J Neurogastroenterol Motil, Vol. 26 No. 4 October, 2020 pISSN: 2093-0879 eISSN: 2093-0887 https://doi.org/10.5056/jnm20080 JNM Journal of Neurogastroenterology and Motility Original Article Fecal Microbiota Alterations and Small Intestinal Bacterial Overgrowth in Functional Abdominal Bloating/Distention Choong-Kyun Noh and Kwang Jae Lee* Department of Gastroenterology, Ajou University School of Medicine, Suwon, Gyeonggi-do, Korea Background/Aims The pathophysiology of functional abdominal bloating and distention (FABD) is unclear yet. Our aim is to compare the diversity and composition of fecal microbiota in patients with FABD and healthy individuals, and to evaluate the relationship between small intestinal bacterial overgrowth (SIBO) and dysbiosis. Methods The microbiota of fecal samples was analyzed from 33 subjects, including 12 healthy controls and 21 patients with FABD diagnosed by the Rome IV criteria. FABD patients underwent a hydrogen breath test. Fecal microbiota composition was determined by 16S ribosomal RNA amplification and sequencing. Results Overall fecal microbiota composition of the FABD group differed from that of the control group. Microbial diversity was significantly lower in the FABD group than in the control group. Significantly higher proportion of Proteobacteria and significantly lower proportion of Actinobacteria were observed in FABD patients, compared with healthy controls. Compared with healthy controls, significantly higher proportion of Faecalibacterium in FABD patients and significantly higher proportion of Prevotella and Faecalibacterium in SIBO (+) patients with FABD were found. Faecalibacterium prausnitzii, was significantly more abundant, but Bacteroides uniformis and Bifidobacterium adolescentis were significantly less abundant in patients with FABD, compared with healthy controls. Significantly more abundant Prevotella copri and F. prausnitzii, and significantly less abundant B. uniformis and B. adolescentis were observed in SIBO (+) patients, compared with healthy controls. Conclusion The fecal microbiota profiles in FABD patients are different from those in healthy controls, particularly in SIBO (+) patients, suggesting a role of gut microbiota in the pathogenesis of FABD. (J Neurogastroenterol Motil 2020;26:539-549) Key Words Faecalibacterium prausnitzii; Abdominal bloating and distention; Microbiota; Prevotella copri Received: April 17, 2020 Revised: June 1, 2020 Accepted: July 3, 2020 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. *Correspondence: Kwang Jae Lee, MD, PhD Department of Gastroenterology, Ajou University School of Medicine, 164, World Cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do 16499, Korea Tel: +82-31-219-5119, Fax: +82-31-219-5999, E-mail: [email protected] ⓒ 2020 The Korean Society of Neurogastroenterology and Motility J Neurogastroenterol Motil, Vol. 26 No. 4 October, 2020 539 www.jnmjournal.org Choong-Kyun Noh and Kwang Jae Lee AJIRB-BMR-SMP-17-472). All participants provided written informed consent. Introduction Patients meeting the Rome IV criteria for FABD were enrolled Abdominal bloating is a common symptom defined as a sub- in the study. The criteria included: (1) recurrent feelings of bloat- jective feeling of abdominal pressure regardless of the presence of ing or visible distention for at least 3 days per month in the last 3 objective abdominal distention.1 Abdominal bloating is commonly months, (2) onset of symptoms at least 6 months prior to presenta- accompanied in patients with functional gastrointestinal disorders tion, and (3) insufficient criteria to establish diagnosis of IBS or such as irritable bowel syndrome (IBS),1-3 functional dyspepsia,4 functional dyspepsia.6 Every subject completed a gastrointestinal and functional constipation.5 According to the Rome IV criteria, symptom questionnaire. Degrees of abdominal distention and functional abdominal bloating and distention (FABD) can be di- bloating sensation were measured by a visual analog scale score. agnosed when there are insufficient criteria for a diagnosis of IBS, General demographic information, past medical and operation his- functional constipation, functional diarrhea, or postprandial distress tory, medications (antibiotics, probiotics, or other medications), and syndrome.6 Bristol stool form scales were recorded.13 All subjects underwent the The pathophysiologic mechanisms of FABD are not fully hydrogen breath test (HBT) and were subsequently classified into understood, but believed to be multifactorial. The plausible mecha- SIBO (−) and SIBO (+) groups. nisms include increased luminal contents or gas in the intestine, Twelve healthy