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Rifaximin is FDA-approved for treatment of irritable bowel syndrome with diarrhea (IBS-D), but poor solubility may limit its efficacy against microbes in the mucus layer, e.g. Escherichia coli. Here we evaluate adding the mucolytic N-acetylcysteine (NAC) to improve rifaximin efficacy. In a resazurin checkerboard assay, combining rifaximin with NAC had significant synergistic effects in reducing E. coli levels. The optimal rifaximin + NAC combination was then tested in a validated rat model of IBS-D (induced by cytolethal distending toxin [CdtB] inoculation). Rats were inoculated with vehicle and treated with placebo (Control-PBS) or rifaximin + NAC (Control-Rif + NAC, safety), or inoculated with CdtB and treated with placebo (CdtB-PBS), rifaximin (CdtB-Rifaximin), or rifaximin + NAC (CdtB-Rif + NAC) for 10 days. CdtB-inoculated rats (CdtB-PBS) developed wide variability in stool consistency (P = 0.0014) vs. controls (Control-PBS). Stool variability normalized in rats treated with rifaximin + NAC (CdtB-Rif + NAC) but not rifaximin alone (CdtB-Rifaximin). Small bowel bacterial levels were elevated in CdtB-PBS rats but normalized in CdtB-Rif + NAC but not CdtB-Rifaximin rats. E. coli and Desulfovibrio spp levels (each associated with different IBS-D microtypes) were also elevated in CdtB-inoculated (CdtB-PBS) but normalized in CdtB-Rif + NAC rats. Cytokine levels normalized only in CdtB-Rif + NAC rats, in a manner predicted to be associated with reduced diarrhea driven by reduced E. coli. These findings suggest that combining rifaximin with NAC may improve the percentage of IBS-D patients responding to treatment., (© 2024. The Author(s).)

Introduction: Gut microbiome changes are linked to obesity, but findings are based on stool data. In this article, we analyzed the duodenal microbiome and serum biomarkers in subjects with normal weight, overweight, and obesity., Methods: Duodenal aspirates and serum samples were obtained from subjects undergoing standard-of-care esophagogastroduodenoscopy without colon preparation. Aspirate DNAs were analyzed by 16S rRNA and shotgun sequencing. Predicted microbial metabolic functions and serum levels of metabolic and inflammatory biomarkers were also assessed., Results: Subjects with normal weight (N = 105), overweight (N = 67), and obesity (N = 42) were identified. Overweight-specific duodenal microbial features include lower relative abundance (RA) of Bifidobacterium species and Escherichia coli strain K-12 and higher Lactobacillus intestinalis , L. johnsonii , and Prevotella loescheii RA. Obesity-specific features include higher Lactobacillus gasseri RA and lower L. reuteri (subspecies rodentium ), Alloprevotella rava , and Leptotrichia spp RA. Escalation features (progressive changes from normal weight through obesity) include decreasing Bacteroides pyogenes , Staphylococcus hominis , and unknown Faecalibacterium species RA, increasing RA of unknown Lactobacillus and Mycobacterium species, and decreasing microbial potential for biogenic amines metabolism. De-escalation features (direction of change altered in normal to overweight and overweight to obesity) include Lactobacillus acidophilus , L. hominis , L. iners , and Bifidobacterium dentium . An unknown Lactobacillus species is associated with type IIa dyslipidemia and overweight, whereas Alloprevotella rava is associated with type IIb and IV dyslipidemias., Discussion: Direct analysis of the duodenal microbiome has identified key genera associated with overweight and obesity, including some previously identified in stool, e.g., Bifidobacterium and Lactobacillus . Specific species and strains exhibit differing associations with overweight and obesity, including escalation and de-escalation features that may represent targets for future study and therapeutics., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Background& Aims: Despite accelerated research in small intestinal bacterial overgrowth (SIBO), questions remain regarding optimal diagnostic approaches and definitions. Here, we aim to define SIBO using small bowel culture and sequencing, identifying specific contributory microbes, in the context of gastrointestinal symptoms., Methods: Subjects undergoing esophagogastroduodenoscopy (without colonoscopy) were recruited and completed symptom severity questionnaires. Duodenal aspirates were plated on MacConkey and blood agar. Aspirate DNA was analyzed by 16S ribosomal RNA and shotgun sequencing. Microbial network connectivity for different SIBO thresholds and predicted microbial metabolic functions were also assessed., Results: A total of 385 subjects with <10 3 colony forming units (CFU)/mL on MacConkey agar and 98 subjects with ≥10 3 CFU/mL, including ≥10 3 to <10 5 CFU/mL (N = 66) and ≥10 5 CFU/mL (N = 32), were identified. Duodenal microbial α-diversity progressively decreased, and relative abundance of Escherichia/Shigella and Klebsiella increased, in subjects with ≥10 3 to <10 5 CFU/mL and ≥10 5 CFU/mL. Microbial network connectivity also progressively decreased in these subjects, driven by the increased relative abundance of Escherichia (P < .0001) and Klebsiella (P = .0018). Microbial metabolic pathways for carbohydrate fermentation, hydrogen production, and hydrogen sulfide production were enhanced in subjects with ≥10 3 CFU/mL and correlated with symptoms. Shotgun sequencing (N = 38) identified 2 main Escherichia coli strains and 2 Klebsiella species representing 40.24% of all duodenal bacteria in subjects with ≥10 3 CFU/mL., Conclusions: Our findings confirm ≥10 3 CFU/mL is the optimal SIBO threshold, associated with gastrointestinal symptoms, significantly decreased microbial diversity, and network disruption. Microbial hydrogen- and hydrogen sulfide-related pathways were enhanced in SIBO subjects, supporting past studies. Remarkably few specific E coli and Klebsiella strains/species appear to dominate the microbiome in SIBO, and correlate with abdominal pain, diarrhea, and bloating severities., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: We recently demonstrated that diarrhea-predominant irritable bowel syndrome (IBS-D) subjects have higher relative abundance (RA) of hydrogen sulfide (H 2 S)-producing Fusobacterium and Desulfovibrio species, and constipation-predominant IBS (IBS-C) subjects have higher RA of methanogen Methanobrevibacter smithii., Aims: In this study, we investigate the effects of increased methanogens or H 2 S producers on stool phenotypes in rat models., Methods: Adult Sprague-Dawley rats were fed high-fat diet (HFD) for 60 days to increase M. smithii levels, then gavaged for 10 days with water (controls) or methanogenesis inhibitors. To increase H 2 S producers, rats were gavaged with F. varium or D. piger. Stool consistency (stool wet weight (SWW)) and gas production were measured. 16S rRNA gene sequencing was performed on stool samples., Results: In HFD diet-fed rats (N = 30), stool M. smithii levels were increased (P < 0.001) after 52 days, correlating with significantly decreased SWW (P < 0.0001) at 59 days (R = - 0.38, P = 0.037). Small bowel M. smithii levels decreased significantly in lovastatin lactone-treated rats (P < 0.0006), and SWW increased (normalized) in lovastatin hydroxyacid-treated rats (P = 0.0246), vs. controls (N = 10/group). SWW increased significantly in D. piger-gavaged rats (N = 16) on day 10 (P < 0.0001), and in F. varium-gavaged rats (N = 16) at all timepoints, vs. controls, with increased stool H 2 S production. 16S sequencing revealed stool microbiota alterations in rats gavaged with H 2 S producers, with higher relative abundance (RA) of other H 2 S producers, particularly Lachnospiraceae and Bilophila in F. varium-gavaged rats, and Sutterella in D. piger-gavaged rats., Conclusions: These findings suggest that increased M. smithii levels result in a constipation-like phenotype in a rat model that is partly reversible with methanogenesis inhibitors, whereas gavage with H 2 S producers D. piger or F. varium results in increased colonization with other H 2 S producers and diarrhea-like phenotypes. This supports roles for the increased RA of methanogens and H 2 S producers identified in IBS-C and IBS-D subjects, respectively, in contributing to stool phenotypes., (© 2023. The Author(s).)

Diarrhea-predominant irritable bowel syndrome (IBS-D), associated with increased intestinal permeability, inflammation, and small intestinal bacterial overgrowth, can be triggered by acute gastroenteritis. Cytolethal distending toxin B (CdtB) is produced by gastroenteritis-causing pathogens and may underlie IBS-D development, through molecular mimicry with vinculin. Here, we examine the effects of exposure to CdtB alone on gut microbiome composition, host intestinal gene expression, and IBS-D-like phenotypes in a rat model. CdtB-inoculated rats exhibited increased anti-CdtB levels, which correlated with increased stool wet weights, pro-inflammatory cytokines (TNFα, IL2) and predicted microbial metabolic pathways including inflammatory responses, TNF responses, and diarrhea. Three distinct ileal microbiome profiles (microtypes) were identified in CdtB-inoculated rats. The first microtype (most like controls) had altered relative abundance (RA) of genera Bifidobacterium, Lactococcus, and Rothia . The second had lower microbial diversity, higher Escherichia-Shigella RA, higher absolute E. coli abundance, and altered host ileal tissue expression of immune-response and TNF-response genes compared to controls. The third microtype had higher microbial diversity, higher RA of hydrogen sulfide (H 2 S)-producer Desulfovibrio , and increased expression of H 2 S-associated pain/serotonin response genes. All CdtB-inoculated rats exhibited decreased ileal expression of cell junction component mRNAs, including vinculin-associated proteins. Significantly, cluster-specific microRNA-mRNA interactions controlling intestinal permeability, visceral hypersensitivity/pain, and gastrointestinal motility genes, including several previously associated with IBS were seen. These findings demonstrate that exposure to CdtB toxin alone results in IBS-like phenotypes including inflammation and diarrhea-like stool, decreased expression of intestinal barrier components, and altered ileal microtypes that influenced changes in microRNA-modulated gene expression and predicted metabolic pathways consistent with specific IBS-D symptoms.

