Association between dietary intake and the prevalence of tumourigenic bacteria in the gut microbiota of middle-aged Japanese adults

The relative contribution of diet to colorectal cancer (CRC) incidence is higher than that for other cancers. Animal models have revealed that Escherichia coli containing polyketide synthase (pks+ E. coli) in the gut participates in CRC development. The purpose of this cross-sectional study was to examine the relationship between dietary intake and the prevalence of pks+ E. coli isolated from the microbiota in faecal samples of 223 healthy Japanese individuals. Dietary intake was assessed using a previously validated brief-type self-administered diet history questionnaire. The prevalence of pks+ E. coli was evaluated using faecal samples collected from participants and specific primers that detected pks+ E. coli. The prevalence of pks+ E. coli was 26.9%. After adjusting for baseline confounders, the prevalence of pks+ E. coli was negatively associated with the intake of green tea (odds ratio [OR], 0.59 [95% confidence interval (CI) 0.30–0.88] per 100 g/1,000 kcal increment) and manganese (OR, 0.43 [95% CI 0.22–0.85] per 1 mg/1,000 kcal increment) and was positively associated with male sex (OR, 2.27 [95% CI 1.05–4.91]). While futher studies are needed to validate these findings, these results provide insight into potential dietary interventions for the prevention of CRC.

intake of food and beverages, and the pks + E. coli status. A comparison of food and beverage intake between participants with and without pks + E. coli is shown in Table 2. The median green tea consumption was significantly lower in the pks + E. coli group than in the pks -E. coli group (107.1 g, interquartile range [IQR] 21.4-324.6 vs 150.0 g, IQR 53.6-375.0, p = 0.019). In addition, after adjusting for energy intake using the density method, the median green tea consumption was significantly lower in the pks + E. coli group compared to the pks -E. coli group (53.6 g/1,000 kcal, IQR 12.4-148.2 vs 86.7 g/1,000 kcal, IQR 29.8-257.7, p = 0.010). The same trend was observed in the total amount of non-alcoholic beverages consumed, which included green tea. The median egg intake was significantly lower in the pks + E. coli group than in the pks -E. coli group (18.3 g/1,000 kcal, IQR 11.1-29.3 vs 22.8 g/1,000 kcal, IQR 14.0-33.2, p = 0.043). No difference was observed in the intake of other diets, beverages, or food groups between the two groups. nutrient intake and the prevalence of pks + E. coli. A comparison of the energy and nutrient intake between participants with or without pks + E. coli is shown in Table 3. All nutrient values are derived from food, not supplements. There was no significant difference observed in the overall crude mean nutrient intake between Table 1. Baseline characteristics of particpants with or without pks + E. coli. The p values shown in bold are statistically significant (p < 0.05). ALT alanine transaminase, AST aspartate transaminase, BMI body mass index, FH family history, FPG fasting plasma glucose, FSI fasting serum insulin, HbA1c haemoglobin A1c, HDL-C high-density lipoprotein-cholesterol, LDL-C low-density lipoprotein-cholesterol, SD standard deviation, γ-GTP γ-glutamyl transpeptidase. 1 Continuous variables are shown as mean with SD and were analysed by unpaired t-test. BMI was calculated as body weight (kg) divided by height squared (m 2 ). 2 Category variables are shown as the number of individuals (%) and were analysed using a Χ 2 test. Step counts (step/day) 1 Table 3. Comparison of the crude or energy-adjusted energy and nutrient intake between participants with and without pks + E. coli. The p values shown in bold are statistically significant (p < 0.05). All variables are shown as the mean with standard deviation and were analysed using an unpaired t-test. 1  Dietary intake variables that showed a significant association with the prevalence of pks + E. coli in the multivariate analysis were used in a restricted cubic spline model to evaluate dose-dependent responses to pks + E. coli (Fig. 1). The analysis of the curves for the dietary intake variables and the prevalence OR of pks + E. coli showed that: (1) the prevalence OR of pks + E. coli was significantly lower when green tea consumption exceeded approximately 430 g/day, or 260 g/1,000 kcal/day (reference: those without green tea consumption), and (2) the prevalence OR of pks + E. coli was significantly lower when the intake of manganese exceeded approximately 2.86 mg/day (reference: 1.05 mg /day), or 2.65 mg/1,000 kcal/day (reference: 0.70 mg/1,000 kcal/day).

