Cindy Boer

Microbiome Composition, Joint Pain and Inflammation | 235 5.2 Streptococcus association not driven by oral medication use Proton Pump Inhibitors (PPI) are among the most widely used over-the-counter drugs in the world. They are used to treat gastro-esophageal reflux and prevent gastric-ulcers. Recent research has shown that the gastrointestinal-microbiome composition of PPI us- ers is profoundly different from non-PPI users, mainly due to a strong increase in Lacto- bacilli abundance, driven by Streptococcus spp.[29]. Another potential over-the-counter medication frequently taken by patients with joint complaints are non-steroidal anti-in- flammatory drugs (NSAIDs), which also affect the gastrointestinal microbiome[29]. To investigate, whether the association between Streptococcus spp. abundance and knee WOMAC pain is mediated by use of these drugs, we included PPI and NSAID use as co- variates in our model (in addition to age, sex, technical covariates, smoking, alcohol con- sumption, and BMI). After adjustment for these covariates, the coefficient was attenu- ated, but the association remains significant (coefficient =3.4×10 −03 , p-value=2.4×10 −04 , MaAsLin, Supplementary Table 4). In line with these results, excluding all current PPI users (n=265) from the analysis, greater Streptococcus spp. abundance was signifi- cantly associated with higher knee WOMAC pain (n=1104, coefficient =2.4×10 −03 , p-val- ue=4.0×10 −03 , MaAsLin). Thus, the association between, Streptococcus spp. and knee WOMAC pain was not due to the confounding effect of PPI use. Since knee WOMAC-pain scores were not normally distributed (left-skewed with an overabundance of zeros), we also assessed the association using a Poisson-regression model, which can account for the non-normal distribution. Here as well, a significant association with Streptococ- cus spp. abundance was observed (beta=1.85, p-value=1.4×10 −04 , Poisson-regression). Further sensitivity analysis excluding individuals who did not report pain (n=1,142) or whose knee WOMAC-pain scores were deemed as outliers (WOMAC-pain score >10, >4SD, n=56) resulted in slight changes in the association coefficients. The association, however, remained significant (Supplementary Table 5). Altogether, these analyses show that the association between relative abundance of Streptococcus spp. and knee WOMAC pain is robust. To adjust for possible spurious collinearity between microbe abundancies[31], we have used the isometric log-ratio transformation (ILR). Using ILR, we have compared the relative Streptococcus spp. abundancy against the geometric mean of the abundancy of all other genera. Results show that the ILR transformed Streptococcus spp. abundancy is associated with knee WOMAC pain (p-value=9.9×10 −06 , MaAsLin, Supplementary Table 6 ). The association remained significant after adjusting for smoking, alcohol con- sumption and BMI (p-value=8.4×10 −04 , MaAsLin, Supplementary Table 6 ), and NSAID and PPI use (p-value=8.5×10 −03 , MaAslin, Supplementary Table 6 ). The qPCR results and ILR transformation demonstrate that the association between Streptococcus spp. abundance and knee WOMAC pain is not an artefact of the 16S rRNA sequencing.