Christopher D. Packey

Commensal Bacteria, Traditional and Opportunistic Pathogens, Dysbiosis and Bacterial Killing in Inflammatory Bowel Diseases

Purpose of review The authors present evidence published during the past 2 years of the roles of commensal and pathogenic bacteria in the pathogenesis of the inflammatory bowel diseases. Recent findings Rodent models conclusively implicate commensal enteric bacteria in chronic, immune-mediated, experimental colitis, and genetically determined defects in bacterial killing by innate immune cells are found in a subset of patients with Crohns disease. There is no evidence that a single pathogen, including Mycobacterium avium subspecies paratuberculosis, causes Crohns disease or ulcerative colitis. However, adherent/invasive Escherichia coli are associated with ileal Crohns disease, with the mechanisms and genetics of adherent/invasive E. coli virulence being elucidated. Molecular characterization of the microbiota in patients with inflammatory bowel diseases reveals decreased biodiversity of commensal bacteria, most notably the phyla Bacteroidetes and Firmicutes, including the clinically relevant Faecalibacterium prausnitzii, and increased E. coli concentrations. VSL#3 is one probiotic preparation shown to be efficacious in certain clinical situations in small clinical trials. Summary Further characterization of altered microbiota in patients with inflammatory bowel diseases and linking dysbiosis with host genetic alterations in immunoregulation, innate microbial killing and barrier function are critical, so that individualized treatments to increase beneficial commensals and their metabolic products (probiotic and prebiotic administration) and diminish deleterious species such as adherent/invasive E. coli can be tailored for defined patient subsets.

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.

Altered enteric microbiota ecology in interleukin 10-deficient mice during development and progression of intestinal inflammation.

Inflammatory bowel diseases (IBD) result from dysregulated immune responses toward microbial and perhaps other luminal antigens in a genetically susceptible host, and are associated with altered composition and diversity of the intestinal microbiota. The interleukin 10-deficient (IL-10−/−) mouse has been widely used to model human IBD; however the specific alterations that occur in the intestinal microbiota of this mouse model during the onset of colonic inflammation have not yet been defined. The aim of our study was to define the changes in diversity and composition that occur in the intestinal microbiota of IL-10−/− mice during the onset and progression of colonic inflammation. We used high throughput sequencing of the 16S rRNA gene to characterize the diversity and composition of formerly germ-free, wild-type and IL-10−/− mice associated with the same intestinal microbiota over time. Following two weeks of colonization with a specific pathogen-free (SPF) microbiota we observed a significant increase in the diversity and richness of the intestinal microbiota of wild-type mice. In contrast, a progressive decrease in diversity and richness was observed at three and four weeks in IL-10−/− mice. This decrease in diversity and richness was mirrored by an increase in Proteobacteria and Escherichia coli in IL-10−/− mice. An increase in E. coli was also observed in conventionally raised IL-10−/− mice at the point of colonic inflammation. Our data reports the sequential changes in diversity and composition of the intestinal microbiota in an immune-mediated mouse model that may help provide insights into the primary vs. secondary role of dysbiosis in human IBD patients.

Molecular detection of bacterial contamination in gnotobiotic rodent units

Gnotobiotic rodents provide an important technique to study the functional roles of commensal bacteria in host physiology and pathophysiology. To ensure sterility, these animals must be screened frequently for contamination. The traditional screening approaches of culturing and Gram staining feces have inherent limitations, as many bacteria are uncultivable and fecal Gram stains are difficult to interpret. Thus, we developed and validated molecular methods to definitively detect and identify contamination in germ-free (GF) and selectively colonized animals. Fresh fecal pellets were collected from rodents housed in GF isolators, spontaneously contaminated ex-GF isolators, selectively colonized isolators and specific pathogen-free (SPF) conditions. DNA isolated from mouse and rat fecal samples was amplified by polymerase chain reaction (PCR) and subjected to quantitative PCR (qPCR) using universal primers that amplify the 16S rRNA gene from all bacterial groups. PCR products were sequenced to identify contaminating bacterial species. Random amplification of polymorphic DNA (RAPD) PCR profiles verified bacterial inoculation of selectively colonized animals. These PCR techniques more accurately detected and identified GF isolator contamination than current standard approaches. These molecular techniques can be utilized to more definitively screen GF and selectively colonized animals for bacterial contamination when Gram stain and/or culture results are un-interpretable or inconsistent.

