Gut microbiota in the early stage of Crohn’s disease has unique characteristics

The balance between beneficial gut commensals and pathogens is crucial to human health. The dysbiosis of gut microbiota contributes to gut inflammation and may be closely related to the onset and progression of CD [4, 5, 7]. However, our understanding of the relationship between gut microbiota and CD is still relatively poor as it is difficult to detect and diagnose CD in the biologic onset or early stage of disease (e.g., preclinical) [5, 25]. In the present study, we explored the characteristics of gut microbiota changes in the early CD of Paris’s definition and advanced CD [22], and found that significant gut microbiota dysbiosis with unique bacterial biomarkers and metabolic pathway changes in the early stage of Chinese CD patients. The observed dysbiosis was associated with disease progression.

Our study revealed that the dysbiosis in early CD mainly manifested in the following three aspects: first, compared with the health subjects, the fecal microflora community abundance in patients with early CD decreased, however, the observed difference was not statistically significant, while that of patients with advanced CD showed a significant decrease in microbial diversity. Secondly, PCoA results could distinguish the bacterial community structure of patients with early CD from that of the health subjects with statistically significant differences. Finally, the abundance and structure of microbiomes of early and advanced CD were similar. Previous studies have shown that fecal microbiota diversity in western and Chinese patients with CD decreased compared with health subjects [19, 20, 26]. However, the gut microbiota of patients with inactive CD and those with active CD were similar in structure and could not be distinguished by PCoA [26]. Furthermore, altered microbiome composition and stability in CD were not associated with disease activity or long-term course [19]. These inconsistencies may be due to the differences in study design, disease stage, drug use, diet, etc., but the reasons for those conditions are not fully understood, and further research is needed.

We detected detailed compositional alterations in the fecal microbiota of patients with early-stage CD at different taxonomic levels and found a significant reduction in multiple short-chain fatty acid (SCFA)-producing bacteria, including Blautia, Clostridium IV, Coprococcus, Dorea, Fusicatenibacter, and a significant increase in Escherichia/Shigella, and Proteus. This trend of gut microbiota imbalance was more obvious in the advanced stage of CD, indicating that gut microbiota imbalance was closely related to the progression of CD inflammation. Although these results were similar to those of previous studies [14, 26,27,28], these studies do not reflect the role of some key microbiota (e.g., Parabacteroides, Escherichia/Shigella) in the early course and progression of CD. The present study overcame this deficiency and provides clues for further researches on the mechanism of gut microbiota–host interaction in CD patients.

In the present study, we found that Bacteroides increased in CD patients and were mainly enriched in early CD, which could cause opportunistic infections when immune dysfunction or intestinal flora was imbalanced. Previous studies indicated that the changes in the abundance of Bacteroides in CD patients were controversial. The increased abundance of Bacteroides in the CD group compared with health participates was related to the maintenance of postoperative remission [4, 29]. However, other studies provided that the abundance of Bacteroides in CD patients was reduced [27, 30]; B. thetaiotaomicron, belonging to the Bacteroides was found to prevent weight loss, colonic histopathological changes and the production of inflammatory factors in a mouse enteritis model induced by dextran sodium sulfate (DSS) [31]. Therefore, more research is needed to explore and explain the potential role of Bacteroides changes in CD patients, especially in the early stages.

In the present study, Parabacteroides were enriched in CD patients, especially patients with early CD. This result was consistent with previous studies, which have shown that Parabacteroides had an important role in the pathogenesis of intestinal inflammation, and the decreased abundance of Parabacteroides in CD patients was related to the reduction of inflammation [12, 32, 33]. Some studies have shown that certain Parabacteroides may aggravate the inflammatory response [34,35,36]. Oral administration of P. distasonis to mice with DSS-induced colitis could significantly aggravate the inflammation [35]. The strain of Parabacteroides distasonis, known as CAVFT-HAR46, isolated from microlesions of cavernous fistulas in the intestinal wall of patients with CD, may be the potential pathogenic cause of CD [35]. In addition, Parabacteroides had an important role in the pathogenesis of intestinal inflammation; the number of Parabacteroides in children with CD under high-level pressure stress was found to increases significantly, suggesting they were related to CD inflammation [36]. However, it remained unclear whether the changes in the abundance of Parabacteroides were the cause or the result of intensified or reduced intestinal inflammation.

