Abdominal bloating is a common symptom defined as a subjective feeling of abdominal pressure regardless of the presence of objective abdominal distention.1 Abdominal bloating is commonly accompanied in patients with functional gastrointestinal disorders such as irritable bowel syndrome (IBS),1-3 functional dyspepsia,4 and functional constipation.5 According to the Rome IV criteria, functional abdominal bloating and distention (FABD) can be diagnosed when there are insufficient criteria for a diagnosis of IBS, functional constipation, functional diarrhea, or postprandial distress syndrome.6
The pathophysiologic mechanisms of FABD are not fully understood, but believed to be multifactorial. The plausible mechanisms include increased luminal contents or gas in the intestine, small intestinal bacterial overgrowth (SIBO), imbalance of gut microbiota, food intolerance, visceral hypersensitivity, and decreased abdominal capacity induced by abnormal viscerosomatic responses or anatomical changes.6-8 Among these mechanisms, increased endogenous gas levels may be associated with dysbiosis and/or SIBO.9,10 However, data regarding SIBO and dysbiosis in patients with FABD are lacking and their association has not been fully investigated. The cause-and-effect relationship between gut microbiota alterations, such as SIBO and fecal dysbiosis, and FABD has not been clearly determined. However, studies have shown that the administration of rifaximin to patients with IBS without constipation improved IBS symptoms and bloating.11 Similarly, administration of probiotics is reported to reduce the symptom severity of patients with functional abdominal bloating.12 Although studies suggest an association of gut microbiota alterations with the pathogenesis of FABD, specific gut microorganisms associated with FABD have not yet been reported.
Therefore, this prospective study was conducted to compare the diversity and composition of fecal microbiota in patients with FABD and healthy individuals, and to evaluate the relationship between SIBO and dysbiosis.
This prospective, single-center study was conducted at Ajou University Hospital (Suwon, Gyeonggi-do, Korea). The study protocol was approved by the institutional review board (Approval No. AJIRB-BMR-SMP-17-472). All participants provided written informed consent.
Patients meeting the Rome IV criteria for FABD were enrolled in the study. The criteria included: (1) recurrent feelings of bloating or visible distention for at least 3 days per month in the last 3 months, (2) onset of symptoms at least 6 months prior to presentation, and (3) insufficient criteria to establish diagnosis of IBS or functional dyspepsia.6 Every subject completed a gastrointestinal symptom questionnaire. Degrees of abdominal distention and bloating sensation were measured by a visual analog scale score. General demographic information, past medical and operation history, medications (antibiotics, probiotics, or other medications), and Bristol stool form scales were recorded.13 All subjects underwent the hydrogen breath test (HBT) and were subsequently classified into SIBO (−) and SIBO (+) groups.
Twelve healthy volunteers without organic or systemic diseases or chronically recurrent abdominal symptoms participated in the study as healthy controls. Exclusion criteria included past history of medical diseases or operation, use of medications affecting gut microbiota in the last 6 months including antibiotics and probiotics, and recurrent abdominal symptoms in the last 3 months. The ethnicity of all study participants including patients with FABD and healthy volunteers was the Korean race.
HBT was performed after an overnight fast. Subjects were asked to abstain from consuming fiber and slowly absorbed carbohydrates (such as bread and potato) the night before the test, as these delay hydrogen excretion in the breath.14,15 Cigarette smoking and exercise were avoided 2 hours before and during the test. Discontinuation of pro-motility and anti-motility medications, antibiotics, and other drugs that can affect gut microbiota for at least 2 weeks prior to the HBT was mandated. Subjects brushed their teeth and rinsed their mouths with antiseptic mouth wash and tap water prior to the test to eliminate an early hydrogen peak due to oral bacterial action on the test sugar.14,15 End-expiratory breath samples were collected in bags. Fasting breath hydrogen concentration was measured 3 to 4 times at the start of the test. The average value was recorded as the basal breath hydrogen level. Subsequently, each subject ingested 10 g of lactulose, and HBT was performed every 15 minutes for 2 to 4 hours. Positive results required an elevated breath hydrogen concentration within 90 minutes, which showed 2 distinct peaks and/or an increase exceeding 20 ppm.15
Fecal samples collected in a centrifuge tube were stored at –20℃ and then transferred to –80℃ freezer within 24 hours for analysis. DNA was extracted from the fecal samples within 48 hours after collection using a DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Extracted DNA was quantified using a Quant-IT PicoGreen assay (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The sequencing library was prepared according to the 16S Metagenomic Sequencing Library protocol (Illumina Inc, San Diego, CA, USA) to amplify the V3 and V4 regions. The methods for library purification, verification, and sequencing have been included in the supplementary materials. Quality filtering and operational taxonomic unit selection are also described in the supplementary materials.