Components of Enteric Dysbiosis Are Present at the Onset of Immune-mediated, Spontaneous Colitis: P-189 YI

BACKGROUND: Patients with inflammatory bowel diseases (IBD) frequently demonstrate intestinal dysbioses, but it is unknown if shifts in the microbiota drive disease or if they are a by-product of inflammation. We utilized multiple cultureindependent techniques to investigate whether enteric dysbiosis ensues prior to or subsequent to the development of inflammation in the interleukin-10 knockout (IL-10 KO) mouse model of immune-mediated, spontaneous colitis. METHODS: 8-10 week old germ-free (GF) wild-type (WT) (n 1⁄4 4) and IL-10 KO (n 1⁄4 9) 129Sv/Ev mice were colonized with identical specific pathogen-free (SPF) feces, and fecal samples were subsequently taken weekly for 4 weeks. Mice were necropsied at 2 and 4 weeks and colonic tissues were harvested. Fecal DNA was isolated and 454 pyrosequencing and qPCR of 16S rRNA genes were performed. QIIME pipeline was utilized for 454 analyses. IL-12p40 secretion from colonic tissue explants was measured by ELISA and Kruskal-Wallis one-way analysis of variance performed. RESULTS: Colonic inflammation in IL-10 KO mice as measured by pro-inflammatory IL-12p40 secretion was seen at 2 weeks after colonization (P<0.02 between groups), whereas WT mice did not develop colitis. QIIME pyrosequencing analyses revealed that microbiota a-diversity (species richness) and b-diversity (species diversity) increased from week 1 to week 2 in WT and IL-10 KO mice, then dramatically decreased at weeks 3 and 4 in IL-10 KO mice (P<0.05) despite increases in total fecal bacterial numbers (P<0.0007). Levels of abundant fecal Bacteroidetes remained unchanged in IL-10 KO mice at week 2, then decreased at weeks 3 and 4. qPCR showed that IL-10 KO mouse fecal Lactobacillus spp. levels were lesser than WT mouse levels at weeks 1 and 2, then significantly increased to exceed WT levels at weeks 3 and 4 (P<0.002). However, at 2 weeks after colonization, some obvious microbial shifts had already commenced in IL-10 KO mice. These included decreases in Firmicutes, (including several members of the Lachnospiraceae family), Verrucomicrobia, (including the abundant human intestinal colonizer Akkermansia muciniphila, which are decreased in IBD patients), and Actinobacteria. There were also dramatic increases in fecal levels of the Proteobacteria phylum in IL-10 KO mice at 2 weeks. E. coli levels, which were lower in IL-10 KO mice than WT mice at week 1, increased to approximate WT levels at 2 and 3 weeks, and then continued to increase until they exceeded WT levels at week 4 (P<0.0001). CONCLUSION(S): GF IL-10 KO mice housed in our gnotobiotic facility develop spontaneous colitis by 2 weeks after colonization with SPF microbiota. While some alterations in the enteric microbiota composition do not occur until after this time point, other changes are already observed by 2 weeks after conventionalization. Though expansion of this line of research is needed, these results suggest that it is possible that some components of intestinal dysbioses precede and drive the development of immune-mediated, spontaneous colitis in the IL-10 KO mouse, and perhaps in humans. If proven, a primary focus in preventing and/or managing IBD would involve avoidance and/or reversal of dysbioses with prebiotics, probiotics, antibiotics, fecal transplant, or anti-microbial peptide induction.