Characterization of the Small Bowel Microbiome Reveals Different Profiles in Human Subjects who are Overweight or have Obesity.

OBJECTIVES: Gut microbiome changes are linked to obesity, but findings are based on stool data. Here, 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. johnsoni, and Prevotella loeschii 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. CONCLUSIONS: 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.

Hydrogen Sulfide Producers Drive a Diarrhea-Like Phenotype and a Methane Producer Drives a Constipation-Like Phenotype in Animal Models.

BACKGROUND: We recently demonstrated that diarrhea-predominant irritable bowel syndrome (IBS-D) subjects have higher relative abundance (RA) of hydrogen sulfide (H2S)-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 H2S 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 H2S 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 H2S production. 16S sequencing revealed stool microbiota alterations in rats gavaged with H2S producers, with higher relative abundance (RA) of other H2S 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 H2S producers D. piger or F. varium results in increased colonization with other H2S producers and diarrhea-like phenotypes. This supports roles for the increased RA of methanogens and H2S producers identified in IBS-C and IBS-D subjects, respectively, in contributing to stool phenotypes.

Defining Small Intestinal Bacterial Overgrowth by Culture and High Throughput Sequencing.

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 <103 colony forming units (CFU)/mL on MacConkey agar and 98 subjects with ≥103 CFU/mL, including ≥103 to <105 CFU/mL (N = 66) and ≥105 CFU/mL (N = 32), were identified. Duodenal microbial α-diversity progressively decreased, and relative abundance of Escherichia/Shigella and Klebsiella increased, in subjects with ≥103 to <105 CFU/mL and ≥105 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 ≥103 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 ≥103 CFU/mL. CONCLUSIONS: Our findings confirm ≥103 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.

Cytolethal distending toxin B inoculation leads to distinct gut microtypes and IBS-D-like microRNA-mediated gene expression changes in a rodent model.

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 (H2S)-producer Desulfovibrio, and increased expression of H2S-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.

Consuming artificial sweeteners may alter the structure and function of duodenal microbial communities.

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.

Coronavirus Disease 2019 (COVID-19) Pandemic Lifestyle Changes May Have Influenced Small Bowel Microbial Composition and Microbial Resistance.

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.

Prevalence of Small Intestinal Bacterial Overgrowth Syndrome in Patients with Non-Alcoholic Fatty Liver Disease/Non-Alcoholic Steatohepatitis: A Cross-Sectional Study.

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 ≥103 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)-1ß, 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-1ß, 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.

Methanogens and Hydrogen Sulfide Producing Bacteria Guide Distinct Gut Microbe Profiles and Irritable Bowel Syndrome Subtypes.

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.

The Response of the Rodent Gut Microbiome to Broad-Spectrum Antibiotics Is Different in Males and Females.

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.

A Smartphone Application Using Artificial Intelligence Is Superior To Subject Self-Reporting When Assessing Stool Form.

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.

Effects of Proton Pump Inhibitors on the Small Bowel and Stool Microbiomes.

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 (> 103 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.

Ultraviolet A light effectively reduces bacteria and viruses including coronavirus.

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.

The duodenal microbiome is altered in small intestinal bacterial overgrowth.

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.

Acute appendicitis is associated with appendiceal microbiome changes including elevated Campylobacter jejuni levels.

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.

Immunization with cytolethal distending toxin B produces autoantibodies to vinculin and small bowel bacterial changes in a rat model of postinfectious irritable bowel syndrome.

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.

Mapping the Segmental Microbiomes in the Human Small Bowel in Comparison with Stool: A REIMAGINE Study.

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.

Optimizing microbiome sequencing for small intestinal aspirates: validation of novel techniques through the REIMAGINE study.

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.

Assessment of Anti-vinculin and Anti-cytolethal Distending Toxin B Antibodies in Subtypes of Irritable Bowel Syndrome.

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).

Metabolic effects of eradicating breath methane using antibiotics in prediabetic subjects with obesity.

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.

Development and validation of a biomarker for diarrhea-predominant irritable bowel syndrome in human subjects.

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.