Discussion
The purpose of this cross-sectional study was to examine the relationship between dietary intake and the prevalence of pks + E. coli in healthy Japanese individuals. After adjusting for confounding factors, our results showed a significant negative association between the prevalence of pks + E. coli and the intake of green tea and manganese. In addition, we showed a significantly higher prevalence of pks + E. coli in males than in females. To our knowledge, this is the first study to show a significant association between dietary intake and the prevalence of pks + E. coli. Furthermore, the results of this study support a hypothesis suggesting an association between dietary intake and CRC risk.
Unlike other cancers, such as lung cancer, no single risk factor accounts for most cases of CRC. Apart from age and male sex, the following risk factors for CRC incidence have been identified and established in previous epidemiological studies: family history of cancer, inflammatory bowel disease, smoking, obesity, diabetes, excessive alcohol consumption, and high consumption of red and processed meat 3 . These established CRC risk factors have been associated with potentially adverse gut microbiome profiles 13,14 , indicating the importance of evaluating the prevalence of tumourigenic bacteria in the gut microbiota. This study indicated that the prevalence of pks + E. coli isolated from faecal matter was 26.9% in our cohort. According to previous studies, the prevalence of pks + E. coli isolated from the colonic epithelium was 20.8% in healthy UK 10 and 22.0% in healthy US individuals 11 . The samples used to evaluate the prevalence of pks + E. coli in previous studies and this current study were tissue and faecal matter, respectively. Although previous studies, as well as the current one, have different evaluation methods for the prevalence of pks + E. coli, the results are relatively similar. Moreover, our results indicating that the prevalence of pks + E. coli was significantly higher in males than in females supports those of previous studies that have reported the risk of CRC to be higher in males compared to females 3,19 . In other words, our results on the prevalence of pks + E. coli are similar to those from previous studies in high-income countries, showing that participant characteristics in our study were unbiased and appropriate.
Taken together the key findings of the current study are as follows: (1) a negative association exists between green tea consumption, and manganese intake and the prevalence of pks + E. coli; (2) a significantly lower OR of pks + E. coli was observed in individuals with a daily intake of 430 g/day green tea compared to those without. Note, the manganese intake derived from green tea contributed to 75.6% of total manganese intake in this cohort, and most of the inter-individual variance in manganese intake was attributed to green tea consumption. Hence, Table 4. Odds ratios of pks + E. coli for the intake of energy-adjusted food, beverage, and nutrients calculated by multivariate logistic regression analysis. Results are shown as odds ratios (ORs) and 95% confidence intervals (CI). Statistical analysis was carried out using the likelihood ratio test for multivariate logistic analysis, and the ORs and 95% CI were estimated. Bold p values are statistically significant (p < 0.05). Adjusted factors included age (continuous), sex (female or male), BMI (continuous), smoking status (never smoker, past smoker, or current smoker), family history (FH) of cancer (yes or no), energy intake (continuous), and step counts (continuous). 1  www.nature.com/scientificreports/ the negative association observed between the intake of manganese and the prevalence of pks + E. coli may be contributory to that of green tea consumption as the results lost significance following adjustment for green tea consumption. The accuracy of estimation of green tea intake by a brief-type self-administered diet history questionnaire (BDHQ) showed a moderate correlation with green tea intake estimated by dietary records (male, r = 0.68; female, r = 0.64). However, it has been reported that the accuracy of the estimated median green tea intake is overestimated by 20-23% 20 . Therefore, a minimum effective amount of green tea, which was significantly associated with reduced prevalence of pks + E. coli in a dose-response relationship, may be 20% lower than approximately 430 g/day (i.e. ~ 340 g [2-3 cups/day]). To our knowledge, there are two systematic reviews 21,22 , five prospective cohort studies [23][24][25][26][27] , and four case and control studies 28-31 that have evaluated the relationship between green tea consumption and CRC risk; however, no consensus has been reached regarding the anti-CRC properties in these studies. The two systematic reviews concluded that the anti-CRC effects of green tea are inadequate and contradictory 21,22 . Only studies targeting East Asians have reported the anti-CRC effects of green tea 26,27,[29][30][31] . Meanwhile, low green tea consumption in non-Asian countries may contribute to the non-significant results of these studies 28 . Green tea consumption for 7 days