This study, using TTGE to study diversity of C. leptum group in IBD patients, provides important information regarding the colonic microbiota in IBD that will augment the existing knowledge in this area. The study shows that there was a reduction of absolute number as well as species number per individual of the C. leptum group in both UC and CD. The reduction in numbers was explained to a large extent by the reduction in F. prausnitzii, and this alteration was present in both quiescent and active IBD. C. leptum group bacteria from IBD patients clustered separately from bacteria present in the healthy controls. Lastly the study provides, for the first time, a catalog of the C. leptum group of bacteria in the feces of healthy adult humans in India. The present study focused solely on the C. leptum group and F. prausnitzii and did not attempt to determine changes in other microbial communities in IBD.
The utilization of culture-independent methods of analysis has significantly enhanced our ability to assess the contribution made by the gut microbiota to human health. In this study, we amplified portions of the 16S rRNA gene that are specific for the C. leptum group of bacteria and used TTGE to identify the numbers of species present in in each individual. TTGE is a very sensitive technique to detect single nucleotide changes in DNA. The primer set used is able to detect species of the C. leptum group including C. leptum, Eubacterium plautii, Clostridium viride, and Ruminococcus albus strains which are present in small proportions in human fecal samples . TTGE revealed multiple bands, which were cloned and sequenced to reveal at least 25 different species or OTU belonging to this group. The clone library that we generated, as well as the real time PCR data, showed that F. prausnitzii was the major constituent (approximately 65%) of this bacterial group. The presence of Subdoligranulum variabile as a part of this bacterial group in our participants is consistent with other reports .
Healthy controls in the present study had a mean of 12 OTUs of the C. leptum group in feces, and this number was significantly reduced in CD and UC patients. However, using the Simpson D index, we did not find any alteration in alpha diversity between HC, UC and CD. Manichanh et al.  reported that the species number of C. leptum, C. coccoides, and certain other Firmicutes was reduced in patients with CD. Their conclusions were based on the analysis of a limited number of clones derived from fecal microbiota of these patients. Other investigators have reported that C. leptum diversity was reduced in patients with CD . Principal components analysis showed that C. leptum from most of the control subjects clustered discretely to one side whereas C. leptum from CD and UC patients clustered separately. This observation needs to be confirmed in more rigorous studies.
Relative abundance of the targeted microbial communities was evaluated using real time PCR in which the specific abundance was expressed relative to amplification of universal bacterial domain sequences of 16S rRNA, i.e. expressed relative to total bacteria. The abundance of C. leptum group bacteria, and of F. prausnitzii, was significantly reduced in both CD and UC compared to HC. Similar findings have been reported earlier [14, 21]. In the present study, there was no significant difference in fecal counts of either C. leptum group or F. prausnitzii between patients with active IBD and those with quiescent IBD (i.e. those who were in remission). There is little literature on this aspect; however one earlier study suggested that the differences in abundance of C. leptum and F. prausnitzii occurred in active IBD but not in IBD in remission .
The significance of these findings can be interpreted varyingly. C. leptum group (cluster IV) is one of the dominant populations of the human fecal microflora [3, 22] and contains a large number of butyrate-producing bacteria. It is not known whether the changes in C. leptum number and diversity antedate the development of IBD. However, by showing that changes occur in the same direction in both CD and UC, we can conclude that these changes are not specific to either disease. The alterations were of greater magnitude in CD patients compared to UC patients. We do not have an explanation for this observation. It was also noted in our study that the changes were noted in both active and quiescent IBD, suggesting that the changes were not secondary to gut inflammation and ulceration and presence of pus and blood in the lumen. Probiotics used in IBD are typically a mixture of lactobacilli and bifidobacteria. Species belonging to the C. leptum group (esp. F. prausnitzii) are also now considered as anti-inflammatory commensal bacteria. Loss of butyrate, which has an anti-inflammatory activity , may result in greater inflammation in the colon.
Our data shows decreased diversity of the C. leptum group in IBD patients. Increased species diversity is an indication of a robust microsystem. Loss of this diversity is likely to impact on gut health, even if it is not the primary phenomenon driving gut inflammation. The intake of prebiotic substrates like inulin can increase both bifidobacteria and F. prausnitzii in the human gut, and this may be one possible intervention to consider in the management of patients with IBD . It is possible that treatment of IBD patients with butyrate producing bacteria such as the C. leptum group bacteria will emerge as another therapeutic option in these patients .