The oral microbiome plays a critical role in the occurrence of oral diseases, 4 , 5 and may also play an important role in maintaining systemic health. 6 , 7 Some studies have suggested an increased risk of CRC 8 in individuals with oral diseases, though null associations were reported in other studies. 9 , 10 Chronic inflammation caused by oral microbes has been suggested to play a role in CRC development. 11 Two research teams reported that the Fusobacterium nucleatum, one of the predominant subgingival microbial species present in periodontitis, 12 is more abundant in colorectal carcinoma tissues than in normal colorectal tissues. 13 , 14 In addition, a recent study 15 proposed that colon conditions could provide an anaerobic environment similar to subgingival space. In this anaerobic environment, periodontal pathogen species, such as Prevotella intermedia , Fusobacterium nucleatum and Prevotella gingivalis , are adept at coaggregation, which may result in consistent and chronic inflammation, and then promote the development of CRC. 15 These results suggest that the oral microbiome may play a direct role in the pathophysiology of CRC. However, to our knowledge, no study has prospectively and systematically investigated the oral microbiome in relation to the risk of CRC. Herein, we carried out a prospective nested case–control study within the Southern Community Cohort Study (SCCS) to investigate the association of the oral microbiome with the subsequent risk of developing CRC.

Colorectal cancer (CRC) is the third leading cause of cancer death in the United States. 1 Epidemiological studies have shown that lifestyle factors, such as obesity, smoking and alcohol drinking are associated with an increased CRC risk. 2 , 3 Systemic inflammation is also associated with an increased risk of CRC. 3

During all of these analyses, smoking amount (pack‐year of smoking) and alcohol consumption status were adjusted. For analyses of prevalence, sequencing depth was additionally adjusted. Considering the fact that taxa of different taxonomic ranks are correlated, we used Galwey's method, 31 implemented in the function “meff” of the R package “poolR” ( https://github.com/ozancinar/poolR/ ) to estimate the total independent tests. Then, Bonferroni correction was used to adjust for multiple testing. Stratified analyses by ethnic group and gender were conducted for both carriage and abundance. In addition, we did sensitivity analyses by excluding the cases diagnosed within 2 years ( N = 47) after enrollment and the corresponding controls ( N = 94). We also conducted analyses based on relative abundance after rarefying the OTU table at a subsampling depth of 20,000 to account for variations in sequencing depth among samples. The results did not change materially. All of these analyses were carried out using R (v3.3.1) and Python (v2.7.8).

We also investigated other bacterial taxa in relationship to CRC risk. Taxa of different taxonomic ranks are correlated evolutionarily through a phylogenetic tree. Taxa with a median relative abundance of >0.01% among control subjects, including seven phyla, 24 families, 35 genera and 71 species, were treated as “common taxa” in our study and included in downstream analyses. At each taxonomic level, the raw sequencing count for each taxon was normalized using the centered log‐ratio transformation after adding 1 as a pseudocount. 29 , 30 Then for each taxon, a conditional logistic regression analysis was performed to test the association of taxa abundance with CRC risk. For taxa with a median relative abundance of ≤0.01% among controls, they were treated as “rare taxa” in our study. Only those rare taxa that were observed in at least 20% of the control subjects were included in the statistical analyses, in which study participants were grouped into carriers and non‐carriers. Conditional logistic regression analyses were conducted for each taxon to evaluate the association of taxon prevalence with CRC risk. This included three phyla, 22 families, 54 genera and 145 species.

To estimate the association between individual bacterial taxon and CRC risk, first we investigated five pre‐defined oral pathogens, including three “red complex” periodontal pathogens, 27 Porphyromonas gingivalis , Treponema denticola and Tannerella forsythia , and two additional periodontal pathogenic species, Fusobacterium nucleatum 12 and Prevotella intermedia . 28 For each pathogen, study participants were categorized into two groups according to whether they carried the pathogen or not. The association between prevalence of the pathogen and CRC risk was analyzed through conditional logistic regression modeling.

To evaluate the relationship between overall oral microbiota composition and CRC risk, we calculated the Bray–Curtis dissimilarity, 24 as well as unweighted and weighted UniFrac distance matrices. 25 PERMANOVA‐S 26 was used to estimate whether there was a difference regarding these three distance metrics between CRC cases and controls. Statistical analyses were conducted within matched case–control sets.

