Fusobacterium nucleatum in biopsied tissues from colorectal cancer patients and alcohol consumption in Korea

The roles of individual bacteria and their relationship in the development of colorectal cancer (CRC) remain unclear. We aimed to determine the prevalence of CRC-associated bacteria using quantitative real-time PCR (qPCR) or 16S rRNA analysis and the statistical correlations of patient demographics and clinical characteristics comprising alcohol consumption with CRC-associated bacteria. We determined the prevalence of five CRC-associated bacterial species in 38 CRC patients (39 samples) and 21 normal individuals using qPCR, and the relative abundance of bacterial taxa in the gut microbiome was assessed using 16S rRNA analysis. Fusobacterium nucleatum was the only bacterium that was significantly (P < 0.0001) more prevalent in the cancer tissue (82.1%) than in the normal tissue (0%) by qPCR. 16S rRNA analysis showed a significant correlation between six operational taxonomic units (OTUs), namely, the genera Fusobacterium, Peptostreptococcus, Collinsella, Prevotella, Parvimonas, and Gemella, in patients with CRC. An integrated analysis using 16S rRNA data and epidemiological characteristics showed that alcohol consumption was significantly correlated with the abundance of Fusobacterium OTUs. The correlation of alcohol consumption with the abundance of Fusobacterium OTUs in cancer tissue discovered using 16S rRNA analysis suggests a possible link between alcohol metabolism and subsequent tumorigenesis caused by F. nucleatum.


Scientific Reports
| (2020) 10:19915 | https://doi.org/10.1038/s41598-020-76467-7 www.nature.com/scientificreports/ be accompanied by shifts in the gut microbiome and metabolome 10 . Furthermore, a recent study showed that the gut microbiome may promote the progression of CRC 11 . Therefore, it is crucial to identify bacteria that may be associated with the development of CRC and to assess which other factors can elicit transformation from a healthy gut microbiome to a tumorigenic microenvironment. Diet is the most well-known factor that influences the gut microbiome, and differences in diet can substantially affect the entire gut microenvironment 12 . In South Korea, CRC is one of the most prevalent types of cancer; however, few metagenomic studies have been conducted to determine the association between the gut microbiome and CRC development 13 . The composition of the gut microbiome may be influenced by factors other than diet, including alcohol consumption 14 . Numerous epidemiological studies have shown that alcohol consumption is strongly associated with the incidence of CRC 15 . Retrospective propensity score matching analysis (adjusted hazard ratio: 1.86) revealed that high alcohol consumption increased the development of CRC 16 . Alcohol consumption is considerably high in South Korea 17 and may be a causative factor underlying the high prevalence of CRC in Korea 18 . However, to our knowledge, the effect of alcohol consumption on the gut microbiome has not been investigated thoroughly in South Korea.
In this study, we aimed to determine the prevalence of five CRC-associated bacteria (ETBF, E. faecalis, colB + E. coli, F. nucleatum, and S. gallolyticus) in the biopsied tissues of patients with CRC using quantitative real-time PCR (qPCR) and the relative abundance of operational taxonomic units (OTUs) by 16S rRNA analysis. Furthermore, we determined the associations between the epidemiological characteristics of CRC and the gut microbiome and alcohol consumption.

