Tamar Ringel-Kulka

Alterations in composition and diversity of the intestinal microbiota in patients with diarrhea-predominant irritable bowel syndrome.

Background  The intestinal microbiota has been implicated in the pathophysiology of irritable bowel syndrome (IBS). Due to the variable resolutions of techniques used to characterize the intestinal microbiota, and the heterogeneity of IBS, the defined alterations of the IBS intestinal microbiota are inconsistent. We analyzed the composition of the intestinal microbiota in a defined subgroup of IBS patients (diarrhea‐predominant IBS, D‐IBS) using a technique that provides the deepest characterization available for complex microbial communities.

Molecular analysis of the luminal- and mucosal-associated intestinal microbiota in diarrhea-predominant irritable bowel syndrome.

Alterations in the intestinal microbiota have been suggested as an etiological factor in the pathogenesis of irritable bowel syndrome (IBS). This study used a molecular fingerprinting technique to compare the composition and biodiversity of the microbiota within fecal and mucosal niches between patients with diarrhea-predominant IBS (D-IBS) and healthy controls. Terminal-restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the bacterial 16S rRNA gene was used to perform microbial community composition analyses on fecal and mucosal samples from patients with D-IBS (n = 16) and healthy controls (n = 21). Molecular fingerprinting of the microbiota from fecal and colonic mucosal samples revealed differences in the contribution of T-RFs to the microbiota between D-IBS patients and healthy controls. Further analysis revealed a significantly lower (1.2-fold) biodiversity of microbes within fecal samples from D-IBS patients than healthy controls (P = 0.008). No difference in biodiversity in mucosal samples was detected between D-IBS patients and healthy controls. Multivariate analysis of T-RFLP profiles demonstrated distinct microbial communities between luminal and mucosal niches in all samples. Our findings of compositional differences in the luminal- and mucosal-associated microbiota between D-IBS patients and healthy controls and diminished microbial biodiversity in D-IBS fecal samples further support the hypothesis that alterations in the intestinal microbiota may have an etiological role in the pathogenesis of D-IBS and suggest that luminal and mucosal niches need to be investigated.

Characterization of the Fecal Microbiota Using High-Throughput Sequencing Reveals a Stable Microbial Community during Storage

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at −80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or −80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or −80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.

Probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 versus placebo for the symptoms of bloating in patients with functional bowel disorders: a double-blind study.

Background Recent data suggest a role for the intestinal microbiota in the pathogenesis of functional bowel disorders (FBDs). Probiotic studies in FBDs generated inconsistent results suggesting a strain-specific and product-specific effect. Aim To investigate the clinical efficacy of Lactobacillus acidophilus NCFM (L-NCFM) and Bifidobacterium lactis Bi-07 (B-LBi07) in nonconstipation FBDs. Methods A double-blind, placebo-control clinical trial of the probiotic bacterias L-NCFM and B-LBi07 twice a day (2×1011CFU/d) versus placebo over 8 weeks. Primary endpoints were global relief of gastrointestinal symptoms and satisfaction with treatment. Secondary endpoints were change in symptoms severity, well-being, and quality of life. Microbiological effect was assessed by quantitative real time polymerase chain reaction on fecal samples. Results Sixty patients (probiotic, n=31; placebo, n=29), 72% females, 84% whites, mean age 37 years. Abdominal bloating improved in the probiotics compared with the placebo group at 4 weeks (4.10 vs 6.17, P=0.009; change in bloating severity P=0.02) and 8 weeks (4.26 vs 5.84, P=0.06; change in bloating severity P<0.01). Analyses on the irritable bowel syndrome subgroup (n=33) showed similar results. Conclusions L-NCFM and B-LBi07 twice a day improve symptoms of bloating in patients with FBDs. These data supports the role of intestinal bacteria in the pathophysiology of FBD and the role for probiotic bacteria in the management of these disorders.

Early life exposures as risk factors for pediatric eosinophilic esophagitis.

Objectives: Few etiologic studies of eosinophilic esophagitis (EoE) have been conducted. Early life exposures have been shown to predispose to other allergic disease, but their role has not been assessed in EoE. The present study sought to explore early life exposures as possible risk factors for developing EoE in the pediatric population. Methods: This was a 2-phase case-control study conducted at the University of North Carolina. The first phase consisted of survey development for early life exposures via cognitive interview. In the second phase, a telephone-based questionnaire was administered to cases with EoE (n = 31) and 2 sets of controls, patients with gastroesophageal reflux disease, and siblings of nonsyndromic cleft lip/palate patients (n = 26 in each). Different controls were explored to identify controls reflective of the source population of the cases. Siblings of cleft lip/palate patients were identified as the more suitable control population. Odds ratios were calculated to evaluate the association between early life exposures and the development of pediatric EoE. Results: Early life exposures were associated with increased odds of developing pediatric-onset EoE. Antibiotic use in infancy was associated with 6 times the odds of having EoE (95% confidence interval 1.7–20.8). Cesarean delivery, preterm birth, and formula-only or mixed (infant formula and breast milk) feeding also have trends toward increased odds for developing EoE. Conclusions: A number of early life exposures may be associated with the development of EoE. These are potentially modifiable risk factors that if confirmed would have implications for improved understanding of EoE pathogenesis and disease prevention.