Raw sequencing data were trimmed and filtered to remove bases and reads of low quality by the use of Sickle. 18 Then, BayesHammer 19 was utilized for sequencing error correction and PANDAseq 20 was used to stitch the paired‐end reads together. 21 Clean reads were then clustered into Operational Taxonomic Units (OTUs) at 97% sequence identity using the closed‐reference OTU picking strategy with the Human Oral Microbiome Database 22 (HOMD) as reference via the Quantitative Insights Into Microbial Ecology (QIIME), v1.9.1. 23 We removed one sample with less than 20,000 sequencing reads. Finally, data from 231 CRC patients and 461 controls were included in the downstream analysis.

DNA was isolated from mouth rinse samples using Qiagen's QIAmp DNA kit. Sequencing libraries were prepared using the NEXTflex 16S V4 Amplicon‐Seq Kit (Bioo Scientific 4,201–05), after the protocol provided by the manufacturer. Sequencing was performed at paired‐end 250 bp using Illumina HiSeq System at the BGI Americas. Each 96‐well plate was sequenced with two duplicate quality control samples (QCs). In total, six duplicate samples from the same single subject were sequenced and very similar microbiome profiles were observed: coefficient of variability (CV) for the Shannon index and the Simpson index (measurement of microbial community diversity within each sample) among the six samples were 1.7% and 0.3%, respectively; CV for the relative abundance of the four most abundant phyla, i.e. Firmicutes , Actinobacteria , Bacteroidetes , Proteobacteria , among the six samples ranged from 5.1% to 9.4%; Pearson correlation coefficients of phylum‐level microbial relative abundance between the six samples ranged from 97.8% to 99.9%.

We conducted a nested case–control study of incident CRC among SCCS participants who donated mouth rinse samples. Individuals who received antibiotics treatment during the 7 days prior to sample collection were excluded. For each of the 231 incident CRC cases, two controls were randomly selected and individually matched to cases by age of enrollment (±5 years), ethnic group (African‐American/European‐American/other), gender, smoking status (current smoker/former smoker/never smoker), season‐of‐study enrollment (spring/summer/fall/winter) and recruitment method (CHC/general population).

During the study enrollment, participants completed a baseline survey with a comprehensive questionnaire, which collected information on demographics, anthropometric characteristics, lifestyle factors, disease history, medication use, and other characteristics. As part of the active follow‐up surveys initiated in 2008, participants were asked about their personal medical histories, including their oral health. Incident CRC cases diagnosed after entry into the SCCS were identified via linkage to state cancer registries operating in the 12‐state study area and national mortality registries. CRC was defined according to the International Classification of Diseases (ICD‐10), codes C18‐C21.

The SCCS is an ongoing prospective cohort study investigating risk factors for cancer and other chronic diseases. Details on the methodology of the study have been described elsewhere. 16 , 17 Briefly, approximately 86,000 adults were recruited between 2002 and 2009 from 12 states in the southeastern US, two‐thirds of whom were African‐Americans. Approximately 86% of them were recruited from community health centers (CHCs), institutions providing basic health care and preventative services in underserved areas. As a result, the cohort includes a substantial number of individuals of low income and educational status. The remaining 14% of the cohort members were recruited through mail‐based general population sampling. Mouth rinse samples were collected from ~34,100 participants at the time of enrollment. The SCCS was reviewed and approved by the institutional review boards at Vanderbilt University Medical Center and Meharry Medical College. Written informed consent was obtained from all study participants.

