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.

Quantitative sequencing clarifies the role of disruptor taxa, oral microbiota, and strict anaerobes in the human small-intestine microbiome.

BACKGROUND: Upper gastrointestinal (GI) disorders and abdominal pain afflict between 12 and 30% of the worldwide population and research suggests these conditions are linked to the gut microbiome. Although large-intestine microbiota have been linked to several GI diseases, the microbiota of the human small intestine and its relation to human disease has been understudied. The small intestine is the major site for immune surveillance in the gut, and compared with the large intestine, it has greater than 100 times the surface area and a thinner and more permeable mucus layer. RESULTS: Using quantitative sequencing, we evaluated total and taxon-specific absolute microbial loads from 250 duodenal-aspirate samples and 21 paired duodenum-saliva samples from participants in the REIMAGINE study. Log-transformed total microbial loads spanned 5 logs and were normally distributed. Paired saliva-duodenum samples suggested potential transmission of oral microbes to the duodenum, including organisms from the HACEK group. Several taxa, including Klebsiella, Escherichia, Enterococcus, and Clostridium, seemed to displace strict anaerobes common in the duodenum, so we refer to these taxa as disruptors. Disruptor taxa were enriched in samples with high total microbial loads and in individuals with small intestinal bacterial overgrowth (SIBO). Absolute loads of disruptors were associated with more severe GI symptoms, highlighting the value of absolute taxon quantification when studying small-intestine health and function. CONCLUSION: This study provides the largest dataset of the absolute abundance of microbiota from the human duodenum to date. The results reveal a clear relationship between the oral microbiota and the duodenal microbiota and suggest an association between the absolute abundance of disruptor taxa, SIBO, and the prevalence of severe GI symptoms. Video Abstract.

Age and the aging process significantly alter the small bowel microbiome.

Gut microbial diversity decreases with aging, but existing studies have used stool samples, which do not represent the entire gut. We analyzed the duodenal microbiome in 251 subjects aged 18-35 (n = 32), 36-50 (n = 41), 51-65 (n = 96), and 66-80 (n = 82). Decreased duodenal microbial diversity in older subjects is associated with combinations of chronological age, number of concomitant diseases, and number of medications used, and also correlated with increasing coliform numbers (p < 0.0001). Relative abundance (RA) of phylum Proteobacteria increases in older subjects, with increased RA of family Enterobacteriaceae and coliform genera Escherichia and Klebsiella, and is associated with alterations in the RA of other duodenal microbial taxa and decreased microbial diversity. Increased RA of specific genera are associated with chronological age only (Escherichia, Lactobacillus, and Enterococcus), number of medications only (Klebsiella), or number of concomitant diseases only (Clostridium and Bilophila). These findings indicate the small intestinal microbiome changes significantly with age and the aging process.

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.

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.

Undiagnosed Kidney Injury in Uninsured and Underinsured Diabetic African American Men and Putative Role of Meprin Metalloproteases in Diabetic Nephropathy.

Diabetes is the leading cause of chronic kidney disease. African Americans are disproportionately burdened by diabetic kidney disease (DKD) and end stage renal disease (ESRD). Disparities in DKD have genetic and socioeconomic components, yet its prevalence in African Americans is not adequately studied. The current study used multiple biomarkers of DKD to evaluate undiagnosed DKD in uninsured and underinsured African American men in Greensboro, North Carolina. Participants consisted of three groups: nondiabetic controls, diabetic patients without known kidney disease, and diabetic patients with diagnosed DKD. Our data reveal undiagnosed kidney injury in a significant proportion of the diabetic patients, based on levels of both plasma and urinary biomarkers of kidney injury, namely, urinary albumin to creatinine ratio, kidney injury molecule-1, cystatin C, and neutrophil gelatinase-associated lipocalin. We also found that the urinary levels of meprin A, meprin B, and two kidney meprin targets (nidogen-1 and monocytes chemoattractant protein-1) increased with severity of kidney injury, suggesting a potential role for meprin metalloproteases in the pathophysiology of DKD in this subpopulation. The study also demonstrates a need for more aggressive tests to assess kidney injury in uninsured diabetic patients to facilitate early diagnosis and targeted interventions that could slow progression to ESRD.

Carnosic acid as a major bioactive component in rosemary extract ameliorates high-fat-diet-induced obesity and metabolic syndrome in mice.

In this study, we investigated the preventive effects of carnosic acid (CA) as a major bioactive component in rosemary extract (RE) on high-fat-diet-induced obesity and metabolic syndrome in mice. The mice were given a low-fat diet, a high-fat diet or a high-fat diet supplemented with either 0.14% or 0.28% (w/w) CA-enriched RE (containing 80% CA, RE#1L and RE#1H), or 0.5% (w/w) RE (containing 45% CA, RE#2), for a period of 16 weeks. There was the same CA content in the RE#1H and RE#2 diets and half of this amount in the RE#1L diet. The dietary RE supplementation significantly reduced body weight gain, percent of fat, plasma ALT, AST, glucose, insulin levels, liver weight, liver triglyceride, and free fatty acid levels in comparison with the mice fed with a HF diet without RE treatment. RE administration also decreased the levels of plasma and liver malondialdehyde, advanced glycation end products (AGEs), and the liver expression of receptor for AGE (RAGE) in comparison with those for mice of the HF group. Histological analyses of liver samples showed decreased lipid accumulation in hepatocytes in mice administrated with RE in comparison with that of HF-diet-fed mice. Meanwhile, RE administration enhanced fecal lipid excretion to inhibit lipid absorption and increased the liver GSH/GSSG ratio to perform antioxidant activity compared with HF group. Our results demonstrate that rosemary is a promising dietary agent to reduce the risk of obesity and metabolic syndrome.

Essential Structural Requirements and Additive Effects for Flavonoids to Scavenge Methylglyoxal.

Reactive dicarbonyl species, such as methylglyoxal (MGO), are considered as the major precursors of advanced glycation end products (AGEs), which are believed to be one of the physiological causes of diabetes and its complications. Scavenging of reactive dicarbonyl species using naturally occurring flavonoids has been proposed as an effective way to prevent diabetic complications. To elucidate the structural requirements of flavonoids in scavenging MGO, seven flavonoids (quercetin, luteolin, epicatechin, genistein, daidzein, apigenin, and phloretin) and five sub-components of the flavonoids (gallic acid, phloroglucinol, pyrogallol, pyrocatechol, and resorcinol) were examined in this study. Our results showed the following: (1) 1,2,3-trihydroxybenzene (pyrogallol) has higher MGO scavenging activity than 1,3,5-trihydroxybenzene and 1,2- and 1,3-dihydroxybenzene, and substitution at position 5 of pyrogallol diminished the scavenging activity, indicating that position 5 is the active site of pyrogallol; (2) the A ring is the active site of flavonoids in contributing the MGO-trapping efficacy, and the hydroxyl group at C-5 on the A ring enhances the trapping efficacy; (3) the double bond between C-2 and C-3 on the C ring could facilitate the trapping efficacy; and (4) the number of hydroxyl groups on the B ring does not significantly influence the trapping efficacy. In addition, we found there is an additive effect in MGO trapping by two common flavonoids, quercetin and phloretin, indicating that flavonoid-enriched foods and beverages hold great promise to prevent the development of diabetic complications.