Intestinal microbiota in healthy U.S. young children and adults--a high throughput microarray analysis.

It is generally believed that the infants microbiota is established during the first 1–2 years of life. However, there is scarce data on its characterization and its comparison to the adult-like microbiota in consecutive years. Aim To characterize and compare the intestinal microbiota in healthy young children (1–4 years) and healthy adults from the North Carolina region in the U.S. using high-throughput bacterial phylogenetic microarray analysis. Methods Detailed characterization and comparison of the intestinal microbiota of healthy children aged 1–4 years old (n = 28) and healthy adults of 21–60 years (n = 23) was carried out using the Human Intestinal Tract Chip (HITChip) phylogenetic microarray targeting the V1 and V6 regions of 16S rRNA and quantitative PCR. Results The HITChip microarray data indicate that Actinobacteria, Bacilli, Clostridium cluster IV and Bacteroidetes are the predominant phylum-like groups that exhibit differences between young children and adults. The phylum-like group Clostridium cluster XIVa was equally predominant in young children and adults and is thus considered to be established at an early age. The genus-like level show significant 3.6 fold (higher or lower) differences in the abundance of 26 genera between young children and adults. Young U.S. children have a significantly 3.5-fold higher abundance of Bifidobacterium species than the adults from the same location. However, the microbiota of young children is less diverse than that of adults. Conclusions We show that the establishment of an adult-like intestinal microbiota occurs at a later age than previously reported. Characterizing the microbiota and its development in the early years of life may help identify ‘windows of opportunity’ for interventional strategies that may promote health and prevent or mitigate disease processes.

Altered Colonic Bacterial Fermentation as a Potential Pathophysiological Factor in Irritable Bowel Syndrome

OBJECTIVES:Dysbiosis leading to abnormal intestinal fermentation has been suggested as a possible etiological mechanism in irritable bowel syndrome (IBS). We aimed to investigate the location and magnitude of altered intestinal bacterial fermentation in IBS and its clinical subtypes.METHODS:IBS patients who satisfied the Rome III criteria (114) and 33 healthy controls (HC) were investigated. Intestinal fermentation was assessed using two surrogate measures: intestinal intraluminal pH and fecal short-chain fatty acids (SCFAs). Intraluminal pH and intestinal transit times were measured in the small and large bowel using a wireless motility capsule (SmartPill) in 47 IBS and 10 HC. Fecal SCFAs including acetate, propionate, butyrate, and lactate were analyzed by capillary gas chromatography in all enrolled subjects. Correlations between intestinal pH, fecal SCFAs, intestinal transit time, and IBS symptom scores were analyzed.RESULTS:Colonic intraluminal pH levels were significantly lower in IBS patients compared with HC (total colonic pH, 6.8 for IBS vs. 7.3 for HC, P=0.042). There were no differences in total and segmental pH levels in the small bowel between IBS patients and HC (6.8 vs. 6.8, P=not significant). The intraluminal colonic pH differences were consistent in all IBS subtypes. Total SCFA level was significantly lower in C-IBS patients than in D-IBS and M-IBS patients and HC. The total SCFA level in all IBS subjects was similar with that of HC. Colonic pH levels correlated positively with colon transit time (CTT) and IBS symptoms severity. Total fecal SCFAs levels correlated negatively with CTT and positively with stool frequency.CONCLUSIONS:Colonic intraluminal pH is decreased, suggesting higher colonic fermentation, in IBS patients compared with HC. Fecal SCFAs are not a sensitive marker to estimate intraluminal bacterial fermentation.

Luminal cysteine-proteases degrade colonic tight junction structure and are responsible for abdominal pain in constipation-predominant IBS.