We further evaluated the associations between the oral taxa and CRC risk, presented in Tables 2-4 , stratified by ethnic group and gender. Most associations were observed among both African‐Americans and European‐Americans, as well as in men and women. In general, the associations were stronger among African‐Americans than among European‐Americans, and among females than among males (Supporting Information Tables 1 – 5 ). For example, the oral pathogen Treponema denticola showed an association with an increased risk of CRC with ORs (95% CIs) of 1.79(1.12–2.86) and 1.55(0.70–3.41) among African‐Americans and European‐Americans, respectively, and 2.02(1.19–3.41) and 1.33(0.73–2.42) among females and males, respectively (Supporting Information Table 1 ). Similar differential associations were also observed for Prevotella intermedia between African‐Americans and European‐Americans, and between females and males (Supporting Information Table 1 ). We also found that some taxa showed stronger associations among European‐Americans than among African‐Americans, e.g. Burkholderiaceae , Lautropia and Lactobacillus salivarius (Supporting Information Table 4 ). However, a formal test of multiplicative interaction failed to show statistical significance. We also did sensitivity analyses to exclude the CRC patients who were diagnosed within 2 years ( N = 47) after enrollment and the corresponding controls ( N = 94), and the results did not change materially.

Among those 224 rare taxa, 16 showed an association with CRC risk at p <0.05, and all of them were associated with an increased risk of CRC (Table 4 ). The most significant taxon was the phylum SR1 . It was observed among ~55% of cases and ~42% of controls. Participants carrying this species had a higher risk than non‐carriers with an OR (95% CI) of 1.76(1.25–2.47) and a P value of 1.09 × 10 −3 . Within this phylum, two species were associated with CRC risk. Carrying SR1_[G‐1] sp. oral taxon 874 was associated with a 75% increased risk of CRC with a P value of 1.58 × 10 −3 . Carrying SR1_[G‐1] sp. oral taxon 345 was associated with a 50% increased CRC risk with a P value of 0.04. In the phylum Actinobacteria , the species Bifidobacterium dentium was associated with an increased risk of CRC. In the phylum Bacteroidetes , in addition to the oral pathogen Prevotella intermedia (Table 2 ), another species, Prevotella sp. oral taxon 304 , also showed an association with increased CRC risk. Three genera and two species in the phylum Firmicutes , namely Peptococcus , Anaeroglobus , Mitsuokella , Lactobacillus salivarius and Eubacterium yurii , were associated with an increased CRC risk. In the phylum Proteobacteria , four taxa were associated with an increased risk of CRC, including Burkholderiaceae , Lautropia , Neisseria oralis and Campylobacter sp. oral taxon 044 . Among these four taxa, strong correlations were observed between Burkholderiaceae and Lautropia . After mutual adjustments, neither of these two taxa showed an association with CRC risk. In the phylum Spirochaetes , in addition to the oral pathogen Treponema denticola , two more species, Treponema lecithinolyticum and Treponema sp. oral taxon 250 , were also associated with an increased CRC risk. Among the 224 rare taxa included in the investigation of taxa prevalence and CRC risk, there were 92 independent tests. After Bonferroni correction, none of these associations reached p <0.05.

We evaluated the differences of abundance for common taxa ( N = 137) between CRC patients and control participants. Multiple taxa were associated with CRC risk at a p <0.05 (Table 3 ). In the phylum Actinobacteria, the family Bifidobacteriaceae was associated with an increased risk of CRC with an OR (95% CI) of 1.10(1.01–1.19) and a P value of 0.03. In the phylum Bacteroidetes , the species Prevotella melaninogenica was associated with a decreased CRC risk with an OR (95% CI) of 0.91(0.84–0.99) and a P value of 0.04. Within this phylum, another two species, Prevotella denticola and Prevotella sp. oral taxon 300 , were associated with an increased risk, with ORs (95% CIs) of 1.11(1.02–1.20) and 1.13(1.01–1.26) respectively, and P values of 0.02 and 0.04, respectively. In the phylum Firmicutes , seven taxa were found to be associated with CRC risk and all of them showed a protective effect on the risk of developing CRC. Among them, the most abundant taxon at the species level, Streptococcus sp. oral taxon 058 , showed the most significant association with an OR (95% CI) of 0.79(0.67–0.94) and a P value of 7.87 × 10 −3 (Table 3 ). Among the 137 common taxa included in association analyses of taxa abundance and CRC risk, there were 53 independent tests and no taxa maintained a significant association with CRC risk after Bonferroni correction (Table 3 ).

