BackgroundThe gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the composition and activities of which influences mucosal development, immunity, nutrition and drug metabolism. It remains unclear whether the composition of the dominant GTM is conserved within animals of the same strain and whether stable GTMs are selected for by host-specific factors or dictated by environmental variables.Methodology/Principal FindingsThe GTM composition of six highly inbred, genetically distinct strains of mouse (C3H, C57, GFEC, CD1, CBA nu/nu and SCID) was profiled using eubacterial –specific PCR-DGGE and quantitative PCR of feces. Animals exhibited strain-specific fecal eubacterial profiles that were highly stable (c. >95% concordance over 26 months for C57). Analyses of mice that had been relocated before and after maturity indicated marked, reproducible changes in fecal consortia and that occurred only in young animals. Implantation of a female BDF1 mouse with genetically distinct (C57 and Agoutie) embryos produced highly similar GTM profiles (c. 95% concordance) between mother and offspring, regardless of offspring strain, which was also reflected in urinary metabolite profiles. Marked institution-specific GTM profiles were apparent in C3H mice raised in two different research institutions.Conclusion/SignificanceStrain-specific data were suggestive of genetic determination of the composition and activities of intestinal symbiotic consortia. However, relocation studies and uterine implantation demonstrated the dominance of environmental influences on the GTM. This was manifested in large variations between isogenic adult mice reared in different research institutions.

ObjectiveColonic mucosa-associated Escherichia coli are increased in Crohn's disease (CD) and colorectal cancer (CRC). They variously haemagglutinate, invade epithelial cell lines, replicate within macrophages, translocate across M (microfold) cells and damage DNA. We investigated genes responsible for these effects and their co-association in colonic mucosal isolates.DesignA fosmid library yielding 968 clones was prepared in E coli EPI300-T1 using DNA from a haemagglutinating CRC isolate, and resulting haemagglutinating clones were 454-pyrosequenced. PCR screening was performed on 281 colonic E coli isolates from inflammatory bowel disease (IBD) (35 patients), CRC (21) and controls (24; sporadic polyps or irritable bowel syndrome).Results454-Pyrosequencing of fosmids from the haemagglutinating clones (n=8) identified the afimbrial adhesin afa-1 operon. Transfection of afa-1 into E coli K-12 predictably conferred diffuse adherence plus invasion of HEp-2 and I-407 epithelial cells, and upregulation of vascular endothelial growth factor. E coli expressing afaC were common in CRC (14/21, p=0.0009) and CD (9/14, p=0.005) but not ulcerative colitis (UC; 8/21) compared with controls (4/24). E coli expressing both afaC and lpfA (relevant to M-cell translocation) were common in CD (8/14, p=0.0019) and CRC (14/21, p=0.0001), but not UC (6/21) compared with controls (2/24). E coli expressing both afaC and pks (genotoxic) were common in CRC (11/21, p=0.0015) and UC (8/21, p=0.022), but not CD (4/14) compared with controls (2/24). All isolates expressed dsbA and htrA relevant to intra-macrophage replication, and 242/281 expressed fimH encoding type-1 fimbrial adhesin.ConclusionsIBD and CRC commonly have colonic mucosal E coli that express genes that confer properties relevant to pathogenesis including M-cell translocation, angiogenesis and genotoxicity.

Crohn's disease (CD) and ulcerative colitis (UC) have features that suggest bacterial involvement, and all genetic models of inflammatory bowel disease (IBD) require the presence of commensal bacteria. CD is associated with innate immune response genes such as NOD2/CARD15 and the autophagy genes ATG16L1 and IRGM. However, IBD responds to immunosuppression, suggesting that any bacteria involved are not acting as conventional pathogens. Molecular techniques are rapidly advancing our knowledge of the gut microbiota. In CD there is reduced diversity, and notably a reduction in the probiotic Faecalibacterium prausnitzii, the presence of which in the terminal ileum is associated with a reduced risk of recurrence following surgery. There is also a consistent increase in mucosa-associated Escherichia coli with an "adherent and invasive" phenotype, which allows them to replicate inside macrophages and induce granulomas. Speculation that CD could be caused by the Mycobacterium avium subspecies paratuberculosis (MAP) continues. The response to antitumor necrosis factor treatments suggests that, if relevant at all, MAP is not acting as a conventional pathogen. However, there is increased colonization by MAP in CD, and there is evidence that it could have an indirect effect mediated by the suppression of macrophage function. UC relapse is frequently associated with infection by pathogens, but there is less evidence for involvement of a specific bacterial species. Poor barrier integrity followed by an inflammatory reaction to bacterial components, with chronicity maintained by an autoimmune process, seems a plausible pathogenic model. Bacterial theories of pathogenesis are now becoming testable by targeted therapeutic interventions.

