Despite the reduction of beneficial microbes, expansion of pathobionts, and the reduced microbial diversity, which all indicate IBD, the dysbiosis remains inconsistent, and the composition of microbiome varies.[23] The reason for variation may be because of heterogeneity in the sampling method and the disease status of the subject.
In this study, although lavage fluid and stool showed some correlation, all three samples (i.e., lavage, stool, and biopsy samples) were significantly different from each other. The results are consistent with those in previous studies. The microbial communities of multiple biopsy sites within the mouth, stomach, duodenum, and colon were largely distinct from those in stool.[4, 24, 25] In addition, the microbiome variation by colonic biopsy between different colonic regions showed the significant effect by sampling site on the abundance of bacterial taxa.[26] In our study, the biopsy and lavage samples revealed that Firmicutes and Bacteroidetes were less abundant, with the concomitant increase of Proteobacteria and fusobacteria. A study participated by Korean patients with CD showed a similar distribution to our study.[27] Thus, biopsy reflects mucosa-associated microbiome, whereas the stool reflects the luminal microbiota in the distal large intestine.
Luminal contents after bowel preparation provide the closest microbiome of the biopsy sample.[9] Colonic lavage samples also provide a relatively accurate representation of biopsy microbiome composition.[10] Bowel preparation appears sufficient to cleanse the lumen to ensure that the luminal contents sampled at colonoscopy—being derived from mucus-adherent microbes rather than the bulk stool stream—contain a microbial community more similar to that of the biopsy samples. The stool material obtained during colonoscopy could be more representative of the mucosal layer. Hence, a noninvasive lavage analysis would be a good alternative method to biopsy. However, in this study, biopsy and lavage samples had no association. Five biopsy samples were not analyzed in this study because of low read counts. The reason is that biopsy procedure is too invasive to acquire enough amount of tissue for microbiome analysis. Furthermore, bowel cleansing washed the entire intestine; thus, the post-bowel preparation fluid could contain loosely attached mucosa-associated microbiome and the whole part of luminal microbiome from the mouth to the colon. The lavage microbes would originate from the whole intestine, oral cavity, and respiratory tract; therefore, colonic lavage samples may have some variances. Meanwhile, another method using local lavage and brush technique for each intestinal part has been reported but still requires validation.[11, 12, 28]
Most of the studies analyzing the mucosa-associated microbiome performed biopsy sampling after bowel cleansing. In general, colonoscopy preparations can result in multiple negative effects on the intestinal microbiome. Standard bowel preparation can alter the diversity of mucosa-associated microbiota.[29] At the class level, Proteobacteria and Coriobacteria increase, whereas Clostridia significantly decrease after colonoscopy.[30] In normal individuals, a high-volume PEG bowel cleansing preparation has a long-lasting effect on the gut microbiota composition and homeostasis. The short-term effects of bowel preparation may be because most of the bacteria are being washed out in a nondiscriminatory manner, reducing the low-abundance taxonomic groups to levels below detection; on average, bowel preparation with PEG can reduce bacterial load.[7] In fresh stool samples and distal colonic mucosal biopsies collected from 24 healthy subjects before and during a flexible sigmoidoscopy of an unprepared bowel, stool samples obtained a significantly higher diversity than the mucosal samples.[31] More endoscopic studies of an unprepared bowel would be necessary to validate the biopsy method.
The stool samples showed significant differences in diversity between UC and healthy controls. Conversely, the microorganism diversity examination in the colonic lavage and biopsy samples had no significant difference between these two groups. However, the sample size in this study is small to acquire accurate verification. Therefore, we cannot conclude that lavage does not differ between the two groups, considering that the association between intestinal microorganisms and metadata does not consistently appear as a change in the overall population. The change of one or some combinations of specific microorganisms may be a feature that distinguishes UC, and in this case, it can be confirmed by modeling such as machine learning. In this study, stool and lavage predicted UC, obtaining the AUC values of 0.85 and 0.81, respectively, for accuracy. Considering that the accuracy of the model is relatively high (AUC > 0.8), lavage also has a diagnostic potential to verify UC. In another study using biopsy and stool, terminal ileum biopsies performed best (AUC = 0.85), closely matched by the rectum biopsies (AUC = 0.78). The classifier based on the stool samples collected during the diagnosis performed less satisfactorily (AUC = 0.66), with low consistency.[6] Thus, microbiome analysis using colonic lavage can distinguish diseases.
Moreover, facultative anaerobes such as Enterococcus and Lactobacillus are UC-related biomarkers in the case of stool. This result correlates with the shift of bacterial communities from obligate to facultative anaerobes, strongly suggesting a disruption in anaerobiosis and pointing to a potential role for oxygen in intestinal dysbiosis.[32] Decreased influence of obligate anaerobes and increased influence of facultative anaerobes and some aerobic bacterial communities generally happen in UC.[33] In addition, Bifidobacterium and the Lactobacillus group were increased in patients with active IBD.[34] Generally, in subjects with infectious diarrhea, Proteobacteria and Enterobacteriaceae increase and lactobacilli decrease.[35] In the case of lavage, Staphylococcus and Leuconostocaceae are the disease markers. Staphylococcus could be one of the candidates involved in IBD pathogenesis. Staphylococcus aureus can be detected in a UC-affected colon.[36] The species of the Enterobacteriaceae family only increased in patients with CD; examples were Escherichia/Shigella species, which commonly invade the gut mucosal epithelium, causing bloody diarrhea and colonic ulceration.[37] The stool bacteria from patients with UC could cause stronger inflammatory responses than those from healthy controls.[38]
A typical gut microbiota in patients with IBD is characterized by a decrease in stool bacteria such as Firmicutes and Bacteroidetes and an increase in Proteobacteria. However, this finding was not significant in this study. Enrolled patients had inactive UC. Hence, their gut microbiome tended to be similar to that of healthy individuals, suggesting that the stool microbiota has different roles in UC pathophysiology.[39]
Meanwhile, this study has several limitations. First, the lavage procedure was not performed at each local intestinal part. Some lavage fluid in the sigmoid colon may not represent the whole intestine microbiome status. Nonetheless, the colonic lavage sample can show the whole gastrointestinal mucosa-associated microbiome status. Second, our sample size is small; we only enrolled a limited number to extensively analyze each stool, lavage, and biopsy sample. Third, our study did not demonstrate longitudinal microbiome variation between patients with UC and the healthy control. Hence, further studies with more subjects and longitudinal samplings are necessary.