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      Irritable Bowel Syndrome, Particularly the Constipation-Predominant Form, Involves an Increase in Methanobrevibacter smithii, Which Is Associated with Higher Methane Production

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          Abstract

          Background/Aims

          Because Methanobrevibacter smithii produces methane, delaying gut transit, we evaluated M. smithii loads in irritable bowel syndrome (IBS) patients and healthy controls (HC).

          Methods

          Quantitative real-time polymerase chain reaction for M. smithii was performed on the feces of 47 IBS patients (Rome III) and 30 HC. On the lactulose hydrogen breath test (LHBT, done for 25 IBS patients), a fasting methane result ≥10 ppm using 10 g of lactulose defined methane-producers.

          Results

          Of 47, 20 had constipation (IBS-C), 20 had diarrhea (IBS-D) and seven were not sub-typed. The M. smithii copy number was higher among IBS patients than HC (Log 105.4, interquartile range [IQR; 3.2 to 6.3] vs 1.9 [0.0 to 3.4], p<0.001), particularly among IBS-C compared to IBS-D patients (Log 106.1 [5.5 to 6.6] vs 3.4 [0.6 to 5.7], p=0.001); the copy number negatively correlated with the stool frequency (R=−0.420, p=0.003). The M. smithii copy number was higher among methane-producers than nonproducers (Log 106.4, IQR [5.7 to 7.4] vs 4.1 [1.8 to 5.8], p=0.001). Using a receiver operating characteristic curve, the best cutoff for M. smithii among methane producers was Log 106.0 (sensitivity, 64%; specificity, 86%; area under curve [AUC], 0.896). The AUC for breath methane correlated with the M. smithii copy number among methane producers (r=0.74, p=0.008). Abdominal bloating was more common among methane producers (n=9/11 [82%] vs 5/14 [36%], p=0.021).

          Conclusions

          Patients with IBS, particularly IBS-C, had higher copy numbers of M. smithii than HC. On LHBT, breath methane levels correlated with M. smithii loads.

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          Most cited references28

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          Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota.

          Fecal bacteria were studied in healthy elderly volunteers (age, 63 to 90 years; n = 35) living in the local community, elderly hospitalized patients (age, 66 to 103; n = 38), and elderly hospitalized patients receiving antibiotic treatment (age, 65 to 100; n = 21). Group- and species-specific primer sets targeting 16S rRNA genes were used to quantitate intestinal bacteria by using DNA extracted from feces and real-time PCR. The principal difference between healthy elderly volunteers and both patient cohorts was a marked reduction in the Bacteroides-Prevotella group following hospitalization. Reductions in bifidobacteria, Desulfovibrio spp., Clostridium clostridiiforme, and Faecalibacterium prausnitzii were also found in the hospitalized patients. However, total 16S rRNA gene copy numbers (per gram of wet weight of feces) were generally lower in the stool samples of the two groups of hospitalized patients compared to the number in the stool samples of elderly volunteers living in the community, so the relative abundance (percentage of the group- and species-specific rRNA gene copies in relation to total bacterial rRNA gene copies) of bifidobacteria, Desulfovibrio spp., C. clostridiiforme, and F. prausnitzii did not change. Antibiotic treatment resulted in further reductions in the numbers of bacteria and their prevalence and, in some patients, complete elimination of certain bacterial communities. Conversely, the numbers of enterobacteria increased in the hospitalized patients who did not receive antibiotics, and due to profound changes in fecal microbiotas during antibiotic treatment, the opportunistic species Enterococcus faecalis proliferated.
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            High Prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae Detected in the Human Gut Using an Improved DNA Detection Protocol

            Background The low and variable prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae DNA in human stool contrasts with the paramount role of these methanogenic Archaea in digestion processes. We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool. Methodology/Principal Findings Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens (15 for M. smithii and 15 for M. stadtmanae) yielded a sequence similarity of 99–100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences. Conclusions/Significance In contrast to previous reports, these data indicate a high prevalence of the methanogens M. smithii and M. stadtmanae in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome.
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              Methane, a gas produced by enteric bacteria, slows intestinal transit and augments small intestinal contractile activity.

              The presence of methane on lactulose breath test among irritable bowel syndrome (IBS) subjects is highly associated with the constipation-predominant form. Therefore, we set out to determine whether methane gas can alter small intestinal motor function. In dogs, small intestinal fistulae were created to permit measurement of intestinal transit. Using a radiolabel, we evaluated transit during infusion of room air and subsequently methane. In this model, small intestinal infusion of methane produced a slowing of transit in all dogs by an average of 59%. In a second experiment, guinea pig ileum was pinned into an organ bath for the study of contractile activity in response to brush strokes applied to the mucosa. The force of contraction was measured both orad and aborad to the stimulus. The experiment was repeated while the bath was gassed with methane. Contractile activities orad and aborad to the stimulus were significantly augmented by methane compared with room air (P < 0.05). In a third experiment, humans with IBS who had undergone a small bowel motility study were compared such that subjects who produced methane on lactulose breath test were compared with those producing hydrogen. The motility index was significantly higher in methane-producing IBS patients (1,851 +/- 861) compared with hydrogen producers (1,199 +/- 301) (P < 0.05). Therefore, methane, a gaseous by-product of intestinal bacteria, slows small intestinal transit and appears to do so by augmenting small bowel contractile activity.
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                Author and article information

                Journal
                Gut Liver
                Gut Liver
                Gut and Liver
                Editorial Office of Gut and Liver
                1976-2283
                2005-1212
                November 2016
                27 July 2016
                : 10
                : 6
                : 932-938
                Affiliations
                [1 ]Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India
                [2 ]Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India
                Author notes
                Correspondence to: Ujjala Ghoshal, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226014, India, Tel: +91-0522-5221, Fax: +91-522-2668129, E-mail: ujjalaghoshal@ 123456yahoo.co.in

                Ujjala Ghoshal and Ratnakar Shukla contributed equally to this work as first authors.

                Article
                gnl-10-932
                10.5009/gnl15588
                5087933
                27458176
                769d5e9e-8eac-45d2-be35-5b3497708319
                Copyright © 2016 by The Korean Society of Gastroenterology, the Korean Society of Gastrointestinal Endoscopy, the Korean Society of Neurogastroenterology and Motility, Korean College of Helicobacter and Upper Gastrointestinal Research, Korean Association the Study of Intestinal Diseases, the Korean Association for the Study of the Liver, Korean Pancreatobiliary Association, and Korean Society of Gastrointestinal Cancer.

                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.

                History
                : 16 November 2015
                : 06 January 2016
                : 30 January 2016
                Categories
                Original Article

                Gastroenterology & Hepatology
                methanogenic flora,real-time polymerase chain reaction,lactulose hydrogen breath test,gut transit

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