Alpha diversity was calculated using Quantitative Insights Into Microbial Ecology (QIIME) to determine species diversity, evenness, and richness of the microbiome in each sample. Chao1, Shannon, and inversed Simpson index were obtained for each sample, and rarefaction curves were generated to examine species diversity information among samples and groups. For beta diversity analysis, relationships between samples were analyzed using principal coordinate analysis and unweighted pair group method with arithmetic mean tree. The results were visualized using FigTree (version 1.4.4) (http://tree.bio.ed.ac.uk/software/figtree/).16 Dissimilarity between samples was calculated based on weighted UniFrac distances. Taxonomic relative abundance was determined by converting operational taxonomic unit taxonomic information using QIIME to relative abundance percentage at the phylum, genus, and species levels.
Principal component analysis (PCA) was conducted for genus and species with a relative abundance ratio (mean value of the group) ≥ 1%, and for phylum at each level. For PCA, the prcomp function of the R package (version 3.1.2) was used to display the relationship between samples on a coordinate plane.17-19 Krona graphs (multi-layered pie charts allowing efficient visualization of taxonomic hierarchy) were used to visualize the relative abundance of each phylum, genus, and species using the Krona Excel Template (version 2.2).20 The Wilcoxon rank-sum test from R package was used for statistical analysis.21,22 To determine biomarkers differentiating healthy controls and FABD patients, linear discriminant analysis coupled with effect size measurements (LEfSe) was conducted.23 From the relative abundance ratio information, major differences between healthy controls and FABD patients were selected using the Kruskal-Wallis test. These differences were estimated through linear discriminant analysis (LDA) scores (log scale). A bar plot (LDA score) and cladogram were generated for items with a
A total of 21 FABD patients and 12 healthy controls were finally enrolled in the present study. The mean age of FABD patients was significantly higher than that of healthy controls (62.1 ± 14.7 years vs 34.5 ± 3.8 years,
Overall fecal microbiota composition of the FABD group differed from that of the control group, based on the PCA results (principal component 1 = 14.9%; Fig. 1A). Overall fecal microbiota composition between the SIBO (+) and (–) groups in patients with FABD did not differ significantly (Fig. 1B). Principal coordinate analysis and 3-dimensional image reconstruction (Analysis of Similarities R = 0.204,
Relative abundance of bacteria at the phylum level differed between healthy controls and patients with FABD, as shown in the Krona chart (Fig. 4). In healthy controls, Firmicutes (44.0%) accounted for the highest proportion, followed by Bacteroidetes (41.2%), Actinobacteria (9.3%), Proteobacteria (2.3%), and Verrucomicrobia (2.4%). The order in FABD patients was Bacteroidetes (52.8%), Firmicutes (35.1%), Proteobacteria (9.8%), Fusobacteria (0.7%), and Actinobacteria (0.3%). The proportion of Proteobacteria in FABD patients was significantly higher than that in the healthy controls (9.8% vs 2.3%,
The relative abundance of microbiota at the genus level revealed that
At the species level,
Our results of the present study demonstrated that overall fecal microbiota composition in the FABD group differed from that in the control group. Fecal microbiota diversity was significantly lower in FABD patients than that in healthy controls. Moreover, relative abundance of bacteria significantly differed between FABD patients and healthy controls. At the phylum level, significantly higher proportion of Proteobacteria and significantly lower proportion of Actinobacteria were observed in FABD patients, compared with healthy controls. At the genus level, significantly higher proportion of
Abdominal bloating is one of the most common symptoms in functional gastrointestinal disorders. FABD is characterized by subjective symptoms of recurrent abdominal fullness, pressure, or a sensation of gas with or without objective increase in abdominal girth.24 Patients enrolled in the present study did not meet the criteria for other functional bowel disorders such as IBS. However, they may also report symptoms of mild abdominal pain and/or minor bowel movement abnormalities. Therefore, the pathophysiology of FABD is considered to be multifactorial. All patients underwent blood tests, endoscopic examinations, abdominal ultrasonography, or computed tomography to exclude organic causes for abdominal bloating or distension. Plausible causes for FABD include visceral hypersensitivity, abnormal intestinal gas transit, impaired evacuation of rectal gas, colonic fermentation, SIBO, and gut microbiota alterations.24 The relevant etiopathophysiological mechanisms for FABD appear to vary from patient to patient.
