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ABSTRACT: 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.
PLoS ONE 01/2012; 7(10):e46953. · 4.09 Impact Factor
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ABSTRACT: 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.
AJP Gastrointestinal and Liver Physiology 07/2011; 301(5):G799-807. · 3.43 Impact Factor
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ABSTRACT: 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.
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.
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 (Margalef's richness, P < 0.01; Shannon diversity, P < 0.01). This decrease in luminal microbial diversity was enhanced in TNBS-treated rats fed VSL#3 (Margalef's richness, P < 0.001; Shannon diversity, P < 0.001) and significantly correlated with reduced clinical colitis scores (Pearson correlation P < 0.05).
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.
Inflammatory Bowel Diseases 01/2011; 17(1):289-97. · 4.86 Impact Factor
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ABSTRACT: Recent 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.
Fecal 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).
Culture 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.
Our culture and molecular data indicate that quantitative differences exist in specific bacterial groups in the microbiota between D-IBS and healthy subjects.
Gut Pathogens 01/2010; 2(1):19. · 2.11 Impact Factor
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ABSTRACT: 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.
Gastrointestinal endoscopy clinics of North America 02/2009; 19(1):141-50, vii.
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ABSTRACT: Emerging evidence has implicated reactive oxygen species (ROS) in the pathogenesis of inflammatory bowel disease (IBD). Although intestinal epithelial cells produce the ROS-neutralizing enzyme superoxide dismutase (SOD), the protein and activity levels of copper/zinc (Cu/Zn) and manganese (Mn) SOD are perturbed in inflamed tissues of IBD patients. Thus we investigated the ability of MnSOD from Streptococcus thermophilus to reduce colitis symptoms in interleukin (IL) 10-deficient mice using Lactobacillus gasseri as a delivery vehicle. Cohorts of 13-15 IL-10-deficient mice were left untreated or supplemented with native L. gasseri or L. gasseri expressing MnSOD for 4 wk. Colonic tissue was collected and inflammation was histologically scored. The presence of innate immune cells was investigated by immunohistochemistry and the host antioxidant response was determined by quantitative PCR. It was demonstrated that L. gasseri was stably maintained in mice for at least 3 days. L. gasseri producing MnSOD significantly reduced inflammation in IL-10-deficient mice compared with untreated controls (P < 0.05), whereas the anti-inflammatory effects of both native and MnSOD producing L. gasseri were more pronounced in males. The anti-inflammatory effects of L. gasseri were associated with a reduction in the infiltration of neutrophils and macrophages. Transcripts of antioxidant genes were equivalent in colonic tissues obtained from control and probiotic-treated IL-10-deficient mice. This study demonstrates that L. gasseri producing MnSOD has significant anti-inflammatory activity that reduces the severity of colitis in the IL-10-deficient mouse.
AJP Gastrointestinal and Liver Physiology 11/2007; 293(4):G729-38. · 3.43 Impact Factor
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ABSTRACT: The gastrointestinal epithelia of mammals are tolerant of their resident gut microbiota but are usually highly responsive to entero-pathogens; the host-specific responses have not been well characterized. To this end, the transcriptional responses of cultured human (Caco-2) and murine (CT-26) colonic epithelial cells were compared after exposure with the microfloral bacterium Lactobacillus reuteri or the human gastrointestinal pathogen Campylobacter jejuni. When in bacterial broth, both species elicit a stronger differential gene expression response in human colonic cells compared with mouse colonic cells. However, when these data are adjusted to remove bacterial broth effects, only human colonic epithelia exposed to C. jejuni show altered gene expression, suggesting that the human pathogen C. jejuni induces a host-specific response. The genes with altered expression are involved in growth, transcription, and steroid biosynthesis. Interestingly, human genes involved in cell polarity and water transport were significantly changed in response to C. jejuni exposure, linking infection with gastrointestinal disease. This study demonstrates that mouse and human colonic epithelia remain relatively unresponsive to commensal bacterial challenge, while the human pathogen C. jejuni elicits a host-specific response.
FEMS Microbiology Letters 10/2006; 262(2):236-43. · 2.04 Impact Factor