All five of the investigated oral pathogens were more prevalent among CRC patients than among controls, but only differences for Treponema denticola and Prevotella intermedia reached a p <0.05 (Table 2 ). Specifically, the carriage of Treponema denticola was associated with an increased risk of CRC with an OR (95% CI) of 1.76 (1.19–2.60) and a P value of 4.45 × 10 −3 . For these five pathogens, there were three independent tests, and after Bonferroni correction, Treponema denticola still showed an association with CRC risk. Carriages of Treponema denticola and Tannerella forsythia were correlated with a spearman correlation coefficient of 0.57. After adjusting for Tannerella forsythia, Treponema denticola was still associated with an increased risk of CRC with an OR (95% CI) of 1.95(1.25–3.04) and a P value of 3.14 × 10 −3 . Similarly, Prevotella intermedia was also associated with an increased risk of CRC with an OR (95% CI) being 1.55(1.08–2.22) and a P value of 0.02 (Table 2 ). Fusobacterium nucleatum was present in most study participants, with a nearly equal prevalence observed in CRC patients and control subjects.

Table 1 presents the distribution of selected demographic characteristics of the study participants. About 74% of the study participants were African‐Americans, 60% female, 30% overweight and over 40% obese. Most participants had a low education level and low income, with only about 18% having a college education and 15% having an annual household income of at least $50,000. The CRC patients and control subjects were generally similar for these characteristics, and for smoking status, because of the matched study design. No significant differences were observed for tooth loss or tooth decay, but CRC cases were slightly more likely to have poor oral health status.

Discussion

In this prospective study of oral microbiome and CRC risk, we found that two of five oral pathogens, Treponema denticola and Prevotella intermedia, were associated with increased CRC risk. In addition, 11 common taxa and 16 rare taxa were also associated with the risk of CRC. Our findings warrant further investigation in larger studies to comprehensively estimate the potentiality of utilizing the oral microbiota for CRC early detection or manipulating it for CRC prevention.

Two studies have reported the increased abundance of the oral pathogen Fusobacterium nucleatum in colorectal carcinoma tissues than in normal colorectal tissues.13, 14 In our study, Fusobacterium nucleatum was nearly universally prevalent in the oral cavity, and neither abundance nor prevalence of this bacteria was associated with CRC risk. Similarly, in two recently published studies with cross‐sectional designs, no associations were observed between oral Fusobacterium nucleatum and CRC risk.32, 33 In our study, sequencing data of the V4 region of the 16S rRNA gene were used to quantify the abundance of Fusobacterium nucleatum. This may cause misclassification of microbial composition,34 hence led to the lowered statistical power to investigate the relationship of this bacteria with CRC risk.

All of the other four periodontal pathogens, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and Prevotella intermedia, were associated with an increased risk of CRC, though only the associations of the latter two taxa reached nominal significance. Although there is no existing direct evidence linking them to CRC risk, several studies suggested that they were associated with other cancers. For example, Treponema denticola was found to be more prevalent in cancerous esophageal tissues than in normal tissues.35 This pathogen was also reported to promote stemness and migration in oral squamous carcinoma.36 In a recent prospective case–control study, Treponema forsythia showed a suggestive significant association with a higher risk of esophageal adenocarcinoma.37 Prevotella intermedia was suggested to cooperate with other oral pathogens, such as Fusobacterium nucleatum and Porphyromonas gingivalis, to colonize and persist as a community in the colon and form an inflammatory microenvironment, which may promote CRC development.15 However, in our study, we did not see any correlation of the Prevotella intermedia with Fusobacterium nucleatum or Porphyromonas gingivalis, with the maximum Pearson correlation coefficient of <0.47. Studies have also suggested that certain oral bacteria may transfer to other body sites, including the gastrointestinal tract,38, 39 while further research will be needed to elucidate the underlying biological mechanism of this transmission.

Multiple studies investigating oral health status in association with CRC risk have been conducted and inconsistent results were reported.8-10 In 2016, we carried out a meta‐analysis of oral health with risk of CRC and did not find any associations.9 However, the results need to be interpreted with caution because there were many limitations in these studies, e.g., an inconsistent definition of oral health, self‐reported oral health (potentially prone to recall errors) and many others. On the other hand, the association of the oral pathogen Fusobacterium nucleatum with CRC has been consistently reported; however, the mechanism underlying the association is not clear. In our study, we found associations for the other oral pathogenic bacteria with CRC, however, the results need to be replicated in larger studies.