Studies using stool samples suggest that non-sugar sweetener (NSS) consumption affects gut microbiome composition. However, stool does not represent the entire gut. We analyzed the duodenal luminal microbiome in subjects consuming non-aspartame non-sugar sweeteners (NANS, N = 35), aspartame only (ASP, N = 9), and controls (CON, N = 55) and the stool microbiome in a subset (N = 40). Duodenal alpha diversity was decreased in NANS vs. CON. Duodenal relative abundance (RA) of Escherichia , Klebsiella , and Salmonella (all phylum Proteobacteria) was lower in both NANS and ASP vs. CON, whereas stool RA of Escherichia , Klebsiella , and Salmonella was increased in both NANS and ASP vs. CON. Predicted duodenal microbial metabolic pathways altered in NANS vs. CON included polysaccharides biosynthesis and D-galactose degradation, whereas cylindrospermopsin biosynthesis was significantly enriched in ASP vs. CON. These findings suggest that consuming non-sugar sweeteners may significantly alter microbiome composition and function in the metabolically active small bowel, with different alterations seen in stool., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: The coronavirus disease 2019 (COVID-19) global pandemic necessitated many severe lifestyle changes, including lockdowns, social distancing, altered food consumption and exercise patterns, and extensive hygiene practices. These extensive changes may have affected the human gut microbiome, which is highly influenced by lifestyle., Aims: To examine the potential effects of pandemic-related lifestyle changes on the metabolically relevant small bowel microbiome., Methods: Adult subjects presenting for upper endoscopy without colonoscopy were identified and divided into two matched groups: pre-pandemic (February 2019-March 2020) and intra-pandemic (April 2021-September 2021, all COVID-19 negative). Duodenal aspirates and blood samples were collected. Duodenal microbiomes were analyzed by 16S rRNA sequencing. Serum cytokine levels were analyzed by Luminex FlexMap3D., Results: Fifty-six pre-pandemic and 38 COVID-negative intra-pandemic subjects were included. There were no significant changes in duodenal microbial alpha diversity in the intra-pandemic vs. pre-pandemic group, but beta diversity was significantly different. The relative abundance (RA) of phylum Deinococcus-Thermus and family Thermaceae, which are resistant extremophiles, was significantly higher in the intra-pandemic vs. pre-pandemic group. The RA of several Gram-negative taxa including Bacteroidaceae (phylum Bacteroidetes) and the Proteobacteria families Enterobacteriaceae and Pseudomonadaceae, and the RA of potential disruptor genera Escherichia-Shigella and Rothia, were significantly lower in the intra-pandemic vs. pre-pandemic group. Circulating levels of interleukin-18 were also lower in the intra-pandemic group., Conclusions: These findings suggest the small bowel microbiome underwent significant changes during the pandemic, in COVID-19-negative individuals. Given the key roles of the small bowel microbiota in host physiology, this may have implications for human health., (© 2023. The Author(s).)

Introduction: Non-alcoholic fatty liver disease (NAFLD) is a multifactorial, wide-spectrum liver disorder. Small intestinal bacterial overgrowth (SIBO) is characterized by an increase in the number and/or type of colonic bacteria in the upper gastrointestinal tract. SIBO, through energy salvage and induction of inflammation, may be a pathophysiological factor for NAFLD development and progression., Aim/methods: Consecutive patients with histological, biochemical, or radiological diagnosis of any stage of NAFLD (non-alcoholic fatty liver [NAFL], non-alcoholic steatohepatitis [NASH], cirrhosis) underwent upper gastrointestinal endoscopy. Duodenal fluid (2cc) was aspirated from the 3rd-4th part of duodenum into sterile containers. SIBO was defined as ≥10 3 aerobic colony-forming units (CFU)/mL of duodenal aspirate and/or the presence of colonic-type bacteria. Patients without any liver disease undergoing gastroscopy due to gastroesophageal reflux disease (GERD) comprised the healthy control (HC) group. Concentrations (pg/mL) of tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, and IL-6 were also measured in the duodenal fluid. The primary endpoint was to evaluate the prevalence of SIBO in NAFLD patients, while the comparison of SIBO prevalence among NAFLD patients and healthy controls was a secondary endpoint., Results: We enrolled 125 patients (51 NAFL, 27 NASH, 17 cirrhosis, and 30 HC) aged 54 ± 11.9 years and with a weight of 88.3 ± 19.6 kg (NAFLD vs. HC 90.7 ± 19.1 vs. 80.8 ± 19.6 kg, p = 0.02). Overall, SIBO was diagnosed in 23/125 (18.4%) patients, with Gram-negative bacteria being the predominant species (19/23; 82.6%). SIBO prevalence was higher in the NAFLD cohort compared to HC (22/95; 23.2% vs. 1/30; 3.3%, p = 0.014). Patients with NASH had higher SIBO prevalence (6/27; 22.2%) compared to NAFL individuals (8/51; 15.7%), but this difference did not reach statistical significance ( p = 0.11). Patients with NASH-associated cirrhosis had a higher SIBO prevalence compared to patients with NAFL (8/17; 47.1% vs. 8/51; 15.7%, p = 0.02), while SIBO prevalence between patients with NASH-associated cirrhosis and NASH was not statistically different (8/17; 47.1% vs. 6/27; 22.2%, p = 0.11). Mean concentration of TNF-α, IL-1β, and IL-6 did not differ among the different groups., Conclusion: The prevalence of SIBO is significantly higher in a cohort of patients with NAFLD compared to healthy controls. Moreover, SIBO is more prevalent in patients with NASH-associated cirrhosis compared to patients with NAFL.

Introduction: Irritable bowel syndrome (IBS) includes diarrhea-predominant (IBS-D) and constipation-predominant (IBS-C) subtypes. We combined breath testing and stool microbiome sequencing to identify potential microbial drivers of IBS subtypes., Methods: IBS-C and IBS-D subjects from 2 randomized controlled trials (NCT03763175 and NCT04557215) were included. Baseline breath carbon dioxide, hydrogen (H 2 ), methane (CH 4 ), and hydrogen sulfide (H 2 S) levels were measured by gas chromatography, and baseline stool microbiome composition was analyzed by 16S rRNA sequencing. Microbial metabolic pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes collection databases., Results: IBS-C subjects had higher breath CH 4 that correlated with higher gut microbial diversity and higher relative abundance (RA) of stool methanogens, predominantly Methanobrevibacter , as well as higher absolute abundance of Methanobrevibacter smithii in stool. IBS-D subjects had higher breath H 2 that correlated with lower microbial diversity and higher breath H 2 S that correlated with higher RA of H 2 S-producing bacteria, including Fusobacterium and Desulfovibrio spp. The predominant H 2 producers were different in these distinct microtypes, with higher RA of Ruminococcaceae and Christensenellaceae in IBS-C/CH 4 + (which correlated with Methanobacteriaceae RA) and higher Enterobacteriaceae RA in IBS-D. Finally, microbial metabolic pathway analysis revealed enrichment of Kyoto Encyclopedia of Genes and Genomes modules associated with methanogenesis and biosynthesis of methanogenesis cofactor F420 in IBS-C/CH 4 + subjects, whereas modules associated with H 2 S production, including sulfate reduction pathways, were enriched in IBS-D., Discussion: Our findings identify distinct gut microtypes linked to breath gas patterns in IBS-C and IBS-D subjects, driven by methanogens such as M. smithii and H 2 S producers such as Fusobacterium and Desulfovibrio spp, respectively., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Introduction: Stool form assessment relies on subjective patient reports using the Bristol Stool Scale (BSS). In a novel smartphone application (app), trained artificial intelligence (AI) characterizes digital images of users' stool. In this study, we evaluate this AI for accuracy in assessing stool characteristics., Methods: Subjects with diarrhea-predominant irritable bowel syndrome image-captured every stool for 2 weeks using the app, which assessed images for 5 visual characteristics (BSS, consistency, fragmentation, edge fuzziness, and volume). In the validation phase, using 2 expert gastroenterologists as a gold standard, sensitivity, specificity, accuracy, and diagnostic odds ratios of subject-reported vs AI-graded BSS scores were compared. In the implementation phase, agreements between AI-graded and subject-reported daily average BSS scores were determined, and subject BSS and AI stool characteristics scores were correlated with diarrhea-predominant irritable bowel syndrome symptom severity scores., Results: In the validation phase (n = 14), there was good agreement between the 2 experts and AI characterizations for BSS (intraclass correlation coefficients [ICC] = 0.782-0.852), stool consistency (ICC = 0.873-0.890), edge fuzziness (ICC = 0.836-0.839), fragmentation (ICC = 0.837-0.863), and volume (ICC = 0.725-0.851). AI outperformed subjects' self-reports in categorizing daily average BSS scores as constipation, normal, or diarrhea. In the implementation phase (n = 25), the agreement between AI and self-reported BSS scores was moderate (ICC = 0.61). AI stool characterization also correlated better than subject reports with diarrhea severity scores., Discussion: A novel smartphone application can determine BSS and other visual stool characteristics with high accuracy compared with the 2 expert gastroenterologists. Moreover, trained AI was superior to subject self-reporting of BSS. AI assessments could provide more objective outcome measures for stool characterization in gastroenterology., (Copyright © 2022 by The American College of Gastroenterology.)