exhibited beneficial effects in improving lymphocytic DNA damage in middle-aged healthy non-smokers 32 . In addition, green tea catechins have anti-inflammatory properties 33,34 , which help to mitigate against oxidative tissue injury 34 . Subsequent changes in the gut microbiota and reduced intestinal inflammation may then be related to the anti-inflammatory properties of green tea and green tea polyphenols 35 . Although there are no studies describing the relationship between green tea consumption and the prevalence of pks + E. coli, one study has described the effects of green tea consumption on the gut microbiome. Yuan et al. performed an intervention study in healthy Chinese adults and found a significant increase in the Firmicutes to Bacteroidetes ratio isolated from faecal matter after 2 weeks of green tea consumption (400 mL/day) 36 . Firmicutes and Bacteroidetes are two major bacterial phyla that dominate the human gut microbiota. The Firmicutes to Bacteroidetes ratio increases from birth to adulthood and is decreased in advanced ages 37 . Interestingly, higher frequencies of colibactinproducing E. coli and enterotoxigenic Bacteroides fragilis in the colonic epithelium were observed in patients with familial adenomatous polyposis compared to healthy individuals 11 . Their study also reported that mice with guts Figure 1. Association of the intake of green tea (a,b) and manganese (c,d) with the prevalence odds ratio (OR) of colibactin-producing E. coli in a restricted cubic spline logistic regression model. Adjusted factors included age (continuous), sex (female or male), BMI (continuous), smoking status (never smoker, past smoker, or current smoker), family history of cancer (yes or no), energy intake (continuous), and step counts (continuous). The solid line represents the OR. The broken lines show the 95% Confidence Intervals (CIs). If the 95% CI for the OR did not include 1.00, the p value was estimated to be < 0.05. If the 95% CI included 1.00, the p value was estimated to be ≥ 0.05.

Scientific RepoRtS
| (2020) 10:15221 | https://doi.org/10.1038/s41598-020-72245-7 www.nature.com/scientificreports/ co-colonised with colibactin-producing E. coli and B. fragilis had a higher tumour growth rate due to increased levels of interleukin-17 in the colon, as well as DNA damage in the colonic epithelium compared to mice with either bacterial strain alone 11 . These results suggest that green tea consumption significantly reduces the prevalence of pks + E. coli by suppressing the growth of certain microorganisms in the gut microbiome. The detailed mechanisms and causal relationships must be clarified with further intervention studies and fundamental studies. The strength of this study is the verified association between the prevalence of pks + E. coli and dietary intake estimated using a validated dietary assessment tool. By using the approach described above, this study generated a new hypothesis for the association between diet and the prevalence of pks + E. coli as a tumourigenic bacteria. However, this study has certain methodological limitations. First, the temporal and direct causal relationship observed between dietary intake and the prevalence of pks + E. coli could not be inferred as this study is a crosssectional study. Second, although our results show that green tea and manganese intake is negatively associated with the prevalence of pks + E. coli, these food and nutrient intakes estimated by BDHQ have not been fully validated against objective biomarkers. The results may have been affected by systematic errors due to body mass index and gender 38 . In addition, when we examined the association between diet and the prevalence of pks + E. coli, we focused exclusively on diet estimated from a validated BDHQ. We have previously reported that yoghurt consumption increases stool frequency 39 . The results suggest that yoghurt consumption may affect the gut microbiome. Therefore, it is necessary to verify our results using a validated dietary assessment tool other than the BDHQ. Further, we conducted an exploratory investigation of the association between the intake of specitic foods, beverages, and nutrients and the prevalence of pks + E. coli; however, multiple testing problems may arise when multiple tests are used to calculate p values. Finally, there is the possibility of selection bias due to higher health awareness of the participants in this study than in the general population. Of the 750 participants in the Nutrition and Exercise Intervention Study (NEXIS) cohort, 259 adults agreed to participate. As the participation rate is relatively low, volunteer bias may occur. In addition, participants are all from Tokyo metropolitan area with an average age of approximately 58 years. These limitations may prevent the generalisation of the results. Therefore, future studies with larger randomised samples should be used to investigate further the association between diet and the prevalence of pks + E. coli. In addition, the effects of green tea consumption and the intake of manganese on the risk of pks + E. coli should be examined by prospective cohort studies and randomised intervention studies.
