SUPPLEMENTARY MATERIAL

Association of coffee and tea intake with the oral microbiome: results from a large cross- sectional study

Brandilyn A. Peters, Marjorie L. McCullough, Mark P. Purdue, Neal D. Freedman, Caroline Y. Um, Susan M. Gapstur, Richard B. Hayes, Jiyoung Ahn

Author affiliations: Division of Epidemiology, Department of Population Health, NYU School of Medicine, New York, NY, USA (BAP, RBH, JA) Epidemiology Research Program, American Cancer Society, Atlanta, GA, USA (MLM, CYU, SMG) Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, USA (MPP, NDF) NYU Perlmutter Cancer Center, New York, NY, USA (RBH, JA) Supplementary Table 1. Association of α- and β-diversity metrics with coffee and tea intake in the CPS-II and PLCO cohorts.

CPS-II (n=457) PLCO (n=481) Shannon index β (95% CI)a Pa β (95% CI)a Pa Coffee 0.003 (-0.036, 0.042) 0.864 -0.004 (-0.022, 0.014) 0.655 Tea 0.071 (0.017, 0.126) 0.011 0.031 (-0.002, 0.065) 0.067 Weighted UniFrac Pb Pb Coffee 0.79 0.84 Tea 0.28 0.003 aParameters are from linear regression models with the Shannon index (averaged over 100 iterations of rarefied OTU table at 3,000 sequence reads/sample) as outcome and continuous coffee or tea intake (cups/day) as the predictor. All models were adjusted for age, sex, study, current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups/day; in tea and coffee models, respectively). bPs from PERMANOVA of weighted UniFrac distance with continuous coffee and tea intake (cups/day) as the predictor, adjusting for age, sex, study, current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups/day; in tea and coffee models, respectively). Supplementary Table 2. Association of α- and β-diversity with coffee and tea intake, using a no coffee/no tea reference group and excluding only tea (n=75) or only coffee (n=177) drinkers from coffee and tea models, respectively.

Categorical Continuous None <1 c/day [1-3) c/day ≥3 c/day Per cup per day Shannon index β (95% CI)a P-trendb β (95% CI)a Pa Coffeec Ref. 0.034 (-0.197, 0.264) -0.025 (-0.242, 0.193) -0.025 (-0.236, 0.186) 0.489 0.002 (-0.015, 0.019) 0.811 Tead Ref. -0.05 (-0.291, 0.192) 0.153 (-0.102, 0.408) 0.148 (-0.117, 0.413) 0.002 0.042 (0.013, 0.07) 0.005

Weighted e b,e e P P-trend P UniFrac Coffeec Ref. 0.57 0.65 0.21 0.17 0.51 Tead Ref. 0.15 0.95 0.72 0.002 0.003 aParameters are from linear regression models with Shannon index (averaged over 100 iterations of rarefied OTU table at 3,000 sequence reads/sample) as outcome. All models were adjusted for age, sex, study (CPS-IIa, CPS-IIb, PLCOa, PLCOb), current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea drinking status (yes/no; in tea and coffee models, respectively). bTrend tests across groups were done by entering the categorical variables into the models as continuous terms. cSample sizes for coffee analysis: none n=54, <1 c/day n=139, [1-3) c/day n=354, ≥3 c/day n=316. dSample sizes for tea analysis: none n=54, <1 c/day n=498, [1-3) c/day n=140, ≥3 c/day n=69. ePs from PERMANOVA of weighted UniFrac distance, adjusting for age, sex, study (CPS-IIa, CPS-IIb, PLCOa, PLCOb), current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea drinking status (yes/no; in tea and coffee models, respectively). Supplementary Table 3. Association of α- and β-diversity with coffee and tea intake in the PLCO cohort, excluding participants who reported adding sugar to their coffee or tea (information on sugar added to coffee/tea was available in PLCO only).

Shannon index β (95% CI)a Pa Coffeeb -0.006 (-0.027, 0.015) 0.583 Teac 0.026 (-0.015, 0.067) 0.215

Weighted UniFrac Pd Coffeeb 0.35 Teac 0.005 aParameters are from linear regression models with the Shannon index (averaged over 100 iterations of rarefied OTU table at 3,000 sequence reads/sample) as outcome and continuous coffee or tea intake (cups/day) as the predictor. All models were adjusted for age, sex, study (CPS-IIa, CPS-IIb, PLCOa, PLCOb), current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups per day; in tea and coffee models, respectively). bParticipants drinking either no coffee or drinking coffee without sugar included in coffee analysis (n=348). cParticipants drinking either no tea or drinking tea without sugar included in tea analysis (n=345). dPs from PERMANOVA of weighted UniFrac distance with continuous coffee and tea intake (cups/day) as the predictor, adjusting for age, sex, study (CPS-IIa, CPS-IIb, PLCOa, PLCOb), current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups per day; in tea and coffee models, respectively).