Gut microbiome composition is different in males and females, but sex is rarely considered when prescribing antibiotics, and sex-based differences in gut microbiome recovery following antibiotic treatment are poorly understood. Here, we compared the effects of broad-spectrum antibiotics on both the stool and small bowel microbiomes in male and female rats. Adult male and female Sprague Dawley rats were exposed to a multi-drug antibiotic cocktail for 8 days, or remained unexposed as controls. Following cessation of antibiotics, rats were monitored for an additional 13-day recovery period prior to euthanasia. Baseline stool microbiome composition was similar in males and females. By antibiotic exposure day 8 (AbxD8), exposed male rats exhibited greater loss of stool microbial diversity compared to exposed females, and the relative abundance (RA) of numerous taxa were significantly different in exposed males vs. exposed females. Specifically, RA of phylum Proteobacteria and genera Lactobacillus , Sutterella , Akkermansia , and Serratia were higher in exposed males vs. exposed females, whereas RA of phyla Firmicutes and Actinobacteria and genera Turicibacter and Enterococcus were lower. By 13 days post antibiotics cessation (PAbxD13), the stool RA of these and other taxa remained significantly different from baseline, and also remained significantly different between exposed males and exposed females. RA of phyla Firmicutes and Actinobacteria and genus Enterococcus remained lower in exposed males vs. exposed females, and genus Sutterella remained higher. However, RA of phylum Proteobacteria and genus Akkermansia were now also lower in exposed males vs. females, whereas RA of phylum Bacteroidetes and genus Turicibacter were now higher in exposed males. Further, the small bowel microbiome of exposed rats on PAbxD13 was also significantly different from unexposed controls, with higher RA of Firmicutes, Turicibacter and Parabacteroides in exposed males vs. females, and lower RA of Bacteroidetes, Proteobacteria, Actinobacteria, Oscillospira , Sutterella , and Akkermansia in exposed males vs. females. These findings indicate that broad-spectrum antibiotics have significant and sex-specific effects on gut microbial populations in both stool and the small bowel, and that the recovery of gut microbial populations following exposure to broad-spectrum antibiotics also differs between sexes. These findings may have clinical implications for the way antibiotics are prescribed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Parodi, Leite, Pimentel, Barlow, Fiorentino, Morales, Pimentel, Weitsman and Mathur.)

Background: Proton pump inhibitor (PPI) use is extremely common. PPIs have been suggested to affect the gut microbiome, and increase risks of Clostridium difficile infection and small intestinal bacterial overgrowth (SIBO). However, existing data are based on stool analyses and PPIs act on the foregut., Aims: To compare the duodenal and stool microbiomes in PPI and non-PPI users., Methods: Consecutive subjects presenting for upper endoscopy without colonoscopy were recruited. Current antibiotic users were excluded. Subjects taking PPI were age- and gender-matched 1:2 to non-PPI controls. Subjects completed medical history questionnaires, and duodenal aspirates were collected using a validated protected catheter. A subset also provided stool samples. Duodenal and stool microbiomes were analyzed by 16S rRNA sequencing., Results: The duodenal microbiome exhibited no phylum-level differences between PPI (N = 59) and non-PPI subjects (N = 118), but demonstrated significantly higher relative abundances of families Campylobacteraceae (3.13-fold, FDR P value < 0.01) and Bifidobacteriaceae (2.9-fold, FDR P value < 0.01), and lower relative abundance of Clostridiaceae (88.24-fold, FDR P value < 0.0001), in PPI subjects. SIBO rates were not significantly different between groups, whether defined by culture (> 10 3  CFU/ml) or 16S sequencing, nor between subjects taking different PPIs. The stool microbiome exhibited significantly higher abundance of family Streptococcaceae (2.14-fold, P = 0.003), and lower Clostridiaceae (2.60-fold, FDR P value = 8.61E-13), in PPI (N = 22) versus non-PPI (N = 47) subjects., Conclusions: These findings suggest that PPI use is not associated with higher rates of SIBO. Relative abundance of Clostridiaceae was reduced in both the duodenal and stool microbiomes, and Streptococcaceae was increased in stool. The clinical implications of these findings are unknown., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Background: Recent data substantiate the importance of acute gastroenteritis in the development of irritable bowel syndrome (IBS). An animal model of postinfectious IBS determined the importance of cytolethal distending toxin B (CdtB) during live Campylobacter jejuni infection and its development of autoimmunity to vinculin. In this study, we examine whether subcutaneous exposure to CdtB alone is sufficient to produce the postinfectious IBS effect and autoimmunity., Methods: Sixty adult Sprague Dawley rats were randomized into 2 groups to receive subcutaneous injection of either CdtB or vehicle and administered a booster injection of the same product 3 weeks later. Serum was collected for anti-CdtB and anti-vinculin titers. Duodenal and ileal luminal contents for total eubacterial qPCR, and ileal bowel segments were harvested for vinculin and ileal expression. In a second experiment, 4 adult, Sprague Dawley rats were injected with either Cy7-labeled anti-CdtB and anti-vinculin antibodies were injected into the tail vein and imaged to determine organ localization of the antibodies., Key Results: Rats that received CdtB increased in serum anti-CdtB after injection. CdtB exposure also precipitated significant elevation in anti-vinculin antibodies (P < .001). This was associated with a reduction in intestinal vinculin expression (P < .001) that negatively correlated with serum anti-CdtB levels. CdtB exposure was also associated with greater levels of duodenal (P < .001) and ileal (P < .01) bacteria by qPCR that positively correlated with anti-CdtB levels., Conclusions and Inferences: Rats injected with CdtB developed a postinfectious IBS-like phenotype and autoimmunity to vinculin with corresponding reduction in intestinal vinculin expression., (© 2020 John Wiley & Sons Ltd.)

Background: Most gut microbiome studies have been performed using stool samples. However, the small intestine is of central importance to digestion, nutrient absorption, and immune function, and characterizing its microbial populations is essential for elucidating their roles in human health and disease., Aims: To characterize the microbial populations of different small intestinal segments and contrast these to the stool microbiome., Methods: Male and female subjects undergoing esophagogastroduodenoscopy without colon preparation were prospectively recruited. Luminal aspirates were obtained from the duodenum, jejunum, and farthest distance reached. A subset also provided stool samples. 16S rRNA sequencing was performed and analyses were carried out using CLC Genomics Workbench., Results: 16S rRNA sequencing identified differences in more than 2000 operational taxonomic units between the small intestinal and stool microbiomes. Firmicutes and Proteobacteria were the most abundant phyla in the small intestine, and Bacteroidetes were less abundant. In the small intestine, phylum Firmicutes was primarily represented by lactic acid bacteria, including families Streptococcaceae, Lactobacillaceae, and Carnobacteriaceae, and Proteobacteria was represented by families Neisseriaceae, Pasteurellaceae, and Enterobacteriaceae. The duodenal and FD microbial signatures were markedly different from each other, but there were overlaps between duodenal and jejunal and between jejunal and FD microbial signatures. In stool, Firmicutes were represented by families Ruminococcaceae, Lachnospiraceae, Christensenellaceae, and Proteobacteria by class Deltaproteobacteria., Conclusions: The small bowel microbiome is markedly different from that in stool and also varies between segments. These findings may be important in determining how compositional changes in small intestinal microbiota contribute to human disease states.

