In this study, we investigated the relationship between the prevalence of pks+ E. coli and stool patterns through a population-based cohort study. Even after adjusting for confounders, we found a stool pattern (factor 1) that was significantly associated with the prevalence of pks+ E. coli. In addition, this stool pattern was correlated with certain plasma and fecal fatty acids. As far as we know, this is the first study to show a stool pattern association with not only prevalence of pks+ E. coli, but also plasma and fecal fatty acids. These associations suggest that stool pattern may reflect the gut microbiota, including the presence of tumorigenic bacteria.
Certain risk factors for CRC incidence have been identified, including smoking, obesity, diabetes, and high consumption of alcohol, as well as consuming red and processed meats, in epidemiological studies [20]. These identified CRC-risk factors have not only been associated with increased CRC incidence, they have also been associated with potentially adverse gut microbiome profiles [21]. Recently, the prevalence of pks+ E. coli isolated from the colonic epithelium has been reported to be higher in patients with familial adenomatous polyposis [22], inflammatory bowel disease [23], and CRC [22] compared to healthy individuals. Thus, it is important to evaluate the association between environmental exposure factors and the prevalence of tumorigenic bacteria in the gut microbiota.
Our results demonstrated a significant association between the prevalence of pks+ E. coli and stool pattern. Animal models mimicking the natural transmission of E. coli producing colibactin from mothers to neonates has shown lower rates of Firmicutes taxa, Proteobacteria taxa, and microbial species richness, as well as higher DNA repair function compared to the sham model [24]. This model has also illustrated an association with gut homeostasis activities, including renewal of the mature epithelium and occurrence of crypt fission [25]. Stool status variables, such as shape [12, 13], frequency [12, 15], and color [16] have been associated with higher microbial species richness profiles from fecal analysis in healthy individuals and patients with acute gastroenteritis. Our results support these findings. A previous study showed that the majority of CRC deaths were attributed to non-screening in the United States [26]. Although a causal relationship between the prevalence of pks+ E. coli as a tumorigenic bacteria and an increased risk of CRC has not been established well, this study may underscore the potential benefits of evaluating the presence of pks+ E. coli as a target for early prognostication in populations with a high risk of CRC. Our results also suggest that stool pattern might be a marker associated with the prevalence of tumorigenic bacteria in healthy individuals. Longitudinal objective monitoring of a person’s stool status from serial samples taken from an individual’s excreta at home, as previously suggested [27], may be the most reasonable and cost-effective method for early detection of risk factors of CRC.
Nutrients derived from our ingested food are utilized by the gut microbiome, with certain preferred energy sources such as short chain fatty acids (SCFAs) for colonocytes [28, 29]. These metabolites can suppress inflammation and carcinogenesis through effects on immunity, gene expression, and epigenetic modulation [28–31]. Some plasma fatty acids have been noted to be inversely or positively associated with the presence of colon adenomas [32] and increased risk of CRC in middle aged adults [33]. In addition, studies in CRC patients have noted lower levels of propionate and butyrate [34] and higher levels of valeric acid, isobutyric acid, and isovaleric acid [35] in SCFAs derived from fecal samples compared to healthy controls. Production of SCFAs has been shown to be reduced in patients with diarrhea compared to those without diarrhea [36]. In addition, inhibition of SCFA synthesis by administration of polyethylglycol and antibiotics has been reported to result in diarrhea [37]. It has been reported that the distal colon transit, reflected in stool frequency, was associated with not only plasma acetate and fecal SCFAs [38], but also with microbiota diversity, especially the Firmicutes taxa (Faecalibacterium, Lactococcus, and Roseburia) [39]. Our results indicated that the stool pattern that showed a relationship with the prevalence of pks+ E. coli was also significantly correlated with certain plasma fatty acids, including α-linoleic acid [40] and certain fecal SCFAs, such as propionate [34] and isovaleric acid [35]. These were also associated with higher incidences of CRC, supporting previous findings. Taking information from previous studies, we speculate that gut microbiota and dietary components interacted to generate biologically active molecules including SCFAs, which influenced gut secretion and motility, and that this could play a fundamental role in stool status [30, 31], as well as affect the prevalence of pks+ E. coli. While detailed mechanisms and causal relationships should be clarified in further studies, we can conclude that fecal matter is not just a simple waste material, but could be a possible tool to assess the gut microbiota, screening for the presence of tumorigenic bacteria and specific fecal fatty acids via comprehensive examination of variables including color, shape, frequency, volume, and odor.
The strength of this study is in finding a verified association between stool patterns and plasma and fecal fatty acids. The multifaceted, self-reported questionnaires used to assess the stool status had previously been validated against objective fecal characteristics as well [41]. In addition, we showed that twice self-reported stool status was highly reproducible and believe that it is unlikely for there to have been misclassification when done in this manner. Thus, this study might generate a new hypothesis for the association between the prevalence of pks+ E. coli as a tumorigenic bacteria and stool pattern.
However, this study has a number of methodological limitations. Even when minimizing the effect of confounders using multivariate analysis to adjust for known covariates, being a cross-sectional study, we are unable to theorize about the temporal and direct causality of the observed association between stool pattern and the prevalence of pks+ E. coli. Second, this study detected the clb gene cluster in DNA extracted from fecal samples, not from the DNA of isolated E. coli pure cultures. Previous study has been evaluated prevalence of pks+ E. coli by the selective cultivation method [23]. However, our previous study [17] demonstrated that prevalence of pks+ E. coli isolated from fecal matter are relatively similar previous reports investigating prevalence of pks+ E. coli by the selective cultivation method [23]. Therefore, it will be necessary to evaluate the concordance rate of prevalence of pks+ E. coli defined using these two different methods for the same subject. Third, although our results showed that a softer stool shape was negatively associated with the prevalence of pks+ E. coli, it is unclear whether participants with diarrhea, who have softer stools, have a lower prevalence of pks+ E. coli or whether the reverse was true. In addition, we were unable to completely exclude systematic error due to self-reporting. In addition, we could not account for unmeasured confounding factors associated with stool status in this observational study. For example, stool color is mainly characterized by stercobilin (urobilin), an orange pigment which is the oxidized metabolite of urobilinogen [42]. Stercobilin derived from bile pigment is responsible for the brown color of human feces. Since we did not measure stercobilin and bile acids directly in all participants, we could not account for their possible effects on the results, though our results were similar after adjusting for bile acids in a subsection of participants with available bile acid data. It is necessary to further verify our results with further studies including patients and community-dwelling residents with symptoms such as diarrhea and constipation. Finally, there is the possibility of sampling bias due to the more health-aware nature of the participants in this study than in the general population. Of 750 participants in the NEXIS cohort study, 259 adults agreed to participate. As the participation rate was relatively low, selection bias may have occurred. In addition, participants were all living in the Tokyo metropolitan area and the mean age was 58 years old in Japan. These limitations may prevent the generalization of our results. Therefore, prospective cohort studies with larger randomized samples should be done to further investigate the association between the prevalence of pks+ E. coli and stool pattern.