In the present study, we comprehensively described the intestinal bacterial and fungal dysbiosis in type 2 SBS rats with ileocecal resection, and found that GLP-2 treatment could partially attenuate the disturbance of intestinal microbiota. Although our previous studies have disclosed severe intestinal bacterial dysbiosis in animal models and patients with ileocecal resection [2, 7], there is still no comprehensive assessment of intestinal fungal dysbiosis accompanied by intestinal bacterial dysbiosis in SBS. Due to the interkingdom interaction between bacteria and fungi, intestinal bacterial dysbiosis is likely to cause intestinal fungal dysbiosis, which may further aggravate the intestinal bacterial dysbiosis, and even exacerbate the intestinal inflammation and thus worsen the prognosis of the disease [19–21]. Limited by previous technology, it was difficult for traditional culture-dependent methods to achieve in-depth understanding of the fungal microbiota. Recently, high-throughput sequencing provides effective technical support for study of intestinal fungi in various diseases [20, 22]. Herein, we used high-throughput ITS sequencing to comprehensively analyze intestinal fungal dysbiosis in type 2 SBS rats. Our previous study found that GLP-2 could promote intestinal mucosal proliferation . However, whether GLP-2 could ameliorate the intestinal microbiota dysbiosis of SBS has not been studied. Yu Hu and colleagues  demonstrated that GLP-2 treatment resulted in the increase of several probiotics and the decrease of some pathogenic bacterial genera in intestine of aged rats. Therefore, we performed this study to investigate the colonic microbiota of SBS rats with or without GLP-2 intervention.
We first characterized the altered composition of intestinal bacterial dysbiosis in SBS rats and evaluated the therapeutic effects of GLP-2 on intestinal bacteria dysbiosis. Significant decrease of OTU counts and α diversity were observed in the colonic contents of SBS and GLP2.SBS rats which revealed that there was significant intestinal bacterial dysbiosis in type 2 SBS rats. Massive resection of small intestine and ileocecal resection lead to fast pass of food, gastric acid and bile acid, which exerts deleterious effect on the luminal microenvironment. This will inevitably affect the living environment of intestinal microbiota, thus leading to intestinal microbiota dysbiosis, presented as reduction of probiotics and an increase of some pathogenic bacteria . Although GLP-2 possess proliferative and anti-inflammatory effects on intestinal mucosa, no significant improvement in OTU counts and α-diversity was found in GLP-2 treated SBS rats. Both PLS-DA and PCoA strongly indicated distinguished bacterial communities between Sham and SBS group, while clusters of SBS and GLP2.SBS group were close to each other, indicating similar bacterial communities. Moreover, notable differences in β-diversity based on weighted UniFrac analysis and Anosim tests suggested that the variation of microbial community contains not only changes in quantity of bacterial species but also changes in specific bacterial abundance . These results demonstrated that massive resection of small intestine and ileocecal resection could severely disrupt normal intestinal bacterial microbiome, while GLP-2 treatment could not restore the intestinal bacterial diversity of SBS rats.
Detailed analysis of the composition and structure of intestinal bacteria in SBS demonstrated that, in comparison with Sham group, the relative abundance of 9 out of 11 phyla changed significantly, suggesting a thorough dysbiosis of intestinal bacteria in SBS rats. In line with previous studies, there existed a dramatic overabundance of Proteobacteria and its family Enterobacteriaceae in colonic contents of SBS rats, which contains a great number of pathogenic bacteria [2, 25, 26].
Proteus, an opportunistic pathogen within the Enterobacteriaceae family, was remarkably prevalent in SBS group in our study. Previous studies have proved Proteus’ potential ability to produce LPS results in the upregulated secretion of pro-inflammatory cytokines and further leads to the destruction of intestinal mucosal barrier and thus inducing bacterial translocation [2, 27]. A remarkable alteration of the Firmicutes phylum in SBS and GLP2.SBS rats was the decrease of the Lachnospiraceae and Ruminococcaceae families, which are able to produce short-chain fatty acids (SCFAs). SCFAs, important metabolites of bacteria, exerted many important biological functions, such as energy-supplying fuel for intestinal epithelial cells, inducing enterocyte proliferation and differentiation, and promoting the production of antimicrobial peptides by epithelial cells [2, 28, 29]. Therefore, deficiency of Lachnospiraceae and Ruminococcaceae families may have adverse effects on intestinal adaptation of SBS. In line with previous research, the relative abundance of Akkermansia genus from Verrucomicrobia phylum decreased significantly and even disappeared completely in SBS rats . Akkermansia is a newly discovered star probiotics, which has a variety of beneficial biological functions, such as anti-tumor effect, intestinal mucosal barrier protection, and alleviating colonic inflammation [30–32]. Yu Xu and colleagues summarized that Akkermansia could protect the intestinal mucosal barrier and inhibit the translocation of intestinal flora, therefore reducing the level of LPS in circulation and thus inhibiting the inflammatory response. . The almost complete loss of Akkermansia in the colon caused by SBS might affect the integrity of the intestinal mucosal barrier of SBS, lead to bacterial translocation and aggravate the systemic inflammatory response, which may have adverse effects on the intestinal adaptation of SBS.
