Study population and material sampling

IBS patients who met the ROME III diagnostic criteria36 were recruited from the outpatient clinic at Sahlgrenska University Hospital Gothenburg, Sweden. Exclusion criteria included microscopic colitis, celiac disease, food allergies, or any other GI disease explaining the symptoms. Other exclusion criteria included abnormal results on standard screening laboratory tests, severe psychiatric, systemic or other chronic diseases, history of drug or alcohol abuse, and the inability to reliably respond to questionnaires in Swedish. Based on recorded bowel movements in a one-week stool diary, IBS patients were subtyped into diarrhea-predominant IBS (IBS-D), constipation-predominant IBS (IBS-C), while IBS patients with mixed loose and hard stools (IBS-M) and those who had unsubtyped IBS (IBS-U) were grouped together (non-constipation-non-diarrhea predominant, nonCnonD)36. Healthy subjects without gastrointestinal (GI) symptoms (assessed for seven days prior to inclusion with a GI symptom screening questionnaire), psychiatric, gastrointestinal, cardiac or metabolic diseases were included. IBS patients and healthy subjects were of Caucasian ethnic origin. Informed consent was obtained from all participants before any study-related procedures. We recorded the use of specific diets and medications in all study subjects (Supplementary Table 1).

From all study subjects, biopsies were obtained from the second part of the duodenum and the sigmoid colon (25–35 cm proximal from the anus) by the same gastroenterologist (IA) using standard biopsy forceps without prior bowel preparation. Once collected all samples were handled by one researcher (JS), biopsies were immediately snap frozen in liquid nitrogen and stored at −80 °C until further analysis or placed in NaCl 0.9% solution and transferred within 2 h to microbiology for culture. Duodenal juice was aspirated prior to biopsy collection from participants where this was possible, i.e. where intestinal liquid was present and could be aspirated through the biopsy channel of the endoscope and collected in a sterile, DNA/RNA free, plastic tube and transferred within 2 h to microbiology for culture. Fecal samples were collected by patients at home, immediately frozen in −20 °C and brought frozen to the laboratory where it was stored frozen in −80 °C until analysis. The study was approved by the Regional Ethical review Board in Gothenburg, and all subjects provided informed consent before any study related procedures were initiated. All experiments were performed in accordance with relevant guidelines and regulations.

Bacterial cultures

Cultures of aerobic and anaerobic bacteria were made on blood agar plates with 4% defibrinated horse blood in aerobic and anaerobic atmospheres of N 2 and 10% CO 2 at 37 °C. Selective cultivation of Gram-negative strains was performed on Drigalski agar under aerobic conditions. Yeast fungus was cultured on Sabouraud’s agar. The minimum incubation time was 48 h. Identification of the microorganisms was based on colony characteristics, Gram staining, biochemical and chromatographic tests and Maldi-tof (Vitek-MS, Biomerieux). Quantification was performed by counting the number of colony-forming units (cfu/ml). Culture-verified SIBO was defined as >105 cfu/ml of colonic bacteria according to the current golden standard15.

Glucose and lactulose breath tests

Breath tests were performed after at least one day with low fiber diet, after fasting overnight, a short resting period in the morning and after careful brushing of the teeth. In addition, the subject was instructed not to consume any laxatives, bulking agents, or bile acid sequestrants the day before the test. If the first baseline value exceeded 15 parts per million (ppm) for methane or hydrogen, the subject performed a mouthwash with tap water before the next baseline value was collected. If the subject had repeatedly high baseline values the subject was rescheduled for an examination another day, after even more stringent instructions about preparation for the test, please see above, and after 24 hours of fiber-free diet. Subjects with high baseline values were rescheduled for a maximum of three visits. After recording two baseline values, subjects ingested 45 gram of glucose (Apoteket Produktion & Laboratorier, Sweden) in 300 ml tap water, or lactulose in the form of syrup (15 mL of syrup solution 670 mg/mL) (Laktulos Meda, Meda AB, Solna, Sweden), respectively. Hydrogen and methane gas in expired air was measured every 15 minutes during the following 120 (glucose) or 180 (lactulose) minutes. The expired air was collected in a two-bag system (Quintron, Milwaukee, WI, USA) with a valve and mouthpiece. The exhaled air was then analyzed with a breath gas analyzer (BreathTracker, Digital microlyzer; Quintron, Milwaukee, WI, USA).

The breath test based definitions of SIBO were based on either hydrogen or methane gas, respectively. The criteria were categorized into: (1) fasting levels (abbreviated fasting criteria of H 2 or CH 4 ≥ 15 ppm37,38; (2) a rise of H 2 or CH 4 above baseline within 90 min (abbreviated increase criteria ≥20 ppm)4,28,39; (3) for the lactulose breath test occurrence of double peaks (two distinct peaks or a characteristic double peak pattern >20 ppm on two consecutive readings40. The criteria for SIBO based on fasting breath levels were evaluated on the basis of the mean of the subject’s baseline values.