Comparison of demographics and clinical characteristics of cases and controls. A total of 39
CRC samples and 21 control samples were examined (Table 1). No significant differences between the groups were observed with respect to epidemiological characteristics, apart from BMI and hypertension. BMI was lower in CRC patients (22.9 ± 2.9) than in controls (24.8 ± 2.7), and hypertension was more prevalent in controls (76.2%, n = 16/21) than in CRC patients (46.2%, n = 18/39). The CRC cases consisted of 71.8% colon cancers and 28.2% rectal cancers, with right-and left-sided cancers accounted for 28.2% and 71.8% of the cases, respectively.
Prevalence of CRC-associated bacteria as assessed by qPCR. The prevalence of the five CRC-associated bacteria was examined in the carcinoma tissues (CTs) of CRC patients and in normal tissues (NTs) of controls using qPCR (Table 2). F. nucleatum was most frequently detected in CRC patients and was not detected in the NTs of controls (82.1% and 0%, respectively; P < 0.0001). However, no significant difference in the prevalence of colB + E. coli, ETBF, E. faecalis, or S. gallolyticus was observed between CRC patients and controls.
Prevalence of F. nucleatum by tumor stage and tissue type. We analyzed the association of F. nucleatum positivity in CRC patients with that in controls based on clinicopathological features. Patients with CRC were classified by tumor stage (early stage [I/II] or late stage [III/IV]). F. nucleatum was detected very frequently in both early (92.3%) and late (76.9%) stages; however, this difference was not significant (Fig. 1A). With respect to the tissue type, the prevalence of F. nucleatum was significantly higher in CTs (82.1%) than in adjacent normal tissues (ATs) (39.9%) as well as distal normal tissues (DTs) (33.3%) in CRC patients; no significant difference was observed between ATs and DTs (Fig. 1B). When the cycle threshold (Ct) values obtained from qPCR were compared among tissue types in F. nucleatum-positive cases, they were found to be significantly lower in CTs (Mann-Whitney U test, P < 0.0001). F. nucleatum was markedly enriched in CTs compared to ATs (Supplementary Fig. S1). The fadA gene, which encodes a virulence factor implicated in adhesion and invasion, was statistically more frequent in the CTs of CRC patients (69.2%) than in the NTs of controls (9.5%) (Fig. 1C).
16S rRNA analysis. Sequencing of the V3 and V4 regions in the 16S rRNA gene produced 171,988 ± 81,412 sequencing reads, on average. After quality filtration, clustering and taxonomy assignment were performed at the genus level by QIIME software 19 . A total of 698 OTUs were generated and used for further statistical analysis. In a diversity analysis, no significant difference in α-or β-diversity was observed between CRC cases and controls ( Supplementary Fig. S2).
When comparing CRC cases with controls, the proportions of six OTUs, namely, Peptostreptococcus, Collinsella, Prevotella, Parvimonas, Fusobacterium, and Gemella, were significantly different after false discovery rate correction for multiple testing (Fig. 2). Among the epidemiological characteristics with continuous values, Fusobacterium-positive CRC patients were significantly younger than Fusobacterium-negative patients (64.0 ± 10.5 vs. 73.1 ± 9.3; P = 0.034), which was also true for Parvimonas-positive patients (60.4 ± 8.5 vs. 69.0 ± 10.9; P = 0.011; Table 3). With respect to epidemiological characteristics with binomial values, Fusobacterium was associated with alcohol consumption and KRAS mutation (P = 0.088 and P = 0.094), and Parvimonas was associated with tumor location and KRAS mutation, although the difference was only marginally significant (P = 0.070, Table 4). Fusobacterium was observed in 14 of the 15 heavy drinkers (93.3%) but not in 11 of the 17 non/light drinkers (64.7%). The proportion of Fusobacterium OTUs was significantly higher in heavy drinkers than in non/ light drinkers (P = 0.003); no corresponding pattern was observed in controls (Fig. 3). With respect to tumor location, the proportion of the Parvimonas OTU was significantly higher in the descending colon and in the rectum (P < 0.001; Supplementary Fig. S3) than in other parts of the colon. Tumor stage and metastasis were not significantly correlated with the presence of specific bacteria. When the Fusobacterium OTU proportions were compared between cases and controls using Ct values, qPCR-positive cases showed higher OTU abundance values than qPCR-negative cases, and the significant positive correlation between these values is shown in Fig. 4.

Discussion
CRC is a major health threat in many countries; however, its etiology and underlying mechanisms are still not comprehensively understood. The determination of a cancer's etiology can result in the development of preventive or therapeutic measures. Several studies have demonstrated enrichment of fecal or tissue samples of CRC patients with specific bacterial pathogens. ETBF is a well-known pathogen of the gastrointestinal tract that can trigger a carcinogenic multistep process through B. fragilis toxin [3][4][5]20 . However, we observed no significant difference in the prevalence of ETBF between CRC cases and controls, and its prevalence (35.9%) was significantly lower than that in a previous study (88.5%) 20 . Table 1. Comparison of demographics and clinical characteristics of CRC patients and controls. NA not applicable. a One patient had two adenocarcinomas: one in the hepatic flexure and one in the rectum. b Four of the 12 controls with tubular adenoma had their tubular adenomas removed during colonoscopy.