In the phylum Actinobacteria, the genus Bifidobacteriaceae was more abundant and the species Bifidobacterium dentium was more prevalent among CRC cases than among controls. No study had linked these two taxa to the risk of developing CRC. In a recent oral microbiome study, the genus Rothia of Actinobacteria was found to be associated with CRC risk.32 However, in our study, this taxon was only slightly more abundant among CRC patients (7.8%) than among controls (7.3%).

The phylum Firmicutes showed a lower abundance in CRC cases than in controls. Specifically, the genus Streptococcus was also found to be significantly less abundant among CRC patients than among controls in a recent oral microbiome study.33 Similarly, in a gut microbiome study, the genus Streptococcus and the species Streptococcaceae and Streptococcus sp. oral taxon 058, showed a lower relative abundance in CRC patients than in normal controls.40 In our study, the family Erysipelotrichaceae, along with one of its genera, Solobacterium, and one of its species, Solobacterium moorei, were also associated with a decreased CRC risk. However, in two gut microbiome studies,41, 42 Erysipelotrichaceae and Solobacterium moorei were significantly enriched in CRC patients compared to healthy controls. This inconsistency may be derived from the different roles of oral and gut microbiota in CRC etiology, considering that for Fusobacterium nucleatum, previous gut microbiome studies had associated this species with increased CRC risk while in the present oral microbiome study no association was found between this species and CRC risk. We also found a lower abundance of Carnobacteriaceae and a higher prevalence of three genera and two species among CRC cases than among controls, while no study has investigated them in association with CRC risk.

We found that in the phylum Bacteroidetes, in addition to the oral pathogen Prevotella intermedia, abundance of Prevotella denticola and Prevotella sp. oral taxon 300 and prevalence of Prevotella sp. oral taxon 304 were also associated with an increased CRC risk, while abundance of Prevotella melaninogenica was associated with a decreased CRC risk. Among them, Prevotella melaninogenica and Prevotella sp. oral taxon 300 showed independent associations based on mutual adjustment analyses. There are no reports available regarding the associations of these four species with any cancers. We also found that prevalence of nine taxa from other three phyla, namely Proteobacteria, Spirochaetes and SR1, were associated with an increased risk for CRC. However, no additional studies have investigated these taxa in relationship with CRC risk.

Most associations were observed in both African‐Americans and European‐Americans, while generally the associations were slightly more significant among African‐Americans, e.g. the oral pathogens Treponema denticola and Prevotella intermedia. Considering that the sample size of African‐Americans (N = 515) was three times that of European‐American participants (N = 159) in our study, these differential associations were not unexpected. However, several studies have demonstrated that there may be racial differences in the microbiome of different human habitats, including the oral cavity.43 Hence, the differential associations among African‐Americans and European‐Americans in our study may be also derived from the racial differences in oral microbiome. For example, despite the smaller sample size of European‐Americans, associations of the genus Lautropia and the species Lactobacillus salivarius with CRC risk were stronger among European‐Americans than among African‐Americans.

To the best of our knowledge, our study is the first prospective study to investigate the influence of the oral microbiome on the risk of developing CRC. We conducted sensitivity analyses to exclude those patients who were diagnosed with CRC within 2 years after the collection of biological samples and the corresponding controls. The results did not change materially, indicating that our findings were not likely the result of reverse causation. In our study, 231 cases and 461 controls were included. We have 89% statistical power to detect an association for CRC risk, with an OR of 1.76 for bacterial taxa with the increment of taxa abundance. However, the power for rare bacteria taxa is limited. Especially for some bacterial species, there is a misclassification based on 16S rRNA sequencing data, which will further lower the statistical power. Nevertheless, the prospective design of our study with stored pre‐diagnostic oral rinse samples provides some of the first opportunities to examine whether the oral microbiota are predictive of near‐term risk of one of the most common cancers afflicting the US population, especially African‐Americans.

In summary, in this prospective nested case–control study, we found that multiple oral bacterial taxa were associated with subsequent CRC risk. These results raise the possibility that the oral microbiome may play an important role in CRC etiology. Further studies with a larger sample size are needed to confirm the identified associations and estimate the potential utilization of the oral microbiota for CRC early detection or prevention.