Microbiota composition assessment in fecal and mucosal samples

DNA was extracted as previously described41 from feces (n = 35), duodenal biopsies (n = 28), sigmoid colonic biopsies (n = 35). In short, 1-step PCR procedure, amplification was carried out by a high fidelity proofreading polymerase for a total of 25 cycles (fecal samples) or 33 cycles (biopsies). For amplification of the sequencing libraries, forward primer 5′-CAAGCAGAAGACGGCATACGAGAT-N8-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′ and reverse primer 5′AATGATACGGCGACCACCGAGATC-N8-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′, where N8 represents an identifying 8-mer and the last 21 and 19 bases in each construct are the sequence specific forward and reverse primers, respectively. Samples were then pooled to equimolar amounts and sequenced in parallel to whole bacterial genomes in a MiSeq instrument (Illumina Inc, San Diego, CA, USA). All controls from the extraction phase, as well as a negative PCR control have been submitted to PCR and consequently sequenced with the respective samples. Raw reads quality filtering and trimming, operational taxonomic units (OTU) clustering- and taxonomic assignment were performed as previously described41. α-diversity was evaluated on Shannon index and number of OTUs following rarefaction at 1099 sequences using the vegan R package. Beta-diversity (diversity between samples) was performed on Bray-Curtis index. Samples exceeding 10000 number of reads for fecal samples, 500 for duodenal biopsies and 15000 for colonic biopsies (Supplementary Table 2) were used. To ensure that the low number of reads did not impact the results from the duodenum, all analysis were additionally performed with only samples exceeding 1000 reads, which gave similar results in all analysis.

Questionnaires

Subjects completed a HADS and IBS-SSS to assess IBS and psychological symptom severity; for further details please see Supplementary Material.

Antibacterial gene expression profile analysis

Human Antibacterial Response RT² Profiler PCR Arrays (Cat No.ID PAHS-148Z, Qiagen) were conducted as previously described12, on biopsies from duodenum and colon. For further details please see Supplementary Material.

Data processing and statistical analysis

Univariate analysis

Univariate analysis was performed in GraphPad Prism 6 (GraphPad Software, La Jolla, CA), Mann-Whitney was used to assess differences between two groups and Kruskal-Wallis followed by Dunn’s test, was implemented for differences between three groups. To evaluate differences between the slopes of the curves of hydrogen and methane production over time in IBS patients and healthy subjects, linear regression analyses were performed on breath test data. If no other specification, results are presented as median ± (25–75% percentile). Group differences in beta-diversity were determined with PERMANOVA. Correlations between bacteria and antibacterial factors were analyzed with non-parametric Spearman correlations.

Multivariate analysis

Principal component analysis (PCA) was performed in R (Version 1.0.153) in the package ropls, to explore potential differences in the global bacterial and antibacterial recognition factor profiles of groups. Clustered correlation matrix analysis was performed on gene expression data using the corrplot-package in R, calculations were performed with Spearman’s Rank Correlation Coefficient.

Multivariate factor orthogonal partial least squares-discriminant analysis (OPLS-DA) was performed to determine the most discriminatory X variables and to discriminate between healthy subjects and IBS patients as well as for IBS patients clustered into subtypes and subjects positive/negative for SIBO according to glucose or lactulose breath tests, using SIMCA software (Version 14.1.3.0, copyright © MKS Data Analytics Solutions) based on bacterial composition (X variables). The R2 parameter represents the goodness of the fit of the OPLS-DA with the best possible fit being R2 = 1. When considering heterogeneous biological variables, a model would be considered to have a good fit with an R2 ≥ 0.5. The Q2 parameter represents an estimate of the predictive ability calculated by cross-validation, with the best possible predictive ability of the OPLS-DA being Q2 = 1, a Q2 value > 0.4 is considered good for biological variables42. In multivariate analysis, outliers were removed if they were both above the Hotelling’s T2 Range Line of 95% and DModX DCrit (0.05). Loading column plots were generated to identify the X-variables most important for the discrimination between subsets. Variable Importance for the Projection (VIP) > 1.4 was used as the number of bacterial genera exceeded the number of subjects by far, except when identifying the bacterial genera that contributed most to the underlying variation in the X variables (bacterial genera) between the different IBS subtypes, few bacterial genera reach a VIP over 1.4, therefore VIP > 1.1 was used for these analysis. To reduce the risk of over-fit post-hoc 100 permutation tests of the OPLS-DA models were performed and only models with permutation indices fulfilling the post-hoc analysis criteria of R2 < 0.4 and Q2 < 0.05 were accepted43. The loading scatter plot may be superimposed over the OPLS-DA score scatter plot to see what X-variables are associated with which individuals. X variables localized further away from the center of the x-axis contribute more to the discrimination of the groups. The results are presented as a score scatter plot, a loading scatter plot or a loading column plot in which the stronger variables with a more reliable contribution to the model are those with a large column and small confidence interval. Multivariate differences of X variables of the OPLS-DA, between patients and healthy subjects, were tested for significance using Hotelling’s T2 test in R (Package “Hotelling”).