P-value Cases (n = 38) Controls (n = 21)
Age (years) 65. 9   www.nature.com/scientificreports/ In a recent study 21 , it was shown by qPCR that only 36.4% of CRC cases were positive for ETBF, and an equal distribution of B. fragilis was found in tumors, paired normal tissue and diverticula. Thus, the prevalence and distribution of ETBF should be determined by follow-up investigations. It has been shown that pathogenic E. coli can synthesize colibactin, which is genotoxic 22 . The prevalence of colB + E. coli in CRC patients might be the link between E. coli and CRC. In our study, the prevalence of colB + E. coli in CRC cases was twice as high as that in controls, but the difference was not statistically significant, probably due to an insufficient sample size. S. gallolyticus and E. faecalis were also regarded as CRC-associated bacteria in previous reports 23,24 ; however, recent studies showed that the roles of S. gallolyticus and E. faecalis in CRC carcinogenesis were controversial due to their low prevalence 21,25,26 . In our study, neither species was frequently detected in cancer patients ( Table 2).  www.nature.com/scientificreports/ In recent decades, F. nucleatum has attracted attention as a potential cause of CRC 4,6,27 . The role of F. nucleatum in CRC pathogenesis has not been comprehensively understood, but at least four mechanisms have been suggested to describe the same 28  Fusobacterium species are strictly anaerobic and are difficult to isolate by culture. In this study, the prevalence of F. nucleatum in the tissues of CRC patients as tested by qPCR (82.1%) was notably higher than that obtained using anaerobic culture (7.7%; data not shown). Therefore, non-culture-dependent detection methods such as  www.nature.com/scientificreports/ qPCR or 16S rRNA analysis may be essential for screening Fusobacterium species or for studying its epidemiology in a population during the development of CRC. The presence of F. nucleatum was significantly correlated with both the early and late stages of CRC (Fig. 1A). This finding suggests that F. nucleatum may be involved in CRC carcinogenesis from an early stage, and one review indicated an association of F. nucleatum with carcinomas throughout the different stages of CRC progression 29 . Analysis of the abundance of F. nucleatum by tissue type showed that the bacteria seemed to be more prevalent in CTs than in ATs and NTs (Fig. 1B). This finding is consistent with previous studies showing that a high level of Fusobacterium colonization is associated with CRC 27,30 .  www.nature.com/scientificreports/ FadA is a well-known oncogenic virulence factor that can induce oncogenic gene expression and promote the growth of CRC cells 6 . Therefore, FadA is regarded as a major virulence factor of F. nucleatum in CRC, and 69.2% of CT samples were positive for the fadA gene. However, some discrepancies between F. nucleatum presence and fadA gene frequency were observed in our study (Fig. 1C). Thus, it may be necessary to evaluate both F. nucleatum presence and fadA gene frequency to accurately determine the presence and virulence of F. nucleatum 31 . Interestingly, a recent study suggested that the fadA gene is not specific to F. nucleatum and that it is also present in other Fusobacterium species 32 . This finding indicates the need for further research regarding the role of other Fusobacterium species in CRC carcinogenesis.
The gut microbiome comprises numerous bacterial species that interact with each other, and it is possible that other bacteria in addition to the five known CRC-associated bacteria that were examined in the present study may affect the carcinogenesis of CRC. Thus, CRC metagenomic analysis is now commonly used to analyze this complex bacterial community. According to our 16S rRNA analysis, six genera, namely, Peptostreptococcus, Collinsella, Prevotella, Parvimonas, Gemella, and Fusobacterium were significantly abundant in CRC patients. Kwong et al. reported that P. anaerobius promoted CRC carcinogenesis in Apc Min/+ mouse models, and a correlation between Peptostreptococcus species and human CRC was reported previously 33 . The role of Collinsella species in CRC has rarely been investigated; however, Ai et al. reported that Collinsella species were specifically correlated with CRC in the Chinese population using metagenomic analysis 34 . The association of Prevotella species with CRC was reported in a previous study 35 , and our results are compatible with this report. Parvimonas and Gemella species were also reported as potential causative agents that may be responsible for CRC carcinogenesis 36 . However, it is difficult to determine which bacteria are the primary cause of CRC, as the gut microbiome is too complex and can vary by population 37 .
Based on an integrated analysis of 16S rRNA data and epidemiologic characteristics, we found a marginally significant correlation between Fusobacterium occurrence and a history of alcohol consumption. It has been shown that alcohol consumption is one of the major contributors to CRC carcinogenesis 38 . The generation of acetaldehyde and other metabolites from alcohol activates cancer-promoting signals 39 . Moreover, it was suggested that ethanol oxidation by intestinal anaerobes, including Fusobacterium species, under aerobic conditions in the colon and rectum may also play an important role in the pathogenesis of ethanol-related CRC 40 . To our knowledge, the present study was the first to determine the relationship between alcohol consumption and the presence of Fusobacterium using 16S rRNA analysis. In the integrated statistical analysis, a cutoff of 1% was used to determine the positivity of each OTU 41 . It is interesting to note that 87.5% (28/32) of qPCR-positive cases and 76.2% (16/21) of qPCR-negative controls showed the same 16S rRNA analysis results when a cutoff of 1% was used. This cutoff seems to be the appropriate threshold to determine abundance in 16S rRNA analysis in CRC tissues, though more investigations are required to confirm the cutoff.
The limitations of this study were the relatively small sample size, which was associated with low statistical significance, and the fact that tubular adenoma could not be analyzed as an independent group due to the small sample size. However, the simultaneous analysis of the presence of specific bacterial pathogens, 16S rRNA analysis, patient demographics and clinical characteristics may be an advantage of this study. The detection of F. nucleatum in stool can be helpful for diagnosing CRC in patients as a biological tumor marker if used concurrently with other tumor markers, and an appropriate testing method should be developed and validated.
In conclusion, F. nucleatum was significantly prevalent in the CTs of patients with CRC and frequently present in both early and late stages. These data support that this bacterium is strongly associated with the development of CRC. The correlation between alcohol consumption and the abundance of Fusobacterium OTUs in cancer tissue discovered using 16S rRNA analysis suggests a possible link between alcohol metabolism and changes in the gut microbiome and subsequent tumorigenesis caused by F. nucleatum.

