Numerous microbes inhabit the human intestine forming diverse and unique microbial communities (Huttenhower et al., 2012; Segata et al., 2012). They play a crucial role in preserving the physiological homeostasis of the host by making essential contributions to the host metabolism and immune system (Round and Mazmanian, 2009; Nicholson et al., 2012). Consistent with this notion, dysbiosis of gut microbiota has been reported to be involved in intestinal disorders such as inflammatory bowel disease (IBD) (Franzosa et al., 2019; Lavelle and Sokol, 2020) and colorectal cancer (CRC) (Wong and Yu, 2019). Remarkably, the abnormal enrichment of oral bacteria has been observed in the guts of patients with intestinal diseases (Strauss et al., 2011; Atarashi et al., 2017; Yachida et al., 2019; Kitamoto et al., 2020a). For instance, Fusobacterium nucleatum, which generally inhabits the oral cavity, is frequently detected in patients with IBD and CRC (Strauss et al., 2011; Yachida et al., 2019). Although the causal association between oral bacterial enrichment and intestinal diseases remains unclear, the possible ectopic colonization of oral bacteria in the gut plays a significant role in the etiology of intestinal diseases. In fact, studies have indicated that intestinal colonization of Klebsiella pneumoniae originating from the saliva of patients with IBD induces gut inflammation in mice (Atarashi et al., 2017).

The oral cavity, the gateway of the digestive tract, hosts a large microbial community distinct from the gut microbiota (Huttenhower et al., 2012; Segata et al., 2012). People constantly swallow such copious amounts of oral microbes with saliva and boluses of food (Humphrey and Williamson, 2001). Considering the antimicrobial barriers along the gastrointestinal tract, such as gastric acidity (Giannella et al., 1972), bile (Begley et al., 2005), and colonization resistance by the gut-resident microbiota, ingested oral bacteria are unlikely to reach and colonize the gut in healthy individuals. In line with this, a previous study identified no evidence of colonization by oral bacteria in the guts of healthy adults (Rashidi et al., 2021). However, metagenomic approaches or full-length 16S rRNA gene sequencing have demonstrated extensive translocation of oral bacteria to the gut in healthy individuals or community-dwelling adults (Schmidt et al., 2019; Kageyama et al., 2023); thus, the argument as to whether oral bacteria can be translocated to the gut remains inconclusive, especially in populations without specific intestinal disorders. To understand the causal association between oral bacteria and intestinal disorders, or their utility as predictors, verification of oral bacterial translocation to the gut in such populations is required.

In this study, we examined paired tongue and rectal microbiota from orthopedic patients without a history of intestinal disorders using 16S rRNA gene amplicon sequencing and the amplicon sequence variant (ASV) approach. Instead of stool samples which are usually used for gut microbiota analysis, rectal swab samples were collected. The bacterial community on the mucosal surface of the rectum is known to display a composition different from that of the stool (Zmora et al., 2018), and rectal swab samples can be used to examine the microbial profile on the mucosal surface of the rectum. This approach enables the evaluation of oral bacterial translocation to the gut from a novel perspective and could provide robust evidence of the translocation. The ASV approach resolves differences of as little as one nucleotide and enhances taxonomic resolution (Callahan et al., 2016). This study aimed to verify oral bacterial detection in the rectal microbiota at the ASV level and identify the relevant factors influencing the increased abundance of oral bacteria in the rectum.

This study examined 49 pairs of tongue and rectal samples collected from orthopedic patients without a history of intestinal disorders using 16S rRNA gene amplicon analysis and the ASV approach, and verified the detection of own oral bacteria in the rectum. The tongue ASVs were detected from the rectal microbiota in 67.3% of participants and demonstrated ≥1% relative abundance in 28.6% of participants. Further, the bacterial composition of the rectum was significantly more similar to the individual’s own tongue than to others’ tongue. These results suggest that oral bacteria can endogenously reach the rectum, transverse the digestive tract, and colonize the mucosal surface, even in the absence of intestinal disorders. This observation supports the findings of a previous study that suggested the oral gut transmission of microbes in healthy participants or community-dwelling adults (Schmidt et al., 2019; Kageyama et al., 2023). Oral bacterial translocation can occur extensively, and not only in populations with specific diseases.

Oral taxa such as S. salivarius, S. parasanguinis, and F. nucleatum were abundantly detected in the rectal microbiota. As stated in the Introduction, F. nucleatum is known to be enriched in the gut of patients with IBD and CRC and is suggested to induce intestinal inflammation and tumorigenesis (Strauss et al., 2011; Kostic et al., 2013; Yachida et al., 2019). Interestingly, this study also detected Solobacterium moorei HMT-678 and Peptostreptococcus stomatis HMT-112 in the rectal microbiota. Their abundances have been reported to increase in the early stages of CRC, similar to that of F. nucleatum (Yachida et al., 2019). Furthermore, network analysis displayed the co-occurrence of S. salivarius and S. parasanguinis with K. pneumoniae, which is reported to induce gut inflammation in mice (Atarashi et al., 2017; Kitamoto et al., 2020b), in the rectum, consistent with our previous study examining salivary and stool samples (Kageyama et al., 2023). Thus, the findings of this study are highly consistent with those of previous reports examining oral gut transmission. Importantly, these findings were observed in a population with no history of intestinal disorders. The observations above suggest that oral bacterial translocation is associated with rectal dysbiosis, characterized by an increase in potentially pathogenic bacteria, which could begin when people are healthy.