volunteers without organic or systemic diseases small intestinal bacterial overgrowth (SIBO), imbalance of gut or chronically recurrent abdominal symptoms participated in the microbiota, food intolerance, visceral hypersensitivity, and decreased study as healthy controls. Exclusion criteria included past history abdominal capacity induced by abnormal viscerosomatic responses of medical diseases or operation, use of medications affecting gut or anatomical changes.6-8 Among these mechanisms, increased microbiota in the last 6 months including antibiotics and probiotics, endogenous gas levels may be associated with dysbiosis and/or and recurrent abdominal symptoms in the last 3 months. The eth- SIBO.9,10 However, data regarding SIBO and dysbiosis in patients nicity of all study participants including patients with FABD and with FABD are lacking and their association has not been fully in- healthy volunteers was the Korean race. vestigated. The cause-and-effect relationship between gut microbio- ta alterations, such as SIBO and fecal dysbiosis, and FABD has not Hydrogen Breath Tests been clearly determined. However, studies have shown that the ad- HBT was performed after an overnight fast. Subjects were ministration of rifaximin to patients with IBS without constipation asked to abstain from consuming fiber and slowly absorbed car- improved IBS symptoms and bloating.11 Similarly, administration bohydrates (such as bread and potato) the night before the test, as of probiotics is reported to reduce the symptom severity of patients these delay hydrogen excretion in the breath.14,15 Cigarette smok- with functional abdominal bloating.12 Although studies suggest an ing and exercise were avoided 2 hours before and during the test. association of gut microbiota alterations with the pathogenesis of Discontinuation of pro-motility and anti-motility medications, FABD, specific gut microorganisms associated with FABD have antibiotics, and other drugs that can affect gut microbiota for at not yet been reported. least 2 weeks prior to the HBT was mandated. Subjects brushed Therefore, this prospective study was conducted to compare their teeth and rinsed their mouths with antiseptic mouth wash and the diversity and composition of fecal microbiota in patients with tap water prior to the test to eliminate an early hydrogen peak due FABD and healthy individuals, and to evaluate the relationship be- to oral bacterial action on the test sugar.14,15 End-expiratory breath tween SIBO and dysbiosis. samples were collected in bags. Fasting breath hydrogen concentra- tion was measured 3 to 4 times at the start of the test. The average value was recorded as the basal breath hydrogen level. Subsequently, Materials and Methods each subject ingested 10 g of lactulose, and HBT was performed every 15 minutes for 2 to 4 hours. Positive results required an el- Study Population and Design evated breath hydrogen concentration within 90 minutes, which This prospective, single-center study was conducted at Ajou showed 2 distinct peaks and/or an increase exceeding 20 ppm.15 University Hospital (Suwon, Gyeonggi-do, Korea). The study pro- tocol was approved by the institutional review board (Approval No. 540 Journal of Neurogastroenterology and Motility Fecal Microbiota and SIBO in Functional Abdominal Boating/Distention Table. Baseline Characteristics of Study Subjects Fecal Sample Collection, DNA Extraction, and HC FABD Variables P-value Sequencing (n = 12) (n = 21) Fecal samples collected in a centrifuge tube were stored at Gender (n [%]) 0.839 –20℃ and then transferred to –80℃ freezer within 24 hours for Male 8 (66.7) 13 (61.9) analysis. DNA was extracted from the fecal samples within 48 Female 4 (33.3) 8 (38.1) hours after collection using a DNeasy PowerSoil Kit (Qiagen, Age (yr) 0.012 Hilden, Germany) according to the manufacturer’s instructions. Mean ± SD 34.5 ± 3.8 62.1 ± 14.7 Range 33-45 22-81 Extracted DNA was quantified using a Quant-IT PicoGreen assay 2 BMI (kg/m ) 0.910 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s Mean ± SD 23.0 ± 2.9 24.2 ± 2.7 instructions. The sequencing library was prepared according to Medical history (n [%]) 0.122 the 16S Metagenomic Sequencing Library protocol (Illumina Inc, Hypertension 0 (0.0) 5 (23.8) San Diego, CA, USA) to amplify the V3 and V4 regions. The Diabetes 0 (0.0) 5 (23.8) methods for library purification, verification, and sequencing have Old CVA 0 (0.0) 0 (0.0) Smoking history (n [%]) 0.839 been included in the supplementary materials. Quality filtering None 9 (75.0) 16 (76.2) and operational taxonomic unit selection are also described in the Past 0 (0.0) 3 (14.3) supplementary