In this study we report the results of multiple regression analysis of the largest 16S rRNA sequence datasets reported thus far on ileal tissues collected from IBD subjects. This analysis demonstrates that IBD phenotype has a predominant effect on microbial composition associated with the macroscopically normal appearing proximal margin of resected ileum. The changes in intestinal microbiota that were observed in ileal CD may have occurred early in the pathogenic process before overt disease was manifest. Alternatively, chronic enteric dysbiosis that arises as a consequence of pathologic inflammation at one location within the GI tract may be propagated to unaffected sites. The observed alterations in ileal microbiota could have an impact on regional inflammation or metabolic properties (e.g., butyrate metabolism), however the functional implications of these shifts remain to be determined.
NOD2 genotype was also selected albeit with more modest effect for all three 16S rRNA sequence datasets and for targeted qPCR assays of the C. coccoides- E. rectales
group. The bacterial 16S rRNA sequences assayed by the C. coccoides – E. rectales
qPCR assay likely overlap with some but not all the 16S rRNA sequences included in Group XIVa and IV. The observation that ileal CD phenotype and NOD2 risk alleles, which presumably contribute to this phenotype, had opposite effects on this microbial group, suggests that the effect of the NOD2 risk allele on the relative frequency of the C. coccoides-E. rectales
group is not simply mediated through an association with ileal CD phenotype. Thus the results of targeted QPCR assays and 16S rRNA sequence analysis both demonstrate a significant effect of NOD2 genotype on ileum associated microbial composition. Our results in disease –unaffected ileal tissues support our previous analysis of an independent set of intestinal tissues that were more heterogeneous with respect to anatomic location and inflammation. Our findings are consistent with the report that the relative frequency of Firmicutes
spp., as determined by targeted qPCR, is higher in NOD2 knockout mice than in wild type mice 
. However, it is important to note that the patient-based studies are not directly comparable to the mouse studies because 1) the NOD2 knockout mouse may differ phenotypically from the NOD2Leu1007fs knock-in mouse 
, and 2) only 4% of the ileal CD patients compared to 28% of colitis and 23% of control non-IBD patients, were homozygous for the ATG16L1 T300A nonrisk allele (ATG16L1NR/NR
). These findings are also consistent with the recent report that the relative frequency of Firmicutes measured by targeted qPCR is higher in three CD patients that were homozygous for the Leu1007fs (SNP13) compared to 11 CD patients that were homozygous for the wild type allele 
. Since only two of the ileal CD patients were homozygous for the Leu1007fs allele in our dataset, a parallel comparison could not be made (see Table S6
infections, particularly recurrent C. difficile
infections have been associated with altered fecal microbial composition 
. Patients with inflammatory bowel diseases are more likely to develop C. difficile
infections, which are also associated with clinical exacerbation of their disease 
. It is possible that the shifts in microbial composition associated with IBD contribute to these patients’ susceptibility to C. difficile
and other infections that may exacerbate inflammation. Alternatively antibiotic treatment of subjects with C. difficile
could contribute to the observed shifts in microbial composition 
Sanger sequencing of the entire 16S rRNA gene permits accurate phylogenetic identification of bacteria, whereas 454 pyrosequencing generates much greater depth of coverage, but with lesser phylogenetic resolution. While the results of all three sequencing methodologies demonstrated associations of disease phenotype, C. difficile
infection and NOD2 genotype to the overall microbial composition in parallel analyses, they differed with respect to the main effects and first order interactions associated with individual phyla/subphyla categories. Methodological differences may reflect biases introduced during the initial PCR amplification step, as noted by Olsen and coworkers for the 27F forward primer 
. In addition, there are likely differences in the phylogenetic resolution/assignment of the complete 16S sequence as opposed to different hypervariable regions (V1–V3 and V3–V5) of the gene sequence. Although the results from each sequencing method may converge with increasing the sample size, differences in the microbial composition data generated by different sequencing methods will make it challenging to compare results from studies using different primers for the initial PCR amplification.
The F. prausnitzii
16 S rRNA sequences assayed by targeted qPCR form a major subset of all the sequences grouped within the Firmicutes.Clostridium Group IV clade. The selection of smoking as a potentially significant covariate is intriguing, since smoking has been associated with ileal CD phenotype and with early postoperative recurrence in ileal CD patients, and is consistent with the observations of Sokol and coworkers that low ileal mucosal concentrations of F. prausnitzii
is associated with early postoperative ileocolonoscopic recurrence of CD 
. Smoking was also selected by analysis of the Sanger and 454V1–V3 datasets as significantly associated with shifts in relative frequency of the Firmicutes.Clostridium Group IV clade (Table S4
). Smoking cessation has clearly been linked to altering the subgingival microbial profile 
, but has not been previously linked to altering the ileum associated microbial profile.
Although pathologic review of the resected tissues provided rigorous phenotyping of the samples, the use of surgically resected tissues may bias the results by sampling of IBD patients with relatively severe disease who have been treated for various lengths of time with antibiotics and different IBD medications. Although the focus of this study was ileal CD, it is likely that other human diseases will exhibit similar links between genetically determined defects in mucosal immunity and alterations of resident microbiota. As we further expand these datasets by analyzing more samples, we anticipate that further associations between microbial composition, IBD subphenotypes, IBD polymorphisms and environmental factors will emerge.