Coaggregation is believed to facilitate the integration of new bacterial species into polymicrobial communities. The aim of this study was to investigate coaggregation between and among human oral and enteric bacteria. Stationary phase cultures of 10 oral and 10 enteric species, chosen on the basis of numerical and ecological significance in their respective environments together with their ease of cultivation, were tested using a quantitative spectrophotometric coaggregation assay in all possible pairwise combinations to provide quantitative coaggregation scores. While 40% of possible partnerships coaggregated strongly for oral strains, strong interactions between oral and gut strains were considerably less common (4% incidence). Coaggregation scores were also weak between members of the intestinal microbiota (7% incidence), apart from Bacteroides fragilis with Clostridium perfringens, and Bifidobacterium adolescentis with C. perfringens. Oral and intestinal bacteria did not strongly interact, apart from B. adolescentis with Fusobacterium nucleatum, Actinomyces naeslundii with C. perfringens and F. nucleatum with Lactobacillus paracasei. Heating and sugar-addition experiments indicated that similar to oral microorganisms, interactions within intestinal bacteria and between intestinal and oral strains were mediated by lectin-carbohydrate interactions.

IntroductionMucosa-associated adherent invasive Escherichia coli (AIEC) are found in colorectal cancer (CRC) and Crohn's disease (IBD).1 They invade intestinal epithelial cells, replicate in macrophages and interact with epithelial cells in vitro to promote proinflammatory cytokine release and NFkB activation.2 The adherent-invasive phenotype strongly correlates with the ability to agglutinate red blood cells (RBCs). We suspect that mucosa-associated E coli may also play a role in CRC pathogenesis by activation of key cellular pathways. Our aim here was to identify and characterise adherence mechanisms for our colonic AIEC isolates.MethodsA fosmid library was constructed containing 30–60 kb DNA fragments from E coli HM358, a CRC mucosa-associated isolate. Clones were screened for the ability to haemagglutinate RBCs. Agglutinating positive (HA+) clones were further assessed for their ability to adhere to epithelial Hep2 cells, to replicate within macrophages and for their ability to translocate across M-cells. They were then subjected to 454 DNA sequencing. Subsequently, 280 E coli isolates from IBD, CRC and healthy control patients were assessed for the presence of a gene cluster identified from sequence analysis.ResultsOf the 964 fosmid clones constructed, 8 displayed a strong HA+ phenotype. Sequence data demonstrated all possessed a diffuse adherence gene cluster (afaA-E). HA+ positive clones were diffusely adherent to Hep2 cells and showed marked ability to replicate within macrophages when compared to HA- clones (p<0.01; ANOVA). In vitro data also suggest that the presence of afa/dr in E coli promotes translocation across M cells, a key portal of mucosal entry. E coli containing afa/dr were more commonly isolated from IBD and CRC patients (57% afa positive) than from healthy controls (28% afa positive, p<0.001 χ2 test).ConclusionAfa/dr E coli are more commonly isolated from IBD and CRC patients than from healthy controls and have properties, including epithelial invasion and replication in macrophages, that overlap with AIEC. Other studies have shown that adherent afa/dr E coli play a key role in the upregulation of angiogenic vascular endothelial growth factor (VEGF) in the intestinal epithelium3 and they may thus have a role in pathogenesis of CRC as well as IBD.