Antimicrobial-resistant and novel pathogens continue to emerge, outpacing efforts to contain and treat them. Therefore, there is a crucial need for safe and effective therapies. Ultraviolet-A (UVA) phototherapy is FDA-approved for several dermatological diseases but not for internal applications. We investigated UVA effects on human cells in vitro, mouse colonic tissue in vivo, and UVA efficacy against bacteria, yeast, coxsackievirus group B and coronavirus-229E. Several pathogens and virally transfected human cells were exposed to a series of specific UVA exposure regimens. HeLa, alveolar and primary human tracheal epithelial cell viability was assessed after UVA exposure, and 8-Oxo-2'-deoxyguanosine was measured as an oxidative DNA damage marker. Furthermore, wild-type mice were exposed to intracolonic UVA as an in vivo model to assess safety of internal UVA exposure. Controlled UVA exposure yielded significant reductions in Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Clostridioides difficile, Streptococcus pyogenes, Staphylococcus epidermidis, Proteus mirabilis and Candida albicans. UVA-treated coxsackievirus-transfected HeLa cells exhibited significantly increased cell survival compared to controls. UVA-treated coronavirus-229E-transfected tracheal cells exhibited significant coronavirus spike protein reduction, increased mitochondrial antiviral-signaling protein and decreased coronavirus-229E-induced cell death. Specific controlled UVA exposure had no significant effect on growth or 8-Oxo-2'-deoxyguanosine levels in three types of human cells. Single or repeated in vivo intraluminal UVA exposure produced no discernible endoscopic, histologic or dysplastic changes in mice. These findings suggest that, under specific conditions, UVA reduces various pathogens including coronavirus-229E, and may provide a safe and effective treatment for infectious diseases of internal viscera. Clinical studies are warranted to further elucidate the safety and efficacy of UVA in humans., Competing Interests: Cedars-Sinai Medical Center has a licensing agreement with Aytu BioSciences. Cedars-Sinai has a patent on internal UV therapy, inventors: AR, MP, GM, RM and GL. SS is an employee of Australian Clinical Labs. The authors declare no other financial conflicts of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Small intestinal bacterial overgrowth (SIBO) is highly prevalent and is associated with numerous gastrointestinal disorders, but the microbes involved remain poorly defined. Moreover, existing studies of microbiome alterations in SIBO have utilized stool samples, which are not representative of the entire gastrointestinal tract. Therefore, we aimed to determine and compare the duodenal microbiome composition in SIBO and non-SIBO subjects, using duodenal aspirates from subjects undergoing standard-of-care esophagogastroduodenoscopy without colon preparation. Using the recently-redefined cutoff for SIBO of >103 colony forming units per milliliter (CFU/mL), 42 SIBO and 98 non-SIBO subjects were identified. Duodenal samples from SIBO subjects had 4x103-fold higher counts than non-SIBO subjects when plated on MacConkey agar (P<0.0001), and 3.8-fold higher counts when plated on blood agar (P<0.0001). Twenty subjects had also undergone lactulose hydrogen breath tests (LHBTs), of whom 7/20 had SIBO. At the 90-minute timepoint, 4/7 SIBO subjects had positive LHBTs (rise in hydrogen (H2) ≥ 20 ppm above baseline), as compared to 2/13 non-SIBO subjects. 16S ribosomal RNA (rRNA) sequencing revealed that SIBO subjects had 4.31-fold higher relative abundance of Proteobacteria (FDR P<0.0001) and 1.64-fold lower Firmicutes (P<0.0003) than non-SIBO subjects. This increased relative abundance of Proteobacteria correlated with decreased α-diversity in SIBO subjects (Spearman R = 0.4866, P<0.0001) Specific increases in class Gammaproteobacteria correlated with the area-under-the-curve for H2 for 0-90 mins during LHBT (R = 0.630, P = 0.002). Increases in Gammaproteobacteria resulted primarily from higher relative abundances of the family Enterobacteriaceae (FDR P<0.0001), which correlated with the symptom of bloating (Spearman R = 0.185, 2-tailed P = 0.028). Increases in family Aeromonadaceae correlated with urgency with bowel movement (Spearman R = 0.186, 2-tailed P = 0.028). These results validate the >103 CFU/mL cutoff for the definition of SIBO, and also reveal specific overgrowth of Proteobacteria in SIBO vs. non-SIBO subjects, coupled with an altered Proteobacterial profile that correlates with symptom severity. Future research may elucidate host-microbiome interactions underlying these symptoms in SIBO patients., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To compare the appendiceal microbiomes and examine the prevalence of Campylobacter species in the appendices of adult subjects with confirmed acute non-perforated appendicitis and controls with healthy appendices., Design: Archived samples of formalin-fixed paraffin-embedded appendiceal tissues were obtained from 50 consecutive female subjects who underwent appendectomy for acute, non-perforated appendicitis, and 35 consecutive female controls who underwent incidental appendectomy during gynaecological surgery., Results: 16S rRNA gene sequencing revealed that the relative abundances (RAs) of the major phyla in appendiceal tissues (Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria) were similar in both groups. Beta diversity was significantly different due to differences in Bacteroidetes and Proteobacteria (p<0.0001). Within Proteobacteria, RAs of classes Alphaproteobacteria (~21%, fold change (FC)=1.31, false discovery rate (FDR) p value=0.03) and Epsilonproteobacteria (~1%, FC=0.25, FDR p value>0.05) were increased in acute appendicitis samples. RAs of unknown genera from families Burkholderiaceae and Enterobacteriaceae were decreased in appendicitis samples, and 14 genera were increased, including Neisseria , Acinetobacter and Campylobacter . Quantitative PCR revealed that levels of Campylobacter jejuni DNA, but not other Campylobacter species or pathogens tested, were significantly higher in appendicitis samples than in controls (p=0.013). Using a cut-off of 0.31 pg/µL, 40% of appendicitis cases and 6% of controls were positive for C. jejuni , indicating specificity of 93.7% (95% Cl 79.2 to 99.2), sensitivity of 40.9% (95% Cl 24.7 to 54.5), and OR of 10.38 (Fisher's p value=0.0006, 95% Cl 2.3 to 47.4)., Conclusions: Our findings indicate that Campylobacter jejuni may be a significant cause of acute appendicitis. This supports earlier studies and suggests that targeted antibiotic therapies could be an alternative treatment for a subset of non-complicated acute appendicitis cases., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The human small intestine plays a central role in the processes of digestion and nutrient absorption. However, characterizations of the human gut microbiome have largely relied on stool samples, and the associated methodologies are ill-suited for the viscosity and low microbial biomass of small intestine samples. As part of the REIMAGINE study to examine the specific roles of the small bowel microbiome in human health and disease, this study aimed to develop and validate methodologies to optimize microbial analysis of the small intestine., Results: Subjects undergoing esophagogastroduodenoscopy without colon preparation for standard of care were prospectively recruited, and ~ 2 ml samples of luminal fluid were obtained from the duodenum using a custom sterile aspiration catheter. Samples of duodenal aspirates were either untreated (DA-U, N = 127) or pretreated with dithiothreitol (DA-DTT, N = 101), then cultured on MacConkey agar for quantitation of aerobic gram-negative bacteria, typically from the class Gammaproteobacteria, and on blood agar for quantitation of anaerobic microorganisms. DA-DTT exhibited 2.86-fold greater anaerobic bacterial counts compared to DA-U (P = 0.0101), but were not statistically different on MacConkey agar. DNA isolation from DA-U (N = 112) and DA-DTT (N = 43) samples and library preparation for 16S rRNA gene sequencing were also performed using modified protocols. DA-DTT samples exhibited 3.81-fold higher DNA concentrations (P = 0.0014) and 4.18-fold higher 16S library concentrations (P < 0.0001) then DA-U samples. 16S rRNA gene sequencing revealed increases in the detected relative abundances of obligate and facultative anaerobes in DA-DTT samples, including increases in the genera Clostridium (false discovery rate (FDR) P = 4.38E-6), Enterococcus (FDR P = 2.57E-8), Fusobacterium (FDR P = 0.02) and Bacteroides (FDR P = 5.43E-9). Detected levels of Gram-negative enteropathogens from the phylum Proteobacteria, such as Klebsiella (FDR P = 2.73E-6) and Providencia (FDR P < 0.0001) (family Enterobacteriaceae) and Pseudomonas (family Pseudomonadaceae) (FDR P = 0.04), were also increased in DA-DTT samples., Conclusions: This study validates novel DTT-based methodology which optimizes microbial culture and 16S rRNA gene sequencing for the study of the small bowel microbiome. The microbial analyses indicate increased isolation of facultative and obligate anaerobes from the mucus layer using these novel techniques.

