Cindy Boer

Microbiome Composition, Joint Pain and Inflammation | 233 5.2 Results Rotterdam Study Microbiome cohort profile For 1,427 participants from the Rotterdam Study (RSIII) we determined the gastroin- testinal-microbiome composition by taking the stool microbiome as a proxy for the in- testinal microbiome. In the Rotterdam Study Microbiome cohort, we sequenced two hypervariable regions of the bacterial 16S rRNA gene, hypervariable regions V3 and V4. After quality control, the 16S reads were directly mapped against the Silva 16S sequence database (v128) using RDP classifier for taxonomic classification. Classification predic- tion was done on multiple taxonomic levels: domain, phylum, class, order, family and genus. Gastrointestinal-microbiome composition of the 1,427 participants in the Rot- terdam Study Microbiome cohort is schematically presented Figure 1 . In total, there are 596 single taxonomies in our cohort, with unknown and unclassified bacteria excluded, as these could not be identified for clinical therapeutic relevance. At phylum level, the dominant phyla are Firmicutes (77.8%) and Bacteriodites (12.5%), followed by Proteo- bacteria (4.9%) and Actinobacteria (4.1%, Figure. 1). This is in concordance with other large-scale population-cohorts of Caucasian adults[26,27]. General characteristics of the Rotterdam Study Microbiome cohort are presented in Table 1 . The study popu- lation (n=1,427) consisted of 57.5% women (n=821) and was slightly obese, with an average body mass index (BMI) of 27.5. A total of 124 individuals had radiographic knee OA, while 285 participants reported knee OA pain (WOMAC pain score>0). The major- ity of participants reporting knee pain was female (n=206). The average WOMAC pain score was also significantly higher in females compared to males (p-value=1.1×10 −07 , Student’s T-test, Table 1 ). Streptococcus abundance is associated with OA knee pain First, we examined whether the overall microbiome composition was different across knee WOMAC pain scores and OA severity (Kellgren-Lawrence radiographic OA sever- ity scores). We found that knee WOMAC pain significantly contributes to the intestinal microbiome β-diversity as evaluated at genus level (Aitchison distance, r2=0.0014, p-value=0.005, PERMANOVA, Supplementary Figure 1 ). This association was attenu- ated when BMI was added to the model and significance was lost (r2=0.00088, p-val- ue=0.143, PERMANOVA). The intra-individual gastrointestinal-microbiome diversity (α-diversity, Shannon index and inverse Simpson) was not associated with knee WOM- AC pain. For knee OA severity (KLsum, n=941), we did not identify an association with gastrointestinal microbiome α- and β-diversity ( Supplementary Table 1 ).