It has been proposed that increased gas production or decreased gas consumption in patients with FABD may result from gut microbiota alterations. Gut microbiota alterations may result in excess gas, abnormal regulation of intestinal gas, and/or altered visceral sensation, which requires further investigation. Abnormal intraluminal fermentation by gut bacteria could lead to excessive gas production and bloating symptoms at least in some patients.25 The association of gut microbiota with abdominal bloating is also supported by the beneficial effect of therapeutic interventions targeting the intestinal microbiota, including antibiotics, probiotics, prebiotics, and diet low in fermentable carbohydrates. A recent study investigated the relationship between intestinal microbiota, abdominal bloating, and altered bowel patterns in a cohort of patients with IBS and healthy controls, demonstrating quantitative differences of species/operational taxonomic units between subgroups of IBS patients with and without bloating.26 A significant association between gut microbiota and bloating symptoms in IBS patients suggests a role of intestinal microbiota in the pathogenesis of bloating in IBS. The fecal microbiota alterations at the phylum, genus, and species levels in patients with FABD observed in the present study also provide evidence suggesting a role of intestinal microbiota in the pathogenesis of FABD.
In the present study, SIBO was diagnosed in 42.8% of patients with FABD. At the genus level, higher proportion of
This prospective study has several limitations. While an increased relative abundance of specific bacteria in FABD patients was evident, laboratory evaluation to examine the mechanism related to fecal microbiota alterations was not performed. Second, the effect of age could not be excluded. Age can affect microbiota composition.34 However, it is very difficult to obtain healthy samples from the elderly aged population. The elderly aged persons have difficulty in strictly controlling diet, medication, and other routine life activities, that may affect gut microbiota. In the present study, diet, medication, and routine life activities of healthy controls were controlled for obtaining healthy samples. Although aging may be a factor for gut microbiota shaping, within-individual variation of gut microbiota is known to be relatively stable over time. Studies indicate that age-related gut dysbiosis may contribute to unhealthy aging and reduced longevity, indicating that samples from the aged people may not be real healthy controls. In the present study, we aimed to know the difference in fecal microbiota between patients with FABD and real healthy controls. Actually, the findings of fecal microbiota composition observed in patients with FABD are different from those related to aging. Moreover, the mean age of patients with FABD is 62, which is relatively below that of the old age group. Third, diet is a major factor affecting gut microbiota as well as a crucial factor in the pathogenesis of abdominal bloating.35,36 Dietary factors may contribute to gut microbiota alterations in patients with FABD. Since habitual diet can be associated with bloating symptoms, they were asked to maintain usual dietary patterns in the present study. Thus, diet was not strictly controlled. Fourth, SIBO is defined by the increased number of microbiota in the small intestine. The compositional change in microbiota related to SIBO remains yet unknown. Fecal microbiota may be affected by the small intestine as well as the large intestine. In order to confirm dysbiosis in the small intestine, the intestinal aspirates may be more appropriate rather than feces.
In conclusion, overall fecal microbiota composition in the FABD group differed from that in the control group. FABD-associated alterations of gut microbiota included lower diversity, increased abundance of
Choong-Kyun Noh: planning the study design, reviewing the data, analyzing the data, and drafting the manuscript; and Kwang Jae Lee: planning the study design, collecting the data, reviewing the data, analyzing the data, interpreting the data, drafting the manuscript, and critically revising the manuscript.