OBJECTIVES:Luminal serine-proteases lead to increased colonic paracellular permeability and visceral hypersensitivity in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). Other proteases, namely cysteine-proteases (CPs), increase airway permeability by digesting epithelial tight junction proteins. In this study, we focused on constipation-predominant IBS (IBS-C) and we aimed to (i) evaluate CP levels in two cohorts of IBS patients, (ii) test if IBS-C fecal supernatant (FSN) affects permeability, and visceral sensitivity after repeated administrations in mice, and (iii) evaluate occludin expression in IBS-C colonic biopsies.METHODS:Fecal CP activity was determined using selective substrate and inhibitor (E64). The effect of papain, as positive control, and IBS-C FSN administrations were evaluated on colonic paracellular permeability and mucosal occludin levels in mice and T84 monolayers. Occludin protein levels were evaluated in IBS-C colonic biopsies. Sensitivity to colorectal distension (CRD) was measured after repeated administrations of IBS-C FSN.RESULTS:We found in a subset of IBS-C patients an enhanced fecal CP activity, in comparison with healthy controls and IBS-D patients. CP activity levels positively correlated with disease severity and abdominal pain scoring. This association was confirmed by receiver operating characteristic curve analysis. In mice, repeated application of IBS-C FSN into colon triggered increased permeability, linked to the enzymatic degradation of occludin, and was associated with enhanced visceral sensitivity to CRD. Finally, occludin levels were found decreased in colonic biopsies from IBS-C patients, and IBS-C FSNs were able to degrade recombinant human occludin in vitro. All these effects were abolished by preincubation of IBS-C FSN with a CP inhibitor, E64.CONCLUSIONS:These data suggest that luminal CPs may represent a new factor contributing to the genesis of symptoms in IBS.

High throughput sequencing reveals distinct microbial populations within the mucosal and luminal niches in healthy individuals

Background: The intestinal microbiota is associated with human health and diseases. The luminal microbiota (LM) and the mucosal-associated microbiota (MAM) are 2 distinct ecosystems with different metabolic and immunological functions. Aim: To characterize the intestinal LM and MAM in humans using high throughput sequencing of the 16S rRNA gene. Methods: Fresh fecal samples and distal colonic mucosal biopsies collected from 24 healthy subjects before (fecal) and during (mucosa) a flexible sigmoidoscopy of an un-prepared bowel. High throughput sequencing of the 16S rRNA gene was used to characterize bacterial communities. Sequences were processed using the QIIME pipeline. Results: LM and MAM populations were significantly different (ANOSIM: R = 0.49, P = 0.001). The LM displayed tighter clustering compared to the MAM (average weighted UniFrac distances 0.27 ± 0.05 vs. 0.43 ± 0.09, P < 0.001, respectively), and showed higher diversity (Shannon diversity index: 4.96 ± 0.37 vs 4.14 ± 0.56, respectively, P < 0.001). The dominant phyla in the LM and MAM were significantly different: Firmicutes (41.4% vs. 29.1%, FDR < 0.0001, respectively), Bacteroidetes (20.2% vs. 26.3%, FDR < 0.05, respectively), Actinobacteria (22% vs. 12.6%, FDR < 0.0001, respectively) and Proteobacteria (9.3% vs. 19.3%, FDR < 0.0001, respectively). The abundance of 56 genera differed significantly (FDR < 0.1) between the 2 niches. All of the genera in the fecal microbiota were present in the MAM while 10 genera were found to be unique to the MAM. Conclusion: The LM and MAM are distinct microbial ecosystems that differ significantly from each other in microbial diversity and composition. These two microbial niches should be investigated independently to better understand the role of the intestinal microbiota in health and disease.

The Rationale and Clinical Effectiveness of Probiotics in Irritable Bowel Syndrome

The pathophysiology of irritable bowel syndrome (IBS) is still unknown. However, several lines of epidemiological, physiological, and clinical data suggest a role for intestinal bacteria in the pathogenesis of the disorder. Recent microbiology studies demonstrated differences in the composition of the intestinal microbiota between patients with IBS and healthy individuals. In addition, physiological studies have shown that manipulation of the intestinal microbiota by antibiotics, prebiotics, or probiotics can affect intestinal functions (eg, motility and sensation) relevant in the pathogenesis of IBS. Several randomized control trials comparing the effects of probiotics versus placebo in IBS have been published. Despite considerable differences in study design, dosing regimens, probiotic species used, and reported clinical end points, the current data indicate improving IBS symptoms and reducing the risk of persistent IBS symptoms. The data on the use of probiotics in children with IBS is more limited but is also suggestive for beneficial effects. The inconsistencies between the studies underline the need to look at each probiotic product separately for specific conditions, symptoms, and patient populations. This review article discusses the rationale for targeting the intestinal microbiota in patient with IBS and provides an overview and a critical evaluation of the currently available clinical data on the use of probiotics in the treatment of patients with IBS.