Background: Antibodies to cytolethal distending toxin B (CdtB) and vinculin are novel biomarkers that rule-in and differentiate irritable bowel syndrome with diarrhea (IBS-D) from other causes of diarrhea and healthy controls., Aim: To determine whether these antibodies can also diagnose and differentiate other IBS subtypes., Methods: Subjects with IBS-D based on Rome III criteria (n = 2375) were recruited from a large-scale multicenter clinical trial (TARGET 3). Healthy subjects without gastrointestinal (GI) diseases or symptoms (n = 43) and subjects with mixed IBS (IBS-M) (n = 25) or IBS with constipation (IBS-C) (n = 30) were recruited from two major medical centers. Plasma levels of anti-CdtB and anti-vinculin antibodies in all subjects were determined by enzyme-linked immunosorbent assay. Optical densities of ≥1.68 and ≥2.80 were considered positive for anti-vinculin and anti-CdtB, respectively. Plasma levels of anti-CdtB and anti-vinculin antibodies were highest in IBS-D and lowest in IBS-C and healthy controls (P < 0.001). Levels in IBS-C subjects were not statistically different from controls (P > 0.1). Positivity for anti-CdtB or anti-vinculin resulted in a statistically significant negative gradient from IBS-D (58.1%) to IBS-M (44.0%), IBS-C (26.7%), and controls (16.3%) (P < 0.001)., Conclusions: Anti-CdtB and anti-vinculin titers and positivity rates differ in IBS subtypes, with higher antibody levels and positivity rates in IBS-D and IBS-M, and lower levels in IBS-C subjects that are similar to those in healthy controls. These antibodies appear useful in the diagnosis of IBS-M and IBS-D, but not IBS-C. Furthermore, these findings suggest that IBS-C is pathophysiologically distinct from subtypes with diarrheal components (i.e., IBS-M and IBS-D).

Objective: Methanogens colonizing the human gut produce methane and influence host metabolism. This study examined metabolic parameters in methane-producing subjects before and after antibiotic treatment., Methods: Eleven prediabetic methane-positive subjects (9F, 2M) with obesity (BMI 35.17 ± 7.71 kg/m(2) ) aged 47 ± 9 years were recruited. Subjects underwent breath testing, symptom questionnaire, oral glucose tolerance test (OGTT), lipid profile, and stool Methanobrevibacter smithii levels, gastric transit, and energy utilization analyses. After a 10-day antibiotic therapy (neomycin 500 mg bid/rifaximin 550 mg tid), all testing was repeated., Results: Baseline stool M. smithii levels correlated with breath methane (R = 0.7, P = 0.05). Eight subjects (73%) eradicated breath methane and showed reduced stool M. smithii (P = 0.16). After therapy, methane-eradicated subjects showed significant improvements in low-density lipoprotein (LDL) (P = 0.028), total cholesterol (P = 0.01), and insulin levels on OGTT (P = 0.05 at 120 minutes), lower blood glucose levels on OGTT (P = 0.054 at 90 minutes), significant reductions in bloating (P = 0.018) and straining (P = 0.059), and a trend toward lower stool dry weight. No changes were detected in gastric emptying time or energy harvest., Conclusions: Breath methane eradication and M. smithii reduction are associated with significant improvements in total cholesterol, LDL, and insulin levels and with lower glucose levels in prediabetic subjects with obesity. The underlying mechanisms require further elucidation., (© 2016 The Obesity Society.)

Diarrhea-predominant irritable bowel syndrome (IBS) is diagnosed through clinical criteria after excluding "organic" conditions, and can be precipitated by acute gastroenteritis. Cytolethal distending toxin B (CdtB) is produced by bacteria that cause acute gastroenteritis, and a post-infectious animal model demonstrates that host antibodies to CdtB cross-react with vinculin in the host gut, producing an IBS-like phenotype. Therefore, we assessed circulating anti-CdtB and anti-vinculin antibodies as biomarkers for D-IBS in human subjects. Subjects with D-IBS based on Rome criteria (n=2375) were recruited from a large-scale multicenter clinical trial for D-IBS (TARGET 3). Subjects with inflammatory bowel disease (IBD) (n=142), subjects with celiac disease (n=121), and healthy controls (n=43) were obtained for comparison. Subjects with IBD and celiac disease were recruited based on the presence of intestinal complaints and histologic confirmation of chronic inflammatory changes in the colon or small intestine. Subjects with celiac disease were also required to have an elevated tTG and biopsy. All subjects were aged between 18 and 65 years. Plasma levels of anti-CdtB and anti-vinculin antibodies were determined by ELISA, and compared between groups. Anti-CdtB titers were significantly higher in D-IBS subjects compared to IBD, healthy controls and celiac disease (P<0.001). Anti-vinculin titers were also significantly higher in IBS (P<0.001) compared to the other groups. The area-under-the-receiver operating curves (AUCs) were 0.81 and 0.62 for diagnosis of D-IBS against IBD for anti-CdtB and anti-vinculin, respectively. Both tests were less specific in differentiating IBS from celiac disease. Optimization demonstrated that for anti-CdtB (optical density≥2.80) the specificity, sensitivity and likelihood ratio were 91.6%, 43.7 and 5.2, respectively, and for anti-vinculin (OD≥1.68) were 83.8%, 32.6 and 2.0, respectively. These results confirm that anti-CdtB and anti-vinculin antibodies are elevated in D-IBS compared to non-IBS subjects. These biomarkers may be especially helpful in distinguishing D-IBS from IBD in the workup of chronic diarrhea.

Background: Acute gastroenteritis can precipitate irritable bowel syndrome (IBS) in humans. Cytolethal distending toxin is common to all pathogens causing gastroenteritis. Its active subunit, CdtB, is associated with post-infectious bowel changes in a rat model of Campylobacter jejuni infection, including small intestinal bacterial overgrowth (SIBO)., Aim: To evaluate the role of host antibodies to CdtB in contributing to post-infectious functional sequelae in this rat model., Methods: Ileal tissues from non-IBS human subjects, C. jejuni-infected and control rats were immunostained with antibodies to CdtB, c-Kit, S-100, PGP 9.5 and vinculin. Cytosolic and membrane proteins from mouse enteric neuronal cell lysates were immunoprecipitated with anti-CdtB and analyzed by mass spectrometry. ELISAs were performed on rat cardiac serum using CdtB or vinculin as antigens., Results: Anti-CdtB antibodies bound to a cytosolic protein in interstitial cells of Cajal (ICC) and myenteric ganglia in C. jejuni-infected and naïve rats and human subjects. Mass spectrometry identified vinculin, confirmed by co-localization and ELISAs. Anti-CdtB antibodies were higher in C. jejuni-infected rats (1.27 ± 0.15) than controls (1.76 ± 0.12) (P < 0.05), and rats that developed SIBO (2.01 ± 0.18) vs. rats that did not (1.44 ± 0.11) (P = 0.019). Vinculin expression levels were reduced in C. jejuni-infected rats (0.058 ± 0.053) versus controls (0.087 ± 0.023) (P = 0.0001), with greater reductions in rats with two C. jejuni infections (P = 0.0001) and rats that developed SIBO (P = 0.001)., Conclusions: Host anti-CdtB antibodies cross-react with vinculin in ICC and myenteric ganglia, required for normal gut motility. Circulating antibody levels and loss of vinculin expression correlate with number of C. jejuni exposures and SIBO, suggesting that effects on vinculin are important in the effects of C. jejuni infection on the host gut.

Objective: Breath testing and duodenal culture studies suggest that a significant proportion of irritable bowel syndrome (IBS) patients have small intestinal bacterial overgrowth. In this study, we extended these data through 16S rDNA amplicon sequencing and quantitative PCR (qPCR) analyses of duodenal aspirates from a large cohort of IBS, non-IBS and control subjects., Materials and Methods: Consecutive subjects presenting for esophagogastroduodenoscopy only and healthy controls were recruited. Exclusion criteria included recent antibiotic or probiotic use. Following extensive medical work-up, patients were evaluated for symptoms of IBS. DNAs were isolated from duodenal aspirates obtained during endoscopy. Microbial populations in a subset of IBS subjects and controls were compared by 16S profiling. Duodenal microbes were then quantitated in the entire cohort by qPCR and the results compared with quantitative live culture data., Results: A total of 258 subjects were recruited (21 healthy, 163 non-healthy non-IBS, and 74 IBS). 16S profiling in five IBS and five control subjects revealed significantly lower microbial diversity in the duodenum in IBS, with significant alterations in 12 genera (false discovery rate < 0.15), including overrepresentation of Escherichia/Shigella (p = 0.005) and Aeromonas (p = 0.051) and underrepresentation of Acinetobacter (p = 0.024), Citrobacter (p = 0.031) and Microvirgula (p = 0.036). qPCR in all 258 subjects confirmed greater levels of Escherichia coli in IBS and also revealed increases in Klebsiella spp, which correlated strongly with quantitative culture data., Conclusions: 16S rDNA sequencing confirms microbial overgrowth in the small bowel in IBS, with a concomitant reduction in diversity. qPCR supports alterations in specific microbial populations in IBS.

