Stool Microbiota Composition Differs in Patients with Stomach, Colon, and Rectal Neoplasms

Gut bacteria form a diverse and complex microbial ecosystem that plays a vital role in health and disease [1]. Bacteria that belong to the Bacteroidetes and Firmicutes phyla [2, 3] form the predominant part of the human gut microbiota, and along with Proteobacteria, Actinobacteria, Synergistetes, and Fusobacteria constitute the majority of the bacterial species found in human gastrointestinal tract (GIT) [4]. Host and factors such as age, genotype, local environment, and dietary habits exert significant effects on gut microbiota [2, 3, 5] including the development of different types of GIT tumors either through the pro-carcinogenic activities of specific pathogens, or due to the effect of microbial metabolites [6]. Short chain fatty acids and butyrate producing bacteria such as Anaerostipes species and, Butyrivibrio species have been reported to suppress inflammation and inhibit neoplastic changes while other bacterial metabolites such as secondary bile acids can induce inflammation, cause DNA damage, and enhance carcinogenesis [6, 7]. The colonization and diversity of bacteria in different regions of the GIT can vary considerably due to the variation in pH and other physiological factors along the GIT. Moreover, the pathogenicity of bacteria can be different for different regions of the GIT. Helicobacter pylori, for instance, has been associated with increased incidence of gastric cancer as well as with a reduced risk of esophageal cancer [8].

Stool specimens represent a conveniently accessible source for investigating the gut microbiota composition. Studies based on 16S rRNA gene sequence analysis in stool samples have revealed an enrichment of certain bacterial taxa in colorectal cancer (CRC) in conjunction with a depletion of others [9‐11]. Bacterial species that have been reported to be linked to CRC include Streptococcus bovis, Bacteroides fragilis, Enterococcus faecalis [9], Clostridium septicum [12], Fusobacterium species [13], and Escherichia coli [14]. Higher abundances of Fusobacterium nucleatum and Bacteroides fragilis have been found to be associated with increased risk of adverse outcomes for CRC, while Faecalibacterium prausnitzii has been associated with a reduced risk [15].

Although the role of H. pylori in gastric carcinoma is well established and it is classified by the International Agency for Research on Cancer as a human carcinogen [1], the knowledge is more limited regarding the diversity of other bacterial species and their functional roles in the development and progression of stomach carcinoma. We here investigated the abundance of gut bacteria in the stool specimens of patients with different GIT malignancies in order to examine differences in taxonomic composition in stool samples based on the location of GIT tumor.

The patients were divided into five non-treated and one treated groups, and the relative abundances of the genera and families in each group were compared to those of the control group. The sample similarities (beta diversities) across the different groups based on genus-level profiles are illustrated with principal coordinates analysis in Fig. 1.

We assessed and compared the stool bacterial profile of patients with GIT neoplasms grouped according to the neoplasm location, with stomach, colon, and rectum being the major groups. In addition to the altered abundances of certain bacterial taxonomic groups seen in the overall group of patients with non-treated GIT neoplasms as compared to controls, further differences were observed depending on the neoplasm location (Table 2).

At the family level, Enterobacteriaceae had significantly higher relative abundance in all non-treated patients, while based on the site of neoplasm, significantly higher abundance of Enterobacteriaceae was observed only in patients with neoplasms of the stomach or small intestine (Table 2). Lower abundances were noted for Lactobacillaceae and Acidaminococcaceae in patients with colon neoplasms, as well as for Bifidobacteriaceae in rectal neoplasms and (Table 2). The family Enterobacteriaceae includes many pathogenic bacteria, in addition to commensal ones, and gut inflammation is thought to initiate increase in Enterobacteriaceae abundance. Previous studies reviewed in Chen et al. [26] have shown increase in pathogenic bacteria together with depletion of normal healthy gut microbiota associated with colorectal cancer. In our study, the increased abundance of this family of bacteria was, however, significant only in patients with neoplasms of the stomach and small intestine.

Lactobacillaceae and Bifidobacteriaceae are the two most prominent families of probiotics that play an important role in the maintenance of GIT homeostasis. The loss of abundance of these bacteria is reported in CRC, while their administration is reported to have a protective effect on CRC development as reviewed in Zou et al. [27]. Lactobacilli are thought to prevent the development of cancer and cancer cell migration and have been reported to inhibit the growth of human colon carcinoma cells [28]. A combination of probiotic Bifidobacterium lactis and prebiotic resistant starch has been shown to prevent the development of CRC in rats and has been proposed as a chemopreventive approach for CRC [29].

The majority of previous studies have considered colorectal cancer as a single group, and there is little information related to bacterial dysbiosis in the colon and rectal cancers separately. In the present study, we found reduced abundance of Lactobacillaceae related to neoplastic growth in colon, while reduced abundance of Bifidobacteriaceae was related to neoplasms of the rectum. Intriguingly, we also observed that at the genus level, while alterations in bacteria were similar between colon and stomach neoplasms (higher abundance of Subdoligranulum; lower abundance of Lachnoclostridium and Oscillibacter), there were no commonly altered bacteria between colon and rectal neoplasms (Fig. 3). This again suggests that there is a distinct difference in the effect of bacterial dysbiosis on neoplastic growth between colon and rectum. Although colorectal tumors are usually considered similar, there are few marked differences between the two types of tumors. At a molecular level, rectal tumors have higher rate of aneuploidy, loss of heterozygosity and TP53 mutations while colon cancers have higher microsatellite instability and more frequent CpG island methylator phenotype (reviewed in Iacopetta 2002) [30]. Bifidobacterium contributes significantly to de novo biosynthesis of folate in gut and to folate store in colon [31]. Humans cannot synthesize folate and are dependent on external sources (from food or bacterial synthesis), and the deficiency of folate can result in chromosomal instability [32, 33] and susceptibility for childhood leukemia [34]. Reduced levels of Bifidobacterium could result in decreased folate levels in the large intestine and thus increase the risk of aneuploidies associated with rectal neoplasms.