Methods
Study population. A total of 38 CRC patients and 21 controls who were diagnosed using colonoscopy at a tertiary-care hospital between June 2015 and November 2016 were enrolled. Patients who received preoperative radiation, chemotherapy, and/or antibiotic treatment within 4 weeks were excluded. One patient had two primary adenocarcinoma lesions; thus, 39 CRC samples were analyzed in this study. Among the 21 controls, 12 individuals had tubular adenomas removed during colonoscopy, and 9 individuals showed no specific findings in colonoscopy. Patients with tubular adenomas were analyzed as controls because most of the polyps could be regarded as low risk due to their pathologic findings and small size (< 10 mm), and the resection margin was clear in all samples. G* power 3.1.9.7 analysis was conducted using the chi-square test to justify the appropriate sample size, and the test showed a significance level of 0.05 and a power of 0.81. Patient demographics and clinical characteristics were investigated by reviewing the medical records and interviews. Total alcohol consumption was calculated based on the history of alcohol intake-determined based on factors such as beverage type, amount, and frequency of consumption-and patients were classified as non/light drinkers (0-1 drink/day) or heavy drinkers (≥ 2 drinks/day). All patients were classified as smokers (persons who are/were smokers) or nonsmokers (persons who never smoked) based on their smoking history.
Ethics approval and consent to participate. This  16S rRNA analysis. The V3 and V4 regions of the 16S rRNA gene (400-450 bp) were PCR amplified using target-specific primers and adapters for Illumina sequencing. Next-generation sequencing was performed on a MiSeq platform (Illumina, San Diego, USA) using the MiSeq Reagent Kit V2 with 500 cycles. Paired-end sequences were merged using PANDASEQ software 46 , and clustering of operational taxonomic units (OTUs) and taxonomy assignment were performed using QIIME software 19 . The observed diversity and Chao1 and Shannon indexes for α-diversity were calculated using QIIME. Principal components were calculated and plotted for β-diversity estimation. The relative proportion of each OTU was compared between cases and controls as well as with respect to epidemiological characteristics. OTUs present in > 1% of all OTUs were regarded as positive 16 . The mock community was analyzed before clinical specimen testing to validate the 16S rRNA analysis results.

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.