Supplementary Table 4. Association of α- and β-diversity with regular and decaffeinated coffee and tea intake.

Regular Decaffeinated Shannon index β (95% CI)a Pa β (95% CI)a Pa Coffee 0.003 (-0.013, 0.019) 0.736 -0.001 (-0.03, 0.027) 0.939 Tea 0.034 (0.001, 0.067) 0.041 0.055 (0.001, 0.11) 0.047

Weighted UniFrac Pb Pb Coffee 0.502 0.317 Tea 0.008 0.054 aParameters are from linear regression models with the Shannon index (averaged over 100 iterations of rarefied OTU table at 3,000 sequence reads/sample) as outcome and continuous regular or decaf coffee or tea intake (cups/day) as the predictor. All models were adjusted for age, sex, study (CPS-IIa, CPS-IIb, PLCOa, PLCOb), current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups per day; in tea and coffee models, respectively). bPs from PERMANOVA of weighted UniFrac distance with continuous regular or decaf coffee and tea intake (cups/day) as the predictor, adjusting for age, sex, study (CPS-IIa, CPS-IIb, PLCOa, PLCOb), current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups per day; in tea and coffee models, respectively).

Supplementary Table 5. Association of α- and β-diversity with coffee and tea intake stratified by smoking status.

Never smokers (n=427) Former smokers (n=439) Current smokers (n=72) Shannon index β (95% CI)a Pa β (95% CI)a Pa β (95% CI)a Pa Coffee -0.016 (-0.042, 0.01) 0.23 0.01 (-0.014, 0.035) 0.41 0.001 (-0.051, 0.052) 0.98 Tea 0.049 (0.012, 0.086) 0.009 0.048 (-0.001, 0.096) 0.054 -0.028 (-0.127, 0.072) 0.59

Weighted UniFrac Pb Pb Pb Coffee 0.525 0.806 0.448 Tea 0.117 0.019 0.083 aParameters are from linear regression models with the Shannon index (averaged over 100 iterations of rarefied OTU table at 3,000 sequence reads/sample) as outcome and continuous coffee or tea intake (cups/day) as the predictor. All models were adjusted for age, sex, study (CPS-IIa, CPS-IIb, PLCOa, PLCOb), BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups per day; in tea and coffee models, respectively). bP from PERMANOVA of weighted UniFrac distance with continuous coffee and tea intake (cups/day) as the predictor, adjusting for age, sex, study (CPS-IIa, CPS-IIb, PLCOa, PLCOb), BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups per day; in tea and coffee models, respectively).

Supplementary Table 6. Association of coffee and tea intake as categorical or continuous variables with presence/absence of oral microbial taxaa.

Categorical Continuous Mean % b c b normalized Fold change (95% CI) P-trend Fold change (95% CI) P carriage count Coffee None <1 c/day [1-3) c/day ≥3 c/day Per cup per day

Synergistetes (Phylum) 1.27 29.74 Ref. 1.08 (0.62, 1.87) 1.16 (0.72, 1.88) 1.53 (0.95, 2.46) 0.06 1.03 (0.98, 1.09) 0.25

Tea None <1 c/day [1-3) c/day ≥3 c/day Per cup per day

Firmicutes; Clostridia; Clostridiales; 0.9 33.48 Ref. 0.91 (0.64, 1.28) 1.48 (0.94, 2.32) 1.61 (0.91, 2.85) 0.03 1.13 (1.03, 1.23) 0.008 Lachnospiraceae_[XIV]; Shuttleworthia (Genus) Firmicutes; Clostridia; Clostridiales; Lachnospiraceae_[XIV]; Shuttleworthia; satelles 0.82 33.48 Ref. 0.91 (0.64, 1.28) 1.48 (0.94, 2.32) 1.61 (0.91, 2.85) 0.03 1.13 (1.03, 1.23) 0.008 () aTaxa included in the table are those from main text Table 4 with mean normalized count<2. bParameters are from logistic regression models adjusted for age, sex, study (CPS-IIa, CPS-IIb, PLCOa, PLCOb), current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups/day; in tea and coffee models, respectively). cTrend tests across groups were done by entering the categorical variables into the models as continuous terms.