Aging was significantly associated with an increased abundance of tongue ASVs in the rectal microbiota ( Figure 4 ). This could be attributed to a diminished barrier function in the gastrointestinal tract with advancing age (Drozdowski and Thomson, 2006), consistent with the results of our previous study (Kageyama et al., 2023). Although distinguishing between pathological alterations and age-induced changes is difficult, the translocation of oral bacteria to the gut can be considered an age-related change that occurs widely in older individuals. Furthermore, considering the enrichment of potential carcinogenic bacteria, this age-related change in the gut microbiota might partially contribute to the high incidence of CRC in older adults. Similarly, PPI use, which reduces gastric acid secretion, contributed to an increase in oral bacteria in the rectum ( Figure 4 ), as some studies using stool samples previously reported (Imhann et al., 2016; Matthew et al., 2016; Jiaying et al., 2023). PPI use is also a known risk factor for Clostridium difficile infection, which is characterized by various gastrointestinal symptoms (Kwok et al., 2012; Inghammar et al., 2021). In this study, the prevalence was not statistically significant; however, C. difficile was only observed in the rectum of PPI users (n=1). Interestingly, the participants with hypertension exhibited a significantly high abundance of oral bacteria in the rectal microbiota ( Figure 4 ). A recent study reported that the transplantation of saliva from individuals with hypertension via oral gavage exacerbated angiotensin II-induced hypertension in mice (Chen et al., 2023). The causal association between hypertension and translocation of oral bacteria should be carefully considered in future studies.

This study has several limitations. First, oral health information such as dental caries, periodontal condition, number of teeth present, and oral hygiene was not obtained. Our previous study suggested that dental plaque accumulation was associated with an increased abundance of oral bacteria in the gut (Kageyama et al., 2023). Therefore, the influence of oral health conditions on oral bacterial occupancy in the rectum should be further considered. Second, our analyses relied on tongue microbiota, despite the fact that oral bacteria are likely transported to the gut via saliva. However, previous studies have reported that the salivary microbiota is mainly composed of tongue microbes (Segata et al., 2012; Kageyama et al., 2017). Meanwhile, further consideration should be given to the translocation of dental plaque-preferring bacteria such as F. nucleatum because the bacterial composition on the tongue is different from that on dental plaque. The translocation of dental plaque-preferring bacteria may have been underestimated in this study. Third, species-level taxonomy was assigned to only an average of 59.0% of each rectal microbiota, unlike 92.9% for tongue microbiota based on a well-developed database for oral taxa, HOMD (Chen et al., 2010). Thus, network analysis of rectal bacterial species may not detect or underestimate the co-occurrence network. Fourth, all the participants were administered antibiotics during surgery. However, we consider that the effect of antibiotics on the bacterial composition of the rectal microbiota examined in this study was minimal because the rectal sample was collected immediately after surgery and the outcome of the 16S rRNA gene analysis is typically independent of whether the bacteria are alive or dead. On the contrary, the fifth limitation is that the 16S rRNA gene analysis could not evaluate whether the tongue bacteria in the rectum were alive or dead. However, considering their dominance in the rectum, they were likely to thrive. A previous study using a culture approach demonstrated that F. nucleatum strains identical to the oral strains were isolated alive from the gut (Komiya et al., 2019). Finally, short-read sequencing of the 16S rRNA gene did not reveal differences between conspecific strains, and the translocation of oral bacteria may have been overestimated. Further studies that utilize long-read sequencing or metagenomic approaches are now required to validate our results.

In conclusion, this study examined tongue and rectal samples using 16S rRNA gene amplicon analysis and the ASV approach and demonstrated the translocation of tongue bacteria to the rectum based on ASV sharing (i.e., identical match of the V1–V2 region of the 16S rRNA gene). Furthermore, this detection was observed in 67.3% of participants without intestinal disorders. These results suggested that oral bacterial translocation occurs extensively in healthy individuals. Moreover, translocation was involved in the increased abundance of potentially pathogenic bacteria, suggesting that translocation might play a role in the initial events of intestinal disorders. Monitoring oral bacteria in the gut could be useful for evaluating intestinal disease risk. These findings could contribute to the development of novel preventive strategies against intestinal diseases by inhibiting the translocation of oral bacteria. Further studies are needed to determine the association between the translocation of oral bacteria and the subsequent risk of disease.

The sequence data presented in the study are deposited in the DDBJ BioProject database, accession number PRJDB17727.

The studies involving humans were approved by The Ethics Committee of Kyushu University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

GL: Investigation, Visualization, Writing – original draft. SK: Conceptualization, Funding acquisition, Visualization, Writing – original draft, Writing – review & editing. AM: Conceptualization, Investigation, Writing – review & editing. ES: Conceptualization, Investigation, Writing – review & editing. JM: Investigation, Writing – review & editing. MA: Investigation, Writing – review & editing. MF: Supervision, Writing – review & editing. YY: Conceptualization, Supervision, Writing – review & editing. TT: Funding acquisition, Supervision, Writing – review & editing.