In addition, our results showed that Enterococcus were also enriched in the feces of CD patients, indicating it may be play a key role in the disease course of CD. Enterococcus, which can produce extracellular peroxide and damage the DNA of mucosal epithelial cells, is an opportunistic human bacterial pathogen [37]. Previous studies have shown that the abundance of Enterococcus in children with CD was significantly increased [38, 39], which was closely related to postoperative recurrence in adult CD patients and was conducive to the fermentation of proteolysis and lactic acid production, while the proteolytic flora was associated with the accumulation of end products known to be toxic to colon cells [40]. In addition, the abundance of Enterococcus faecalis in the faeces of CD patients was related to the location of the lesion. Usually, the abundance of Enterococcus faecalis in patients with ileal CD is higher than that in patients with ileocolon type [20]. Therefore, we hypothesized that Enterococcus may be involved in impairing the intestinal mucosal barrier at the early stage of CD through its secretion or its metabolites, promoting the occurrence of inflammation.

Noteworthy, Escherichia/Shigella and Proteus were enriched in patients with advanced CD compared with the early patients in the present study, suggesting that these bacteria may be the key factors in the progression of the disease. Escherichia/Shigella, a gram-negative bacillus, which could spread between intestinal mucosal cells and eventually colonize, protecting itself from the destruction of innate immunity in the intestine, could cause inflammatory destruction of the intestinal epithelial barrier and lead to the apoptosis of macrophage [20, 41, 42]. Previous studies [20, 27, 43, 44] have also found that Escherichia was enriched in CD patients, and some Escherichia coli strains with adhesion and invasiveness increased in the ileal mucosa of CD patients, which indicated that Escherichia was related to the pathogenesis of CD. Moreover, a cross-sectional study of a large sample showed that Escherichia could be used as a landmark to distinguish CD from non-CD [20]. Other studies also found that Proteus was related to the severity of colitis in mice [45]. Therefore, Escherichia/Shigella and Proteus have great significance for understanding the progression and prognosis of CD; however, further research is needed. Our research finding and the above reports indicated that opportunistic pathogens such as Parabacteroides, Bacteroides, Enterococcus, Escherichia/Shigella and Proteus, which dynamically which changed in the natural course of CD, might play a key role in the occurrence and development of CD. The opportunistic pathogens are expected to become the microbial biomarkers for early CD diagnosis.

The gut microbiota in the early course of CD is characterized not only by the increased opportunistic pathogens but also by the decreased abundance of some common beneficial bacteria, such as SCFA-producing bacteria, which is consistent with the results of previous studies [46]. However, the present study also confirmed that the dynamic reduction of beneficial bacteria in gut microbiota was related to the process of intestinal inflammation and could be used as the potential marker to predict the evolution of CD. The abundance of Firmicutes in early CD patients decreased significantly compared with the controls, mainly due to the significant decrease in genera Coprococcus, Ruminococcus 2, Butyricicoccus, Dorea, Clostridium IV, etc. This decreasing trend was even more pronounced in patients with advanced CD, especially for Roseburia, Lachnospiracea_incertae_sedis, Fusobacterium, Blautia, and Anaerostipes, which in advanced CD decreased more significantly compared with patients in early CDs. Previous studies have shown that these bacteria had an important role in maintaining the dynamic balance of intestinal mucosal immune regulation through their metabolites [7, 10, 47]. Coprococcus not only produces butyrate but is also related to the dopamine metabolic pathway, and dopamine is a key brain signal in the pathogenesis of depression, which may explain the higher depression status in CD patients compared to normal people [48]. Moreover, recent studies have pointed to changes in gut microbiota composition and host processing of bacterial-derived metabolites associated with CD, particularly a reduction in the taxa of Roseburia, Lachnospiraceae, and Ruminococcus 2, etc. [49]. We also found the dynamics of characteristic flora using Spearman correlation heat maps, and our results showed that there was some unknown interaction between these characteristic microbiotas, but further studies are needed to confirm this interaction mode of microbiota in order to deeply understand the dynamic changes of intestinal microbiota in CD patients, and to provide a basis for the regulation of intestinal microbiota. Moreover, these characteristic bacteria were correlated with CRP, FC and ESR etc., which reflect the inflammatory state of CD, further suggesting that the gut microbiota dysbiosis may have cascade amplification in CD, thereby promoting the progression of CD and providing clues for exploring the application of existing CD-related biomarkers and intestinal micro-flora in the early diagnosis of CD in future. Taken together, our results further improve insights into the mechanism of gut microbiota in CD, considering that gut microbiota dysbiosis may be more reversible in the early stage of CD than in the progressed patients. Therefore, targeting intestinal microbiota in the early stage of CD may be more meaningful.