Supplementary Table 7. Association of regular and decaffeinated coffee and tea intake as continuous variables with abundance of oral microbial taxa. Taxa that were significant in the main analysis (total coffee and tea intake) are included here. Parameters are from DESeq2 models adjusted for age, sex, study (CPS‐IIa, CPS‐IIb, PLCOa, PLCOb), current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups/day; in tea and coffee models, respectively). Per cup per day Per cup per day Taxon ID Level (Regular) p‐value q‐value (Decaf) p‐value q‐value Coffee 534_2027 Firmicutes; Bacilli; Lactobacillales; Carnobacteriaceae Family 1.03 (1.02, 1.05) 1.60E‐05 0.00058 0.98 (0.96, 1.01) 0.27 0.58 534_2027 Firmicutes; Bacilli; Lactobacillales; Carnobacteriaceae; Granulicatella Genus 1.03 (1.02, 1.05) 0.00012 0.0083 0.99 (0.97, 1.02) 0.7 0.97 363_W028 Synergistetes Phylum 1.02 (1.00, 1.04) 0.072 0.51 1.00 (0.98, 1.03) 0.73 0.87 Tea 588_6183 Actinobacteria; Actinobacteria; Bifidobacteriales Order 0.96 (0.92, 1.00) 0.067 0.24 0.91 (0.84, 0.99) 0.021 0.15 588_6183 Actinobacteria; Actinobacteria; Bifidobacteriales; Bifidobacteriaceae Family 0.94 (0.89, 1.00) 0.04 0.19 0.87 (0.78, 0.97) 0.012 0.2 775_7609 ; Class 0.93 (0.90, 0.97) 0.00051 0.0041 1.00 (0.95, 1.07) 0.87 0.98 775_7609 Bacteroidetes; Flavobacteriia; Order 0.94 (0.91, 0.98) 0.0011 0.011 0.96 (0.90, 1.03) 0.28 0.58 775_7609 Bacteroidetes; Flavobacteriia; Flavobacteriales; Flavobacteriaceae Family 0.93 (0.89, 0.97) 0.001 0.037 0.97 (0.90, 1.04) 0.38 0.69 322AK152 Bacteroidetes; Flavobacteriia; Flavobacteriales; Flavobacteriaceae; Bergeyella Genus 0.94 (0.90, 0.99) 0.014 0.16 0.92 (0.85, 0.99) 0.034 0.43 707_3932 Firmicutes; Bacilli Class 0.94 (0.91, 0.97) 0.00019 0.0031 1.02 (0.96, 1.07) 0.56 0.86 626_4326 Firmicutes; Bacilli; Bacillales Order 0.96 (0.92, 0.99) 0.02 0.1 0.95 (0.90, 1.02) 0.15 0.35 626_4326 Firmicutes; Bacilli; Bacillales; Gemellaceae Family 0.94 (0.90, 0.98) 0.0033 0.049 0.95 (0.89, 1.02) 0.18 0.51 707_3932 Firmicutes; Bacilli; Lactobacillales Order 0.96 (0.93, 0.99) 0.0043 0.03 0.99 (0.94, 1.05) 0.79 0.87 457F0268 Firmicutes; Clostridia Class 1.02 (0.99, 1.05) 0.13 0.43 1.06 (1.01, 1.11) 0.014 0.14 457F0268 Firmicutes; Clostridia; Clostridiales Order 1.03 (1.00, 1.06) 0.051 0.21 1.05 (1.00, 1.11) 0.071 0.26 095_9956 Firmicutes; Clostridia; Clostridiales; Lachnospiraceae_[XIV]; Shuttleworthia Genus 1.16 (1.09, 1.23) 3.80E‐06 0.00027 1.06 (0.95, 1.18) 0.29 0.83 095_9956 Firmicutes; Clostridia; Clostridiales; Lachnospiraceae_[XIV]; Shuttleworthia; satelles Species 1.21 (1.12, 1.30) 2.10E‐06 0.00053 1.07 (0.95, 1.21) 0.28 1 201BS011 Fusobacteria; Fusobacteriia Class 1.04 (1.01, 1.07) 1.90E‐02 0.1 1.03 (0.99, 1.08) 0.17 0.45 201BS011 Fusobacteria; Fusobacteriia; Fusobacteriales Order 1.06 (1.03, 1.09) 0.0004 0.0084 1.01 (0.96, 1.07) 0.67 0.83 706_6164 Proteobacteria; Betaproteobacteria; Neisseriales; Neisseriaceae; Kingella; oralis Species 0.91 (0.85, 0.97) 3.90E‐03 0.19 0.88 (0.79, 0.97) 0.014 0.42 Supplementary Table 8. Association of coffee and tea intake as continuous variables with abundance of oral microbial