Aim: Rifaximin is a non-absorbed antibiotic relative of rifampicin. The location of effect and staphylococcal resistance are two recent potential concerns with rifaximin. In this study we evaluate the location of effect of rifaximin as well as the development of staphylococcal rifampicin resistance., Methods: Rats were divided into three groups. Group 1 gavaged for 10 days with PBS, group 2 gavaged with rifaximin for 10 days, and group 3 gavaged with rifaximin for 10 days and housed for 30 days. In each group, stool was collected daily for quantitative culture of Staphylococcus spp. and coliforms. After euthanasia luminal bacterial counts were determined at multiple gut locations by qPCR. Rifampicin susceptibility was tested on Staphylococcus pre and post rifaximin., Results: At baseline, rats had a median of 2.90 × 10(6) cfu/ml Staphylococcus spp. in stool. After 10 days of rifaximin, this dropped to 1.20 × 10(5) cfu/ml (P < 0.01). With coliform counts, rats had a median of 1.86 × 10(4) cfu/ml at baseline which dropped to 2.2 × 10(3) cfu/ml (P < 0.01) after rifaximin. After cessation of rifaximin, coliform counts recovered within 3 days. When examining the total bacterial counts by qPCR, rifaximin reduced small bowel bacterial levels, but not colon. This reduction was sustained for 30 days. No colonies of Staphylococcus became resistant and only one colony was intermediate. The mean inhibitory concentration for rifampicin was not different before and after rifaximin., Conclusion: Staphylococcal spp. fail to demonstrate resistance to rifampicin after rifaximin. The transient reductions in stool coliform counts recover while rifaximin appears to produce durable reductions in duodenal bacteria.

Unlabelled: It is increasingly understood that gastrointestinal (GI) methanogens, including Methanobrevibacter smithii, influence host metabolism., Objective: Therefore, we compared M. smithii colonization and weight gain in a rat model under different dietary conditions., Design and Methods: Sprague-Dawley rats were inoculated with M. smithii or vehicle (N = 10/group), fed normal chow until day 112 postinoculation, high-fat chow until day 182, then normal chow until day 253. Thereafter, five rats from each group were fed high-fat and normal chow until euthanasia., Results: Both groups exhibited M. smithii colonization, which increased following inoculation only for the first 9 days. Change to high-fat chow correlated with significant increases in weight (P < 0.00001) and stool M. smithii (P < 0.01) in all rats, with stool M. smithi decreasing on return to normal chow. Rats switched back to high-fat on day 253 further increased weight (P < 0.001) and stool M. smithii (P = 0.039). Euthanasia revealed all animals had higher M. smithii, but not total bacteria, in the small intestine than in the colon. Rats switched back to high-fat chow had higher M. smithii levels in the duodenum, ileum, and cecum than those fed normal chow; total bacteria did not differ in any bowel segment. Rats which gained more weight had more bowel segments colonized, and the lowest weight recorded was in a rat on high-fat chow which had minimal M. smithii colonization., Conclusions: We conclude that M. smithii colonization occurs in the small bowel as well as in the colon, and that the level and extent of M. smithii colonization is predictive of degree of weight gain in this animal model., (Copyright © 2012 The Obesity Society.)

Purpose: Among irritable bowel syndrome (IBS) patients, breath methane producers overwhelmingly have constipation predominance (C-IBS). Although the most common methanogen in humans is Methanobrevibacter smithii, incidence and type of methanogenic bacteria in C-IBS patients are unknown., Methods: By use of a questionnaire and lactulose breath testing, subjects with Rome II C-IBS and methane (>3 ppm) were selected (n = 9). The control group included subjects with IBS who had no breath methane (n = 10). Presence of bacterial DNA was assessed in a stool sample of each subject by quantitative-PCR using universal 16S rDNA primer. M. smithii was quantified by use of a specific rpoB gene primer., Results: M. smithii was detected in both methane and non-methane subjects. However, counts and relative proportion of M. smithii were significantly higher for methane-positive than for methane-negative subjects (1.8 × 10(7) ± 3.0 × 10(7) vs 3.2 × 10(5) ± 7.6 × 10(5) copies/g wet stool, P < 0.001; and 7.1 ± 6.3 % vs 0.24 ± 0.47 %, P = 0.02 respectively). The minimum threshold of M. smithii resulting in positive lactulose breath testing for methane was 4.2 × 10(5) copies/g wet stool or 1.2 % of total stool bacteria. Finally, area-under-curve for breath methane correlated significantly with both absolute quantity and percentage of M. smithii in stool (R = 0.76; P < 0.001 and R = 0.77; P < 0.001 respectively)., Conclusions: M. smithii is the predominant methanogen in C-IBS patients with methane on breath testing. The number and proportion of M. smithii in stool correlate well with amount of breath methane.

Background/aims: Campylobacter jejuni infection is a leading cause of acute gastroenteritis, which is a trigger for post-infectious irritable bowel syndrome (PI-IBS). Cytolethal distending toxin (CDT) is expressed by enteric pathogens that cause PI-IBS. We used a rat model of PI-IBS to investigate the role of CDT in long-term altered stool form and bowel phenotypes., Methods: Adult Sprague-Dawley rats were gavaged with wildtype C. jejuni (C+), a C. jejunicdtB knockout (CDT-) or saline vehicle (controls). Four months after gavage, stool from 3 consecutive days was assessed for stool form and percent wet weight. Rectal tissue was analyzed for intraepithelial lymphocytes, and small intestinal tissue was stained with anti-c-kit for deep muscular plexus interstitial cells of Cajal (DMP-ICC)., Results: All 3 groups showed similar colonization and clearance parameters. Average 3-day stool dry weights were similar in all 3 groups, but day-to-day variability in stool form and stool dry weight were significantly different in the C+ group vs both controls (P < 0.01) and the CDT- roup (P < 0.01), but were not different in the CDT- vs controls. Similarly, rectal lymphocytes were significantly higher after C. jejuni (C+) infection vs both controls (P < 0.01) and CDT-exposed rats (P < 0.05). The counts in the latter 2 groups were not significantly different. Finally, c-kit staining revealed that DMP-ICC were reduced only in rats exposed to wildtype C. jejuni., Conclusions: In this rat model of PI-IBS, CDT appears to play a role in the development of chronic altered bowel patterns, mild chronic rectal inflammation and reduction in DMP-ICC.

Unlabelled: Recent evidence suggests a role for gut bacteria and antibiotics in the pathophysiology and treatment of irritable bowel syndrome (IBS), respectively. While the benefits of the antibiotic rifaximin have demonstrated efficacy and durable improvement in symptoms over 3 months, the long-term need for retreatment using this approach is mostly unknown. In this retrospective study, subjects with nonconstipated IBS who were retreated with rifaximin were examined., Methods: Charts of patients who were seen at a tertiary care medical center between 2007 and 2011 were reviewed. After exclusion criteria were applied, subjects who had received rifaximin and were seen for retreatment were fully reviewed. During review, demographic information, duration of response, and success of treatment and retreatment were evaluated., Results: A total of 522 charts were reviewed. Of these 522 charts, 71 subjects were nonconstipated IBS subjects who had received at least one retreatment. Of these, 48 had a second, 22 had a third, 7 had a fourth, and 4 had a fifth treatment. More than 75% of subjects who initially responded to rifaximin also responded to any further retreatment, with no significant reduction in benefit for successive retreatments. Furthermore, there was no change in the duration of benefit (median time between treatments) for successive retreatments., Conclusions: Retreatment with rifaximin for subjects with nonconstipated IBS in a real-world clinical practice was successful up to five times without decrease in duration or effect.

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with an estimated worldwide prevalence of 10-20%. IBS can be associated with severe gastrointestinal symptoms, including abdominal pain, bloating, and altered bowel function. Although the causes of IBS remain undefined, recent research has increasingly suggested roles for gut flora in IBS. These roles involve postinfectious IBS, which can occur after a single episode of acute gastroenteritis, and small intestinal bacterial overgrowth, in which elevated populations of aerobic and anaerobic bacteria cause abdominal pain and altered bowel function. More recently, potential roles for methanogens in contributing to IBS subtypes have also been identified. In this paper, we review the different mechanisms by which gut flora may contribute to IBS and also discuss the efficacy and safety of various antibiotic therapies for treating IBS symptoms.

