Cindy Boer

234 | Chapter 5.2 Table 1: General characteristics of the Rotterdam Study Microbiome cohort Rotterdam Study Microbiome Females Males Total Cohort participants 821 606 1.427 Age (years) 56.8 (5.9) 56.9 (5.9) 56.9 (5.9) BMI (kg/m2) 27.4 (4.9) 27.6 (4.0) 27.5 (4.5) Alcohol (g/day) 1.3 (2.7) 1.3 (2.3) 1.3 (2.6) Smoking (y/n) 98 smokers/721 non smokers 97 smokers/507 non smokers 195 current smokers PPI (y/n) 182 users/638 non-users 114 users/492 non-users 296 current PPI users NSAIDs (y/n) 127 users/693 non-users 51 users/555 non-users 178 current NSAID users Knee phenotypes Knee OA (y/n) 84 cases/456 controls 40 cases/361 controls 124 cases/817 controls KLSum score 1.0 (1.4) 0.7 (1.2) 0.8 (1.3) WOMAC-Pain score 1.2 (2.6) 0.6 (1.9) 0.9 (2.3) WOMAC-Pain score>0 206 79 285 α-diversity metrics Shannon Index 4.0 (4.1) 4.0 (4.0) 4.0 (0.5) Inverse Simpson Index 26.0 (12.1) 25.5 (12.2) 25.8 (12.2) Depicted are the mean and the SD (standard deviation) in parenthesis PPI oral use of proton pump inhibitors, NSAIDs oral use of non-steroidal anti-inflammatory drugs, OA osteoarthritis, WOMAC Western Ontario and McMaster Osteoarthritis Index To gain more insight into which gut-microbiome taxonomies drive the associ- ation with knee WOMAC pain, we performed multivariate association analysis on the 256 taxonomies remaining after additional QC. After adjusting for age, sex and tech- nical covariates, we found four microbiome abundances significantly (FDR<0.05) as- sociated with knee WOMAC-pain severity ( Table 2 ). These were all in the same clade; from class, order, family leading to the bacterial genus of Streptococcus (genus: coef- ficient =5.0×10 −03 , FDR p-value=1.2×10 −05 , MaAsLin, Table 2 ; See Supplementary Data 1 for the full summary statistics). After additional correction for possible con- founders (smoking and alcohol consumption)[28], Streptococcus spp. abundance re- mained significantly associated with knee WOMAC pain (coefficient =4.8×10 −03 , FDR P-value=3.8×10 −05 , MaAsLin, Supplementary Table 2 ). This association is largely in- dependent of BMI (coefficient =4.1×10 −03 , FDR p-value=2.1×10 −03 , MaAsLin, Supple- mentary Table 2 ). Ethnicity has recently been put forward as a possible confounder for gastrointestinal-microbiome composition26. However, after exclusion of individuals with non-European ancestry (n=163) from our analysis, Streptococcus spp. remained associated with knee WOMAC pain (coefficient =5.0×10 −03 , FDR p-value=5.8×10 −05 , MaAsLin, Supplementary Table 3 ), also, after adjustment for confounders (coeffi- cient =4.3×10 −03 , FDR p-value=1.9×10 −03 , MaAsLin). Altogether, we found that the gut microbiome, and in particular a greater relative abundance of Streptococcus spp. , is sig- nificantly associated with higher knee WOMAC pain independent of smoking, alcohol consumption and BMI.