Given the rapid Westernisation of diet around the world, there is an urgent need to highlight the importance of diet in the prevention of CRC. Furthermore, the difference in dietary intake between groups may explain the large global differences in cancer burden 40 . Therefore, to develop sustainable, comprehensive, and effective public health programmes for CRC prevention, our study data will provide useful insights into the development effective preventative intervention strategies for CRC. The questionnaire for the lifestyle survey and a kit for faecal collection and storage were mailed to the participants. We used a triaxial accelerometer (Actimarker; EW4800; Panasonic Co., Ltd, Japan) to measure daily step counts as an objective index of physical activity. The participants were instructed to complete the questionnaire for the lifestyle survey (e.g. dietary survey) and collect lumps of faeces of ~ 2 cm diameter (approximately 3 g) at home. The collected faeces were immediately placed in a sealed container and stored in a − 20 °C freezer. The participants were instructed to bring the questionnaire and faecal samples to the National Institute of Health and Nutrition, NIBIOHN within 5 days after faecal collection, at which point they received health examinations, such as anthropometric and blood tests. The investigators, qualified as registered dieticians or nurses, checked the questionnaires and interviewed those with unanswered questions or unclear responses to confirm their answers. A portion of the frozen faeces was transported to the University of Shizuoka by a refrigerated truck and tested for the presence of pks + E. coli. Blood samples were used to measure conventional risk factors for lifestyle-related diseases, such as haemoglobin A1c, triglyceride, and low-density lipoprotein-cholesterol. The study protocol was reviewed and approved by the Research Ethical Review Committee of NIBIOHN (approval number: kenei102 and kenei 3-04). Study procedures, as well as the risks associated with participation, were explained and written informed consent was obtained from all participants. Moreover, all study procedures were performed in accordance with relevant standard international guidelines/regulations.

Methods
Of the participants included in the baseline analysis (n = 259) who provided informed consent, those with a history of cancer (n = 12), gastrointestinal disease (n = 3), diabetes mellitus (n = 13), renal failure (n = 1), and cardiovascular disease (n = 6) were excluded from subsequent analyses. In addition, a participant with extremely low/high mean energy intake estimated by the BDHQ (n = 1) was excluded from the analysis as an outlier (< 600 or > 4,000 kcal/day) 41 . As a result, 223 participants were ultimately included in this study.
Dietary assessment. Dietary 20,43 . Food and beverage items listed in the BDHQ consist of food and beverages commonly consumed in Japan, according to the national health and nutrition survey 44 . This study examined only the frequency of intake of the 58 food and beverage items in the past month. Participants with unanswered questions or unclear responses were asked to confirm their responses during a face-to-face interview. Dietary and nutrient intakes were calculated from the weight of food intake (i.e. calculated according to the portion size and frequency of dietary intake) and nutritional information listed in the Standard Tables of Food Composition in Japan 45 .
Statistical analysis. All data were compared between participants with or without the pks + E. coli. Categorical variables were expressed as numbers and percentages. The chi-square test was used to compare variables between the two groups. Descriptive statistics for continuous variables were expressed as mean and SD or median and IQR, and differences in continuous variables between the two groups were evaluated using the unpaired t test or the Mann-Whitney U test. Forty-two nutrient items 43 and 28 items of food, beverages, and food groups 20 calculated from the BDHQ were used in this study. The details of the food groups are described elsewhere 20 . In accordance with studies on the validation against the BDHQ, food and beverage consumption was expressed as median and IQR 20 , whereas nutrient intake was expressed as mean and SD 43 and we conducted the descriptive statistics in accordance with these studies. In this study, to adjust for energy intake, food and nutrient intakes per 1,000 kcal were calculated using the density method 46 . Crude values and energy-adjusted values calculated using the density method for all food and nutrient intake variables were compared between with-without the pks + E. coli. Multivariate logistic regression analysis was used to adjust for potential confounding factors related to food and nutrient intake and the prevalence of pks + E. coli. We adjusted for age (continuous), sex (female or male), body mass index (continuous), smoking status (never smoker, past smoker, or current smoker), family history of cancer (yes or no), energy intake (continuous), and step counts (continuous). These variables were decided with reference to covariates used in previous studies that examined the association between CRC and green tea consumption [23][24][25][26][27][28][29][30][31] . Participant characteristics, as well as food and nutrient intake that were significantly associated with the prevalence of pks + E. coli (p < 0.05) were used in the analysis. The results of the analysis were expressed as OR and 95% CI. The OR and 95% CI were calculated for food and nutrient intake per unit increment. Food and nutrient intake variables that showed a significant association with pks + E. coli prevalence in the multivariate analysis were used in a restricted cubic spline model, with three knots placed at the 5th, 50th, and 95th percentiles to evaluate dose-dependent responses to pks + E. coli 47 . If the 95% CI for the OR did not include 1.00, the p value was estimated to be < 0.05. If the 95% CI included 1.00, the p value was estimated to be ≥ 0.05. p < 0.05 (two-tailed) was considered statistically significant. All analyses were performed using Stata/MP 15.0 Statistical Software (StataCorp, College Station, TX, USA).