However, effective therapies for intestinal bacterial dysbiosis are still lacking. The first drug we thought of and used was antibiotics. However, a previous study had found that several days of antibiotics may lead to a reduction of potentially anti-inflammatory Clostridia and an increase of pro-inflammatory Proteobacteria . According to previous studies, GLP-2 may alleviate intestinal microbiota dysbiosis . As we expected, GLP-2 treatment significantly downregulated the relative abundance of pro-inflammatory Proteus genus in SBS rats, and increased the relative abundance of potentially anti-inflammatory inhibitory Clostridium genus from Clostridia class in SBS rats. Notably, some Clostridium spp. could also be unbeneficial, and were even increased in patients with inflammatory bowel disease when compared with healthy people .
As we expected, the colonic fungi of SBS rats were abnormal, indicated by increased OTU Counts index, Chao and ACE index. There was obvious overlap between the PCoA cluster of Sham group and the GLP2.SBS group, suggesting that their fungal communities were similar. On the contrary, there was no overlap between the PCoA clusters of Sham group and SBS group, suggesting that their fungal structures were quite different. Therefore, GLP-2 treatment not only remarkably inhibited the abnormal reproduction of colonic fungi, but also potentially improved the SBS colonic fungal communities, making it partially restored to the Sham rats. Kelly B. Flett and Hector Chavez [35, 36] demonstrated a high proportion of blood fungal infections in SBS patients using culture-dependent methods, but did not use high-throughput sequencing to analyze intestinal fungal dysbiosis. To our knowledge, our study comprehensively analyzed the fungal dysbiosis in SBS by high-throughput sequencing for the first time, and the results will provide preliminary data support for relevant studies on SBS fungal dysbiosis.
In terms of phyla, we found the relative abundance of Ascomycota phylum in Sham, SBS and GLP2.SBS was over 90%, indicating Ascomycota was the most predominant phylum in the intestine, followed by Basidiomycota phylum. This result is consistent with many previous studies [8, 9, 37]. Furthermore, we comprehensively analyzed the composition of colonic fungi at the class level. The results showed that 3/5 of the top five abundant fungi classes (including Saccharomycetes, Eurotiomycetes, Sordariomycetes) were significantly different in SBS rats in comparison with the Sham rats. Though the other 2/5 classes (including Tremellomycetes, Dothideomycetes) were increased in SBS rats, they did not reach significant difference (probably because of the small sample size). These results indicated that severe intestinal fungal dysbiosis occurred in SBS rats. LEfSe analysis was used to deeply analyze the different fungi in the 3 groups, and some interesting results were found. For example, the relative abundance of Penicillium genus from Eurotiomycetes class decreased significantly in the SBS rats, but GLP-2 treatment could reverse its decrease in SBS rats. In addition, the abundance of Debaryomyces and Meyerozyma genera from Saccharomycetes class increased significantly in the SBS group, while GLP-2 treatment could inhibit its overgrowth. In fact, at the genus level, SBS group exhibited significant changes in six fungal genera (including Aspergillus, Penicillium, Xerochrysium, Wickerhamomyces, Debaryomyces and Meyerozyma) as compared with Sham group, while GLP-2 treatment could partially reverse the significant changes of 3/6 fungal genera (including Penicillium, Debaryomyces and Meyerozyma) at 22nd days after surgery. Penicillium genus could secrete penicillin G and griseofulvin, which have antibacterial and antifungal effects respectively. These compounds may also inhibit the abnormal growth of some bacteria and fungi in the intestinal tract, thus maintaining intestinal microbiota homeostasis [22, 38]. A decrease of the relative abundance of Penicillium may further worsen intestinal microbiota dysbiosis in SBS rats. GLP-2 treatment could partially reverse the decrease of the relative abundance of Penicillium in SBS rats, which may have beneficial effects on the balance of intestinal microbiota. Coexisting in the gut, intestinal fungi and bacteria interact directly or indirectly with each other [11, 39]. A growing number of studies confirmed that the bacterial-fungal interactions played an important role in the occurrence and exacerbation of many diseases, such as IBD, primary sclerosing cholangitis, neurological disease, and even cancer [8–10, 20, 40]. We herein investigated the bacterial-fungal correlation patterns in SBS rats and found disrupted bacteria–fungi correlations existed in the colonic content of SBS rats. Fortunately, GLP-2 treatment might partially re-build the interkingdom interaction in SBS rats.
However, our study also has some limitations, such as small sampling size and lack of sampling over time. Our team's previous published results confirmed that the 16S sequencing data were reliable under consistent sampling process and the difference was small within the group but significant between groups [2, 7]. From the Anosim consistency analysis in this study, we found that the intra group differences of our samples were small, but the inter group differences were large. Therefore, we still found a lot of bacterial and fungal taxa with significant differences in case of existing small sample size. This also indirectly indicated that the intestinal flora of SBS rats was in severe dysbiosis and GLP-2 might attenuate the intestinal flora dysbiosis caused by SBS to a certain extent. We set the sampling time at the 22nd day after operation, because at this time point the intestinal adaptation of SBS rats had reached the peak while the residual intestinal internal environment and the intestinal flora of SBS rats has reached a relatively stable state so that the results would be more reliable. However, if we could enrich the sampling time point to declare the effects of whole process of intestinal adaptation and GLP-2 treatment on intestinal flora of SBS, this study would be more meaningful.