 0.05 were considered as statistically significant .
Availability of data and materials
The sequence data supporting the results of this article are available in the European Bioinformatics Institute Sequence Read Archive under accession number PRJEB19253.
Extract preparation from specimens for multiplex cytokine assay
Colonic and duodenum specimens were obtained in cold RPMI1640 medium supplemented with penicillin and streptomycin. Samples were transferred to cryotubes, snap frozen, and stored in liquid nitrogen until sample preparation. All samples were then individually thawed on ice and immediately disrupted in 300 µl extraction buffer for 2 min on ice with a pellet pestle (Kimble Kontes, USA). Extraction buffer comprised DPBS (Dulbecco’s phosphate buffered saline without calcium and magnesium, Lonza) and EDTA-free protease inhibitors (cOmplete mini, Roche). After further disrupting them mechanically by pipetting, biopsies were passed through a 70 µm cell strainer. All samples were then incubated on ice for 5 min. Finally, supernatants were obtained by centrifugation at 10,000×g for 10 min at 4 °C, snap frozen in liquid nitrogen, and stored at − 80 °C until analysis. Cytokine analysis included IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-17A, IL-23, and TNF-α. Multiplex immunoassay kits (ProcartaPlex) used for analysis were obtained from eBioscience and were run according to manufacturer’s instructions using magnetic beads. Standards for each cytokine were assayed in duplicates to generate standard curves using the reference concentrations as provided by the manufacturer. All samples were individually thawed on ice and wash steps were performed using a hand-held magnetic block. Data were obtained on a validated and calibrated Bio-Plex 200 system (Bio-Rad) and analyzed with Bio-Plex Manager 6.1 software (Bio-Rad). BCA Protein Assay (Pierce) was used to determine total protein concentration and to normalize cytokine concentrations for each sample.
GC–EI/MS of short-chain fatty acids
SCFAs (acetic acid, propionic acid, iso-butyric acid, butyric acid, iso-valeric acid, and valeric acid) were extracted from stool frozen at − 80 °C. SCFA concentrations were measured by a GC–MS equipped with a PEG DB-WAXetr. (30 m; 0.25 mm ID; and 0.25 µm film) column. SCFA were extracted from feces by sequential addition of 1 ml phosphoric acid (0.5%) and 1 ml methyl-tert-butyl-ether, 10 min shaking, 10 min centrifugation, and removal of the upper organic layer. Before extraction 100 nmol of d-acetic acid, d-propionic acid, d-butyric acid, and d-valeric acid were added as internal standards. Calibration curves by stable isotope dilution were performed from 0.1 to 2.000 µM for acetic acid, propionic acid, iso-butyric acid, butyric acid, iso-valeric acid, and valeric acid. A 7890B/5977A MSD GC–MS (Agilent, Waldbronn, Germany) equipped with a PEG DB-WAXetr. (30 m; 0.25 mm ID; and 0.25 µm film) column was used. Helium was used as carrier gas at 1.3 ml/min in splitless mode at 250 °C injector temperature. The initial oven temperature of 60 °C was held for 2 min, and then, the temperature first was ramped up to 150 °C at a rate of 15°C/min. This was followed by a ramp of 5°C/min up to 170 °C and 20°C/min up to 250 °C, where the temperature was held for another 2 min. The mass spectrometer was run in electron impact (EI) mode, where the SCFAs were detected in SIM mode on m/z 60, 63, 73, 74, 76, 79, and 80. The source temperature was set to 250 °C and the transfer line temperature was 280 °C. Data analysis was performed by Mass Hunter (Agilent, Waldbronn, Germany).
A ready-to-use solid-phase sandwich ELISA (Immundiagnostik AG, Bensheim, Germany) was used to detect zonulin (zonulin Stool ELISA) in fecal samples. The tests were performed according to the manufacturer’s instructions. For stool sampling, the Stool Sample Application System (Immundiagnostik AG, Bensheim, Germany) was used according to the manufacturer’s manual .
Statistical analyses were carried out using SPSS 22 (IBM® Corporation USA) and GraphPad Prism® 6.0 (GraphPad Software, Inc., USA). Values are presented as number (%) or median [interquartile range]. For the comparison of categorical variables, we applied Fisher’s exact test. Group differences of continuous variables were determined by Mann–Whitney U test or t test depending on the data distribution (non-gaussian or gaussian). Boxplots are depicted according to Tukey.