Ian M. Carroll

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.

Luminal and mucosal-associated intestinal microbiota in patients with diarrhea-predominant irritable bowel syndrome

BackgroundRecent studies have suggested a role for an altered intestinal microbiota in the pathophysiology of irritable bowel syndrome (IBS). However, no consensus has been reached regarding the association between specific enteric bacterial groups and IBS. The aim of this study was to investigate the fecal and mucosal-associated microbiota using two independent techniques in intestinal samples from diarrhea-predominant IBS (D-IBS) and healthy controls.MethodsFecal and colonic mucosal biopsy samples were obtained from 10 D-IBS patients and 10 healthy controls. Colonic tissue was collected during a un-sedated un-prepped flexible sigmoidoscopy. Fecal and tissue samples were processed immediately upon collection for culture under aerobic and anaerobic conditions or frozen for further molecular analysis. DNA was extracted from all frozen samples and used to enumerate specific bacterial groups using quantitative real-time PCR (qPCR).ResultsCulture analysis of intestinal samples demonstrated a significant reduction in the concentration of aerobic bacteria in fecal samples from D-IBS patients when compared to healthy controls (1.4 × 107 vs. 8.4 × 108 CFUs/g feces, P = 0.002). qPCR analysis demonstrated a significant 3.6 fold increase (P = 0.02) in concentrations of fecal Lactobacillus species between D-IBS patients and healthy controls.ConclusionsOur culture and molecular data indicate that quantitative differences exist in specific bacterial groups in the microbiota between D-IBS and healthy subjects.

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.

Gut Microbial Diversity is Reduced by the Probiotic VSL#3 and Correlates with Decreased TNBS-Induced Colitis

Background: Compositional changes within the normal intestinal microbiota have been associated with the development of various intestinal inflammatory disorders such as pouchitis and inflammatory bowel diseases (IBD). Therefore, it has been speculated that manipulation of a dysbiotic intestinal microbiota has the potential to restore microbial homeostasis and attenuate inflammation. Methods: We performed community composition analyses by terminal restriction fragment length polymorphism (T‐RFLP) of the bacterial 16S ribosomal RNA gene to investigate the impact of the probiotic VSL#3 on colonic microbial community composition and development of trinitrobenzene sulfonic acid (TNBS)‐induced colitis in rats. Results: TNBS‐induced chronic colitis was significantly reduced in VSL#3‐fed rats compared to controls (P < 0.05). T‐RFLP analysis revealed distinct microbial communities at luminal versus mucosal sites. Within the luminal microbiota, chronic colitis was associated with an overall decrease in bacterial richness and diversity (Margalefs richness, P < 0.01; Shannon diversity, P < 0.01). This decrease in luminal microbial diversity was enhanced in TNBS‐treated rats fed VSL#3 (Margalefs richness, P < 0.001; Shannon diversity, P < 0.001) and significantly correlated with reduced clinical colitis scores (Pearson correlation P < 0.05). Conclusions: Our data demonstrate that the probiotic VSL#3 alters the composition of the intestinal microbiota and these changes correlate with VSL#3‐induced disease protection. (Inflamm Bowel Dis 2011;)

Mouse Paneth cell antimicrobial function is independent of Nod2

Objective Although polymorphisms of the NOD2 gene predispose to the development of ileal Crohns disease, the precise mechanisms of this increased susceptibility remain unclear. Previous work has shown that transcript expression of the Paneth cell (PC) antimicrobial peptides (AMPs) α-defensin 4 and α-defensin-related sequence 10 are selectively decreased in Nod2−/− mice. However, the specific mouse background used in this previous study is unclear. In light of recent evidence suggesting that mouse strain strongly influences PC antimicrobial activity, we sought to characterise PC AMP function in commercially available Nod2−/− mice on a C57BL/6 (B6) background. Specifically, we hypothesised that Nod2−/− B6 mice would display reduced AMP expression and activity. Design Wild-type (WT) and Nod2−/− B6 ileal AMP expression was assessed via real-time PCR, acid urea polyacrylamide gel electrophoresis and mass spectrometry. PCs were enumerated using flow cytometry. Functionally, α-defensin bactericidal activity was evaluated using a gel-overlay antimicrobial assay. Faecal microbial composition was determined using 454-sequencing of the bacterial 16S gene in cohoused WT and Nod2−/− littermates. Results WT and Nod2−/− B6 mice displayed similar PC AMP expression patterns, equivalent α-defensin profiles, and identical antimicrobial activity against commensal and pathogenic bacterial strains. Furthermore, minimal differences in gut microbial composition were detected between the two cohoused, littermate mouse groups. Conclusions Our data reveal that Nod2 does not directly regulate PC antimicrobial activity in B6 mice. Moreover, we demonstrate that previously reported Nod2-dependent influences on gut microbial composition may be overcome by environmental factors, such as cohousing with WT littermates.

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.

Alterations in the intestinal microbiota and functional bowel symptoms.

Functional gastrointestinal disorders (FGIDs) are highly prevalent in Western countries yet no single mechanism or etiological agent that initiates IBS has been identified. Current research has implicated the intestinal microbiota with FGIDs. This article reviews the available literature/data regarding the intestinal microbiota and FGIDS. The possible relationships between the intestinal microbiota and the intestinal function and functional bowel symptoms are discussed.

The Intestinal Microbiota in Acute Anorexia Nervosa and During Renourishment: Relationship to Depression, Anxiety, and Eating Disorder Psychopathology

Objective The relevance of the microbe-gut-brain axis to psychopathology is of interest in anorexia nervosa (AN), as the intestinal microbiota plays a critical role in metabolic function and weight regulation. Methods We characterized the composition and diversity of the intestinal microbiota in AN, using stool samples collected at inpatient admission (T1; n = 16) and discharge (T2; n = 10). At T1, participants completed the Beck Depression and Anxiety Inventories and the Eating Disorder Examination–Questionnaire. Patients with AN were compared with healthy individuals who participated in a previous study (healthy comparison group; HCG). Genomic DNA was isolated from stool samples, and bacterial composition was characterized by 454 pyrosequencing of the 16S rRNA gene. Sequencing results were processed by the Quantitative Insights Into Microbial Ecology pipeline. We compared T1 versus T2 samples, samples from both points were compared with HCG (n = 12), and associations between psychopathology and T1 samples were explored. Results In patients with AN, significant changes emerged between T1 and T2 in taxa abundance and beta (between-sample) diversity. Patients with AN had significantly lower alpha (within-sample) diversity than did HCG at both T1 (p = .0001) and T2 (p = .016), and differences in taxa abundance were found between AN patients and HCG. Levels of depression, anxiety, and eating disorder psychopathology at T1 were associated with composition and diversity of the intestinal microbiota. Conclusions We provide evidence of an intestinal dysbiosis in AN and an association between mood and the enteric microbiota in this patient population. Future directions include mechanistic investigations of the microbe-gut-brain axis in animal models and association of microbial measures with metabolic changes and recovery indices.