Leptin is secreted by adipocytes and exerts its effects by interacting with the long form of the leptin receptor, OB-RB. The leptin protein and leptin receptors have been localized in the ovary, and acute leptin treatment directly inhibits ovulation in the rat ovary. It was hypothesized that expression of the leptin receptor gene varies throughout the oestrous cycle to modulate the sensitivity of the ovary to leptin. In this study, expression of genes for the long and short isoforms of the leptin receptor in the adult ovary was investigated at different stages of the rat oestrous cycle. Vaginal cytology was used to determine the stage of the oestrous cycle. Ovaries were collected and RNA was extracted for real-time RT-PCR analysis of leptin receptor gene expression. OB-RB gene expression was low in pro-oestrus (3.13 +/- 0.18 fg RNA per microg total DNA) and dioestrus II (2.52 +/- 0.19 fg RNA per microg total DNA) of the oestrous cycle, whereas expression was high in oestrus (5.9 +/- 0.27 fg RNA per microg total DNA) and dioestrus I (4.6 +/- 0.24 fg RNA per microg total DNA) (P < 0.001). Expression of the gene for the short form of the leptin receptor (OB-RA) was at a maximum in dioestrus I (65.5 +/- 0.8 fg RNA per ng total DNA), high in oestrus (39.0 +/- 0.8 fg RNA per ng total DNA) and low at pro-oestrus (5.0 +/- 0.2 fg RNA per ng total DNA) and dioestrus II (1.1 +/- 0.09 fg RNA per ng total DNA) (P < 0.001). Plasma oestradiol concentrations (pg ml-1) were highest at pro-oestrus (19.38 +/- 1.3), and similar at the remaining three stages studied (oestrus: 13.7 +/- 1.9; dioestrus I: 12.4 +/- 1.0; dioestrus II: 10.3 +/- 0.9) (P < 0.05). Plasma progesterone concentrations (ng ml-1) were higher in the luteal phases of the oestrous cycle (dioestrus I: 18.6 +/- 2.3; dioestrus II: 14.7 +/- 2.5) than during pro-oestrus (5.12 +/- 0.6) and oestrus (5.9 +/- 0.8) (P < 0.05). Plasma leptin concentrations were detectable only in pro-oestrus (0.35 +/- 0.05 ng ml(-1)) and were below the detection limit of the assay at other stages of the oestrous cycle. In summary, mRNA content for the long and short isoforms of the leptin receptor is lower in pro-oestrus and dioestrus II than in oestrus and dioestrus I of the rat oestrous cycle. The fluctuations in leptin receptor mRNA content may be a response to the concentrations of circulating steroid hormones and leptin. This research supports the initial hypothesis and shows that ovarian leptin receptor concentrations vary throughout the oestrous cycle in response to the changing environment of the ovary.

Recent data suggest that steroidogenic enzyme messenger ribonucleic acids (mRNAs) may be overexpressed in thecal cells, and LH receptors may be prematurely expressed in granulosa cells in women with polycystic ovaries. The purpose of this study was to determine whether there is abnormal gene expression in thecal and granulosa cells from polycystic ovaries. Ovarian tissue specimens were obtained from 12 women with PCOS and 24 regularly cycling control women. The granulosa cells and the theca interna were microdissected from individual follicles. LH receptor, steroidogenesis acute regulatory protein (StAR), cholesterol side-chain cleavage cytochrome P450 (CYP11A), and 17alpha-hydroxylase/C(17-20) lyase cytochrome P450 (CYP17) mRNAs were measured by RT-PCR. There was no difference between 3- to 7-mm control follicles and dominant follicles with respect to LH receptor mRNA expression in either thecal or granulosa cells. CYP11A and CYP17 mRNAs were higher in thecal cells from 3- to 7-mm follicles than in dominant follicles, but StAR expression was not different. In granulosa cells, StAR and CYP11A mRNA expression was higher in dominant follicles than in 3- to 7-mm follicles. The mean levels of LH receptor, StAR, CYP11A, and CYP17 mRNA expression were higher in thecal cells from PCOS follicles than in size-matched control follicles. In granulosa cells, the mean levels of LH receptor and CYP11A, but not StAR, mRNA expression were higher in PCOS than in control follicles. These data demonstrate that regulatory protein and steroidogenic enzyme mRNAs are overexpressed in thecal and granulosa cells from polycystic ovaries and support the conclusions that the thecal cells are hyperstimulated and the granulosa cells may be prematurely luteinizing.

The signal initiating ovarian theca cell (TC) differentiation is gonadotropin independent because theca precursor cells do not contain LH receptors. Previously we demonstrated that preantral follicles produce paracrine TC differentiating factors that promote androgen production by an LH-independent mechanism. This study tested the effects of two granulosa cell-produced peptides, insulin-like growth factor-I (IGF-I) and stem cell factor (SCF), on TC differentiation and androgen production. Neutralizing antibodies to either IGF-I or SCF blocked the stimulatory effects of follicle-conditioned medium on TC precursor differentiation more than 90%. The TC isolated from the ovaries of hypophysectomized immature rats by percoll gradient centrifugation were cultured (48 h) with and without SCF (0-100 ng/ml) and IGF-I (0-100 ng/ml) to test their effects on TC differentiation. Androsterone in the medium was measured by RIA. Luteinizing hormone receptor, steroidogenesis acute regulatory protein (StAR), CYP11A, CYP17, and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNAs were measured by specific reverse transcriptase polymerase chain reaction assays. Stem cell factor or IGF-I alone did not stimulate androsterone production but in combination caused a concentration-dependent increase in androsterone levels. Maximum androsterone levels were less than those stimulated by LH (0.1 ng/ml) alone. Although IGF-I synergistically augmented LH stimulation of androsterone production, SCF did not alter LH-stimulated androsterone production in the presence or absence of IGF-I. Stem cell factor alone had no effect on LH receptor, StAR, CYP11A, and 3beta-HSD mRNA expression but decreased CYP17 mRNA levels. Insulin-like growth factor-I alone had no effect on StAR or CYP17 mRNA expression but increased LH receptor, CYP11A, and 3beta-HSD mRNA levels. In combination, SCF plus IGF-I increased the expression of all five mRNAs. These data support the conclusion that IGF-I and SCF are important regulators of TC differentiation.

Leptin blocks the insulin-like growth factor-I-induced increase in FSH-dependent estradiol-17beta (E(2)) production by rat ovarian granulosa cells (GC) in vitro. To determine whether the leptin effect extended to another positive modulator of FSH-dependent E(2) production, the direct ovarian effects of leptin on transforming growth factor beta (TGF-beta) were investigated. Reverse transcription-polymerase chain reaction demonstrated that theca-interstitial cells (TIC) from hypophysectomized rats expressed only a nonsignal-transducing isoform (OB-Ra) of leptin receptor mRNA. Leptin had no effect on TIC androgen production. In contrast, mRNAs for OB-Ra and the signal-transducing (OB-Rb) leptin receptor isoforms were expressed in GC. When GC obtained from 26-day-old rats were cultured (48 h) with FSH and androstenedione, both estrone (E(1)) and E(2) levels increased over those in untreated controls. In the presence of FSH (0.1 IU/ml), TGF-beta (10 ng/ml) potentiated E(2) and E(1) accumulation by 2.7- and 1.45-fold, respectively. Leptin did not alter basal or FSH-stimulated E(2) and E(1) levels. However, leptin suppressed the effect of TGF-beta on FSH-dependent E(2) and E(1) production by 39% and 29%, respectively. Aromatase cytochrome P450 (P450(arom)) mRNA expression and P450(arom) activity were increased by FSH and further augmented by the addition of TGF-beta. Leptin abolished the TGF-beta effect on P450(arom) mRNA expression, and it decreased P450(arom) activity by approximately 27%. These data support the hypothesis that leptin antagonizes the stimulatory effects of TGF-beta on FSH-dependent estrogen production by a mechanism involving the leptin-induced attenuation of P450(arom) activity and mRNA expression in GC.

The recent demonstration of high concentrations of 5alpha-androstane-3,17-dione in the follicular fluid of polycystic ovaries suggests a potential role for 5alpha-reduced androgens in the etiology of polycystic ovary syndrome (PCOS). The purpose of the present study was to determine whether there is increased 5alpha-reductase activity or messenger ribonucleic acid (mRNA) expression in polycystic ovaries. 5alpha-Reductase 1 and 5alpha-reductase 2 mRNAs were measured in thecal (TC) and granulosa (GC) cells from individual follicles of 18 women with PCOS and 26 regularly cycling control women. Both 5alpha-reductase 1 and 2 mRNA expression was higher in GC than in TC, and 5alpha-reductase 2 mRNA levels were approximately 3-fold higher than 5alpha-reductase 1 mRNA. 5alpha-Reductase 1 and 2 mRNA expression were similar in GC from PCOS and control women, but 5alpha-reductase mRNA was decreased in TC from PCOS follicles. In control women, 5alpha-reductase 2 mRNA was highest in GC from 3- to 5-mm follicles and decreased to undetectable levels in GC from 7-mm follicles. A similar pattern of expression was present in GC from PCOS follicles, but detectable levels of 5alpha-reductase 2 mRNA were present in GC from 7-mm follicles. 5alpha-Reductase activity was measured in whole follicles by measuring the conversion of radiolabeled testosterone to dihydrotestosterone. Kinetic analysis of total 5alpha-reductase activity at physiological pH revealed a Km of 1.46 micromol/L and a maximal velocity of 0.31 nmol/min x mg protein, indicating predominantly type 1 activity. The total 5alpha-reductase activity was approximately 4-fold higher in PCOS follicles than in control follicles. These data demonstrate elevated 5alpha-reductase activity in polycystic ovaries and support the hypothesis that 5alpha-reduced androgens may play a role in the pathogenesis of PCOS.

