The mucosa is the anatomical site at which the host first encounters gut microorganisms 
, and it plays a key role in intestinal homeostasis 
. We inferred that microorganisms that are associated with the colonic mucosa would play a significant role in the modulation of host health, and are worthy of a closer examination. To date, our understanding of mucosal-associated microbiota has been derived primarily from culture-based analysis as well as from earlier studies utilizing microbial profiling methods (e.g., DGGE and T-RFLP) 
. A limited number of deep sequencing studies have been conducted to elucidate the phylogenetic identities of colonic mucosal-associated microbes, in particular, the microbiota from healthy individuals (). This study therefore utilized 16S rRNA-based pyrosequencing technology to provide a comprehensive insight to the baseline microbial community that is closely associated with the colonic mucosa.
List of selected publications that utilized sequencing to examine mucosal-associated microbiota in healthy individuals.
It is interesting to note that two of the five individuals in this study were spousal partners living in the same household. Yet, the mucosal-associated microbiota in both individuals clustered apart on the MDS, indicating that each host harbors a unique mucosal-associated microbiota and that the host effect is a significant confounder on the gut microbiota. Our data demonstrate that despite interindividual differences, the five individuals harbored consistently high predominance of Firmicutes (50.9%) and Bacteroidetes (40.2%), and the abundances of both phyla closely approximate findings from previous studies ( and ). Closer examination at a finer taxonomical resolution however revealed slight differences in the abundance of bacterial groups that were ubiquitously observed in the mucosa. To illustrate, this study showed a high abundance of Bacteroides spp. and lactic-acid bacteria (LAB) including Leuconostoc spp., Weissella spp. and Lactococcus spp. among the five individuals. In contrast, previous studies reported Bacteroides spp. and microbial groups belonging to Ruminococcaceae and Lachnospiraceae to be more commonly found in mucosa-associated microbiota ( and ). Besides interindividual differences, the variations in our findings may also be explained by slight differences in experimental technicalities such as sequencing depth and DNA extraction protocols.
List of selected publications that utilized sequencing to examine mucosal-associated microbiota in diseased individuals.
Regardless, because of their high abundance and persistence in the five individuals, Bacteroides
spp. and LAB are postulated to form the keystone groups in the breakdown of carbohydrates to provide to the metabolic needs of these five individuals. In the human intestinal tract, Bacteroides
spp. and related species hydrolyze complex dietary polysaccharides 
, and can also degrade endogenous secretions such as exfoliated epithelial cells and mucus fragments as well as residual dietary polysaccharides in the distal colon and rectum 
. Their versatility in utilizing a broad array of polysaccharides may account for their predominance, and possibly, their role in sustaining the presence of other microbes in the intestinal tract. In contrast, Leuconostoc
, primarily utilize readily fermentable sugars (e.g., hexoses and pentoses) to produce lactic acid 
. Given the relatively low abundance of such simple carbohydrates in the distal colon and rectum, the provision of substrates for LAB may depend on the degradation of complex polysaccharides by Bacteroides
spp. The utilization of different substrates and the resulting micro-niches may explain the inverse relationship in the abundance of Bacteroides
and LAB that was observed in the biopsy samples in close proximity to each other (Figure S4
Our findings further indicate that some individuals exhibited a certain extent of micro-heterogeneity, both along the colon (i.e., 45% similarity for individual E) and in between locations that were 1 cm apart (i.e., on average 53% similarity for biopsy duplicates that differed in individual A, C and D). Numerous factors possibly account for the presence of micro-heterogeneity in the mucosal-associated microbiota. For example, diverticulosis in the right colon of individual E () may have perturbed the localized microbial populations, and resulted in the relatively higher micro-heterogeneity along the colon for this individual. Alternatively, host genetics and dietary preferences are also major factors influencing the differentiation of gut microbiota 
. In addition, mucins (i.e., neutral mucins and acidomucins) are a source of endogenous substrates for the mucosal-associated bacterial fermentation, and interindividual differences in the mucin content may therefore be a factor to explain for differences in gut microbiota 
. Among the mucins, acidomucins are the predominant mucin type in the colon 
. The presence of sulfate and sialic acids on the carbohydrate chains of acidomucins result in higher viscosity and acidity that may be resistant to mucosal-associated bacterial fermentation 
. We inferred that varying concentration of acidomucins in an individual can in turn lead to differences in the bioavailability of endogenous substrates, which may correlate to the micro-heterogeneity of colonic mucosal-associated microbiota.
To address this inference, we observed localized differences in the abundance of acidomucin-positive goblet cells, along and within the colon of an individual. We further found that the abundance of acidomucin in an individual is weakly correlated with the mucosal-associated microbiota (Mantel correlation
0.04). Although weak, this correlation indicates that the micro-heterogeneity in mucosal-associated microbiota may relate, in part, to localized variation in the biochemical environment associated with the duplicate biopsies. This observation is in agreement with published findings, which reported that resident mucolytic bacteria may differ among individuals according to the specific carbohydrate composition of intestinal mucins 
. In this study, the weak correlation and relatively small sample size did not permit a conclusive identification of the bacterial groups that were associated with variations in the acidomucins abundance. However, past studies have found that sulfate-reducing bacteria (e.g., Desulfotomaculum
spp. and Desulfobacter
spp.) were more abundant in gastrointestinal sites with greater numbers of sulfomucin-containing goblet cells 
. In addition, Bacteroides fragilis
which can cleave sulfate from sulfomucin and in turn utilize the remaining desulfated mucins as carbon and energy sources, is also likely to correlate to the abundance of acidomucins 
. Future studies involving a larger sample size would have to be conducted to verify the occurrence of micro-heterogeneity in colonic mucosa, as well as the potential correlation of microbial micro-heterogeneity to localized biochemical characteristics.
In recent years, comparative studies which aim to evaluate for the role of gut microbiota in relation to gastrointestinal diseases are increasingly common. One of the common approaches to addressing this aim is to examine differences in the gut microbiota of diseased and adjacent healthy mucosal samples. To access the mucosal samples, it is necessary to perform bowel cleansing prior colonoscopy, particularly when sampling the right and transverse colon. The bowel cleansing preparation may have altered the loosely adherent microbiota and the lumen microbiota, and in turn led to underestimation of the diversity or abundance of the examined microbial communities. However, standardized cleansing methodologies were used for collection of all biopsy samples, hence validating the relative differences observed in this study. Our findings revealed that certain individuals have micro-heterogeneity in their gut microbiota at localized colonic sites, and this micro-heterogeneity may potentially confound subsequent comparative analysis. To overcome these issues, it is likely that multiple biopsy duplicate pairs would have to be sampled at each particular colonic site, alongside the stool samples from the same individual, to better evaluate the extent to which dysbiosis affects host health status.
In summary, our examination with 16S rRNA-based pyrosequencing provided insight into the mucosal-associated microbiota in five healthy individuals. The data further emphasize that each individual is unique in their abundance of particular microbial taxa, and that micro-heterogeneity is in part due to localized biochemical differences, which may exist in some host individuals.