Gut bacteria are most abundant in colon compared to other regions of the GIT, and disturbances in bacterial abundances would be expected to associate most closely with neoplastic growth located in the colorectal region. However, in our study, there were more bacteria genera altered in gastric neoplasms, which are similar to those detected in colon neoplasms, than in rectal neoplasms. Interestingly, the bacterial genera associated with gastric or colon neoplasms are related to inflammation, or metabolic diseases. Increased levels of Ruminococcus and Subdoligranulum have been reported in the stools of children with food sensitivity compared to healthy children [35]. Subdoligranulum has been found to be associated with chronic inflammation and poor metabolic control [36]. Its levels have been reported to be associated with blood markers of inflammation (C-reactive protein, CRP) and endotoxemia (lipopolysaccharide-binding protein, LBP) in Type 1 diabetes [37]. Subdoligranulum is also reported to inhibit fermentation of inulin by bifidogenic bacteria in colon, which is considered beneficial in the prevention of colon cancer [38]. Oscillibacter are known to produce anti-inflammatory metabolites and have effect on the maintenance of gut barrier integrity in mice [39]. Their abundance is reported to be affected by the presence of other gut bacteria [5]. Reduced abundance of Lachnoclostridium has been reported in stools of Hashimoto’s thyroiditis patients [40]. Increased abundance of Ruminococcus, seen exclusively in gastric neoplasms in our study, is reported in prediabetic patients and associated with impaired fasting plasma glucose, BMI, and waist circumference [41]. Moreover, their higher abundance has been reported in stools of high fat diet-induced obese rats compared to lean rats [42].

On the other hand, in patients with rectal neoplasms, Bifidobacterium had significantly lower abundance as compared to healthy individuals in our study. These bacteria have a role in maintenance of healthy gut bacterial profile by inhibiting growth of pathogens by competitive exclusion; immune function; breakdown of indigestible food component by enzyme secretion; and folate synthesis in gut (reviewed in O’Callaghan et al. 2016) [43].

In patients with neoplasms of the small intestine, higher abundance of Enterobacteriaceae and lower abundance of Lachnoclostridium were similar to those seen in stomach. Patients with pancreatic neoplasms showed, in our study, a similarly low abundance of Lactobacillaceae as seen in those with colon neoplasms and also decreased abundance of Parabacteroides. Gut Parabacteroides distasonis is reported to have anti-inflammatory and anticancer role, acting through reducing TLR4 signaling/Akt activation in mice [44], and TLRs are known to play a significant role in various pancreatic diseases, including pancreatic cancer [45]. Robust conclusions could not be drawn regarding the pancreatic and small intestinal neoplasm groups due to their small sample size.

The gut microbiota is diverse and highly individual [46]. Changes in microbiota richness have been reported in association with cancer development [47], whereas reduced alpha diversity and richness in the gut microbiota have been linked with a Western or urban lifestyle [7] and compromised health in humans [48], as well as with colon cancer in mouse models [49]. There are also reports of increased richness and diversity at the taxonomic and functional levels linked to gastrointestinal cancers [50]. We did not observe significant differences among the groups with respect to alpha diversity or taxonomic richness. The increased levels of Enterobacteriaceae and Subdoligranulum in overall GIT neoplasms could, however, indicate increase in more pathogenic bacteria in GIT neoplasms. Altered abundance of most of the bacterial genera seen in individual gastric, colon, or rectal neoplasm groups was also seen in the overall non-treated GIT neoplasm group, suggesting that a similar pattern of changes in bacterial abundance is related to development of neoplastic growth in GIT with certain taxa having more profound effect in certain specific GIT locations. Chronic inflammation, obesity, and high BMI are some of the common risk factors associated with gastric and colon neoplasms.

Whereas the heterogeneity of histological subtypes of the tumors and the small sample size of each subtype form limitations for statistical conclusions, this is, to the best of our knowledge, the first study investigating the differences in microbiota in different GIT tumor locations. The number of controls was 13, which is similar to the number of patients in each neoplasm subgroup. One limitation in our study is that the healthy control group is on average younger than the patient groups. Therefore, some of the differences in the healthy controls could be associated with their younger mean age. However, the different patient groups had relatively similar mean ages, and hence not a likely confounder in our key analysis task, which was the analysis of differences between the taxonomic compositions in fecal samples between the different patient groups.

In conclusion, we found significant differences in the abundances of specific bacterial taxonomic groups in stool specimens from patients with various GIT neoplasms; the differences were dependent on the location of neoplasia in the GIT. This finding could be of significance in the future as a tool for assessing neoplastic alterations in different parts of the GIT. Studies on the stool abundances of Lactobacillaceae, Bifidobacteriaceae, and Enterobacteriaceae could potentially lead to the development of a noninvasive approach to GIT disease monitoring and treatment follow-up.