taxa in the CPS‐II and PLCO cohorts. Taxa that were significant in the main analysis (total coffee and tea intake) are included here. Parameters are from DESeq2 models adjusted for age, sex, study (CPS‐IIa, CPS‐IIb, or PLCOa, PLCOb), current smoking, BMI (kg/m2), energy intake (kcal/day), ethanol intake (grams/day), and coffee or tea intake (cups/day; in tea and coffee models, respectively). Per cup per day Per cup per day Cochran's Taxon ID Taxonomy Level (CPS‐II) p‐value q‐value (PLCO) p‐value q‐value Q p‐value Coffee 534_2027 Firmicutes; Bacilli; Lactobacillales; Carnobacteriaceae Family 1.03 (1.02, 1.05) 1.60E‐05 0.00058 0.98 (0.96, 1.01) 0.27 0.58 0.002 534_2027 Firmicutes; Bacilli; Lactobacillales; Carnobacteriaceae; Granulicatella Genus 1.03 (1.02, 1.05) 0.00012 0.0083 0.99 (0.97, 1.02) 0.7 0.97 0.024 363_W028 Synergistetes Phylum 1.02 (1.00, 1.04) 0.072 0.51 1.00 (0.98, 1.03) 0.73 0.87 0.33 Tea 588_6183 Actinobacteria; Actinobacteria; Bifidobacteriales Order 0.96 (0.92, 1.00) 0.067 0.24 0.91 (0.84, 0.99) 0.021 0.15 0.24 588_6183 Actinobacteria; Actinobacteria; Bifidobacteriales; Bifidobacteriaceae Family 0.94 (0.89, 1.00) 0.04 0.19 0.87 (0.78, 0.97) 0.012 0.2 0.22 775_7609 Bacteroidetes; Flavobacteriia Class 0.93 (0.90, 0.97) 0.00051 0.0041 1.00 (0.95, 1.07) 0.87 0.98 0.038 775_7609 Bacteroidetes; Flavobacteriia; Flavobacteriales Order 0.94 (0.91, 0.98) 0.0011 0.011 0.96 (0.90, 1.03) 0.28 0.58 0.49 775_7609 Bacteroidetes; Flavobacteriia; Flavobacteriales; Flavobacteriaceae Family 0.93 (0.89, 0.97) 0.001 0.037 0.97 (0.90, 1.04) 0.38 0.69 0.37 322AK152 Bacteroidetes; Flavobacteriia; Flavobacteriales; Flavobacteriaceae; Bergeyella Genus 0.94 (0.90, 0.99) 0.014 0.16 0.92 (0.85, 0.99) 0.034 0.43 0.57 707_3932 Firmicutes; Bacilli Class 0.94 (0.91, 0.97) 0.00019 0.0031 1.02 (0.96, 1.07) 0.56 0.86 0.012 626_4326 Firmicutes; Bacilli; Bacillales Order 0.96 (0.92, 0.99) 0.02 0.1 0.95 (0.90, 1.02) 0.15 0.35 0.93 626_4326 Firmicutes; Bacilli; Bacillales; Gemellaceae Family 0.94 (0.90, 0.98) 0.0033 0.049 0.95 (0.89, 1.02) 0.18 0.51 0.77 707_3932 Firmicutes; Bacilli; Lactobacillales Order 0.96 (0.93, 0.99) 0.0043 0.03 0.99 (0.94, 1.05) 0.79 0.87 0.23 457F0268 Firmicutes; Clostridia Class 1.02 (0.99, 1.05) 0.13 0.43 1.06 (1.01, 1.11) 0.014 0.14 0.21 457F0268 Firmicutes; Clostridia; Clostridiales Order 1.03 (1.00, 1.06) 0.051 0.21 1.05 (1.00, 1.11) 0.071 0.26 0.57 095_9956 Firmicutes; Clostridia; Clostridiales; Lachnospiraceae_[XIV]; Shuttleworthia Genus 1.16 (1.09, 1.23) 3.80E‐06 0.00027 1.06 (0.95, 1.18) 0.29 0.83 0.16 095_9956 Firmicutes; Clostridia; Clostridiales; Lachnospiraceae_[XIV]; Shuttleworthia; satelles Species 1.21 (1.12, 1.30) 2.10E‐06 0.00053 1.07 (0.95, 1.21) 0.28 1 0.1 201BS011 Fusobacteria; Fusobacteriia Class 1.04 (1.01, 1.07) 1.90E‐02 0.1 1.03 (0.99, 1.08) 0.17 0.45 0.91 201BS011 Fusobacteria; Fusobacteriia; Fusobacteriales Order 1.06 (1.03, 1.09) 0.0004 0.0084 1.01 (0.96, 1.07) 0.67 0.83 0.16 706_6164 Proteobacteria; Betaproteobacteria; Neisseriales; Neisseriaceae; Kingella; oralis Species 0.91 (0.85, 0.97) 3.90E‐03 0.19 0.88 (0.79, 0.97) 0.014 0.42 0.6