There is increasing evidence that leptin is a physiological link between obesity and infertility. Although leptin receptors have been demonstrated in human ovaries, there is no information regarding the effects of leptin on cells from developing ovarian follicles. To test the direct effects of leptin on human ovarian cells, granulosa cells (GC) and theca cells were isolated from the ovaries of regularly cycling women. Serum was obtained at the time of surgery, and follicular fluid was aspirated from the follicles before isolation of the ovarian cells. Leptin concentrations were similar in follicular fluid and serum. RT-PCR analysis demonstrated that the long, signaling form of the leptin receptor was expressed in both theca and GC. In cultured GC, leptin had no effect on estradiol production, alone or in the presence of FSH, but caused a concentration-related inhibition of the insulin-like growth factor I (IGF-I) augmentation of FSH-stimulated estradiol production. The effect of leptin was specific, because there was no effect on progesterone production. In cultured theca cells, leptin did not alter androstenedione production, alone or in the presence of LH. Leptin caused a concentration-related inhibition of the IGF-I augmentation of LH-stimulated androstenedione production. These data demonstrate that leptin can directly inhibit IGF-I action in ovarian theca and GC at concentrations commonly present in obese women.

Objectives and Design: Leptin, a product of adipocytes, is a cytokine with multiple effects on the reproductive axis. Leptin causes the activation of STAT proteins within target cells. The aromatase gene promoter in adipose stromal cells contains a functional STAT binding region, leading to the hypothesis that leptin may regulate aromatase activity in fat tissue. To test this hypothesis, adipose stromal cells were isolated from subcutaneous abdominal fat or breast fat then placed into tissue culture., Materials and Methods: The cells were treated for three days with increasing concentrations of recombinant human leptin. Aromatase activity in the stromal cells was measured by the release of 3H2O from radiolabeled androstenedione precursor., Results: Basal aromatase activity varied markedly between, but there were no differences between abdominal fat and breast fat. Leptin concentrations in the physiological range of normal weight or thin women (10 ng/ml) had no effect on aromatase activity. In 2 of 8 abdominal fat cultures and 1 of 2 breast fat cultures, a high obese concentration of leptin (100 ng/ml) stimulated a significant increase in aromatase activity. In the remaining subjects there was no effect of leptin, even at high concentrations., Conclusions: These data demonstrate that in approximately 30 percent of our subject population leptin was able to stimulate aromatase activity in adipose stromal cells at high concentrations. The elevated levels of aromatase activity may contribute to increase circulating estrogen levels in certain obese women and suggest that elevated leptin concentrations in obese women may cause locally elevated estrogen concentrations in the breast and thereby promote tumor formation.

During ovarian follicle growth, precise regulation of the onset of androgen production by ovarian theca-interstitial cells (TIC) is necessary for maintaining follicle viability. Thus, temporary suppression of TIC androgen production in preantral follicles is the key to promoting follicle development. Evidence indicates that this process is coordinated via intraovarian growth factors. Hepatocyte growth factor (HGF) can induce granulosa cell (GC) proliferation and suppress follicular atresia, indicating a role for HGF in promoting follicle growth and viability. To determine whether HGF could reversibly suppress androgen production, this study investigated the effect of HGF on TIC differentiation and steroid production. Twenty-six-day-old rats were used in all studies. HGF messenger RNA (mRNA) expression in TIC and GC was determined by reverse transcription-PCR. Agarose gel electrophoresis of the PCR products yielded a single band corresponding to the 290-bp HGF product for both TIC and GC. HGF expression in cultured TIC and GC was not blocked by gonadotropins or HGF. To investigate the effects of HGF on TIC steroidogenesis, TIC were isolated from the ovaries of hypophysectomized rats. TIC (3.0 x 10(4) cells/well) were cultured with LH (0-3 ng/ml) and/or HGF (0-100 ng/ml) for 48 h, and androsterone levels were measured by RIA. HGF did not alter androsterone levels in the absence of LH; however, HGF reversibly impaired LH-dependent androsterone production by as much as 57% (IC50 = 1.5 +/- 0.01 ng/ml). LH (0.3 ng/ml) stimulated progesterone (P4) synthesis by TIC (1201 +/- 190 pg/ml) compared to that by control cells (210 +/- 30 pg/ml). HGF stimulated basal P4 production, and LH-dependent P4 synthesis was augmented 2.6-fold by HGF (ED50 = 0.3 +/- 0.01 ng/ml). The DNA content and cell viability in TIC cultures were not affected by HGF. The effect of HGF on steroidogenic enzyme gene expression in TIC was also investigated via PCR. HGF did not alter the level of basal or LH-induced P450 side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase mRNAs; however, LH-dependent P45017 alpha hydroxylase/C17,20 lyase mRNA content was reduced 4.5 fold in the presence of HGF. Thus, HGF is expressed in both TIC and GC obtained from the immature rat ovary, suggesting its presence in growing follicles. In TIC, HGF stimulated P4 synthesis, but impaired androgen production, concurrent with a down-regulatory effect on P45017 alpha hydroxylase/C17,20 lyase gene expression. Collectively, these results indicate that HGF reversibly impairs LH-stimulated androgen production in TIC. Such effects may help promote folliculogenesis.

There has been considerable interest in rat ovarian insulin-like growth factor binding proteins IGFBPs because they are potent inhibitors of FSH action.In situ, IGFBP-2 and -4 and IGFBP-3 mRNAs are expressed in rat theca interstitial (TIC) and theca lutein cells respectively. Although much is known about IGFBPs in rat TIC at the mRNA level, the synthesis and regulation of IGFBP proteins remain poorly understood. The purpose of this study was to identify the species of IGFBPs produced by TIC and to determine the effects of LH and IGF-1 on their expression. This was accomplished by culturing rat TIC for 2 days in serum-free medium with graded doses of LH and/or IGF-I, and measuring IGFBP mRNAs in the cells and IGFBP proteins in the conditioned media by RT-PCR and Western immunoblotting respectively. The RT PCR analysis identified strong bands for IGFBP-2 and -4 mRNAs in TIC. In some treatments, the mRNAs for IGFBP-3 and -6 were also identified, but transcripts for IGFBP-1 and -5 were undetectable. Two species of IGFBPs were detected in the conditioned media of control (untreated) TIC, the 31 kDa IGFBP-2 and the 24 kDa (non-glycosylated) and 28 kDa (glycosylated) forms of IGFBP-4. There was no detectable IGFBP-5 and barely detectable amounts of IGFBP-3 and -6 in the conditioned media. Treatment with LH (0.2-20 μU/ml) caused no significant changes in the levels of the 31 kDa IGFBP-2 and the 24 kDa and 28 kDa IGFBP-4 bands, and there was no detectable IGFBP protease activity. In contrast, IGF-I (100 ng/ml) stimulated the expression of IGFBP-2, IGFBP-4 and a 17.5 kDa IGFBP-4 fragment. The immunoreactive IGFBP-4 fragment suggests the media contained an IGFBP-4 protease. The IGF-I effects were dose dependent (ED(50)=12.4±3.3 ng/ml). Co-treating TIC with LH (0.2-20 μU/ml) caused no significant change in the activity of IGF-I in stimulating the expression of IGFBP-2, IGFBP-4 and IGFBP-4 protease. We have demonstrated that IGF-I acts directly on rat TIC to stimulate the expression of the intrinsic IGFBP system. LH, either alone or together with IGF-I, did not significantly change the expression of TIC IGFBP proteins. Therefore, we hypothesize that IGF-I, but not LH, may be a physiologically important regulator of the IGFBP system in rat TIC. Because IGF-I is a potent stimulator of theca function, changes in the expression of this intrinsic IGFBP system could have new implications for ovarian androgen production, both at the physiologic and pathophysiologic levels.

We have previously demonstrated that TGFα inhibits theca-interstitial cell (TIC) androgen production by specifically blocking LH stimulation of 17α-hydroxylase/C(17-20) lyase (P450(17α)) activity. The purpose of the present studies was to examine the mechanism by which this block occurs. TIC were isolated from hypophysectomized immature rats by Percoll gradient centrifugation and cultured up to 6 days in serum-free medium with LH (0-100 ng/ml) and TGFα (0-100 ng/ml). When freshly isolated TIC were treated with TGFα alone (100 ng/ml) there was no change in PKA activity from basal levels. LH (100 ng/ml) stimulated a significant increase in PKA activity that was abolished by TGFα. TGFα did not diminish LH stimulation of cAMP production. TGFα alone did not alter the basal expression of cholesterol side-chain cleavage (P450(scc)), 3β-hydroxysteroid dehydrogenase (3β-HSD) or P450(17α) mRNAs. LH stimulated dose-related increases in P450(scc) (80-fold), 3β-HSD (5-fold) and P450(17α) (35-fold) mRNAs. Concomitant treatment with TGFα (100 ng/ml) inhibited LH stimulation of P450(17α) mRNA >90% and P450(scc) mRNA 35% while 3β-HSD mRNA was stimulated 2-fold. Time course studies demonstrated that the effects of TGFα were present at 2 days in culture. At 4 and 6 days in culture there were small, if any, increases in mRNA levels stimulated by LH. There were no significant effects of TGFα at 4 or 6 days. Our data demonstrate that TGFα inhibition of TIC androgen production involves suppression of P450(scc) and P450(17α) mRNA expression by inhibiting LH stimulation of PKA activity.