Although many bacteria and metabolites associated with CD have been identified, understanding the mechanisms through which microbes influence the occurrence and development of CD requires an extension from association to causation, and the functional analysis of gut microbiota provides a perspective. Functional analysis which has unique significance in the differentiation between groups is often more important than species composition analysis in biological value. PICRUSt (Phylogenetic Investigation of Communities by reconstruction of unobserved States) studies community phyloevolution through recessive state reconstruction, the software predicts metagenomic functional composition based on 16S rDNA and reference sequence databases [23, 24]. By comparing the results of functional analysis of metagenomic sequencing data and corresponding 16S predicted functional analysis, it is found that the accuracy of this method is 84–95%, and the functional analysis of intestinal microbiota and soil microbiota is close to 95%, which can greatly reflect the functional gene composition in the sample [24]. Our PICRUST results suggested that an imbalance in gut microbiota was involved in the progression of CD by changing their gene function, which could provide important reference information for the study of downstream microbiota interaction/response mechanism.

There were some limitations in this study. First, this was a cross-sectional study, but it was nested within a longitudinal prospective Chinese CD cohort [50], which to some extent eliminates possible confounding factors to further understand and validate the dynamic changes of gut microbiota during natural CD processes, and to develop suitable microbial biomarkers for early diagnosis. However, it should be pointed out that the Paris’s definition of early CD is a disease stage defined to optimize treatment strategies [22], which can only partly reflect the early stage of the disease course. Therefore, it is necessary to capture CD patients at an earlier stage or preclinical or even biological stage to truly reveal the natural history of CD through prospective follow-up cohort studies, and establish a biobank to disclose the dynamic changes and roles of gut microbiota in the pathogenesis and disease progression of CD. Second, the sample size of the current study was relatively small, further large-sample multi-omics studies were needed to clarify the potential mechanisms and pathways of gut microbiota imbalance involved in the occurrence and development of CD in order to provide a basis for updating the prevention and treatment strategies of CD. Third, this study only analyzed the fecal flora which was similar to the mucosal associated flora of patients with CD [8]. More tests for analyzing the intestinal mucosal associated flora are needed to further elucidate the role of gut microbiota in the biologic onset or early clinical stage of CD. Furthermore, despite the widespread use of the 16s rDNA sequencing method, it was often challenged that use of different sequence processing pipelines may bring ambiguous results, such as different alignment methods, OTU binning procedures, different kits used to extract DNA, the 16S rDNA regions amplified, and reference databases, etc. [51]. Recently, discussions of standards and pipelines have helped researchers improve the data quality, such as the standard for human fecal sample processing [52], normalization strategies for data characteristics [53] and the future use of metagenomic sequencing as a replacement. In this study, we have used the state-of-the-art pipeline to ensure the reliable interpretation of the 16S sequencing data, and we also look forward to more remarkable finding in our future studies with metagenomic sequencing and integrated multi-omics strategies.

Despite these limitations, the present study demonstrated that the progressive consumption of SCFAs producing bacteria during the course of CD significantly affects the functional metabolism, which is not conducive to the maintenance of intestinal epithelial integrity and the regulation of inflammatory response, and may be a potential factor of etiology in the early stage of CD. In addition, the progressive rise of opportunistic pathogens exacerbates gut dysbiosis during CD, which in turn affects disease progression.

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