Not All Microbiomes Reflect Chronic Pain: Evidence from the Urinary Tract in a Case-Control Study

Background/Objectives: Chronic pain is increasingly conceptualized as a systemic condition characterized by central sensitization, autonomic dysregulation, and persistent neuroimmune and neuroendocrine alterations. These systemic changes have been linked to microbial dysbiosis, most prominently within the gut microbiome. In contrast, the relevance of the urinary microbiome outside primary urological disease remains poorly understood, particularly in non-urological chronic pain conditions. The objective of this study was to determine whether patients with chronic low back pain exhibit differences in urinary microbial diversity, community composition, or taxon-specific abundance compared with pain-free controls. Methods: In this age- and sex-matched case-control study, midstream urine samples were collected from ten patients with chronic low back pain and ten pain-free controls and analyzed using 16S rRNA gene sequencing (V4 region). Sequence data were processed using nf-core/ampliseq and DADA2. Alpha diversity, beta diversity, and differential abundance were assessed using depth-adjusted models, compositional and phylogenetically informed distance metrics, and ANCOM-BC2, with multiple sensitivity analyses to account for the low-biomass nature of urinary microbiome data. Results: After accounting for sequencing depth, no significant differences in alpha diversity were observed between patients and controls for any metric. Beta diversity analyses revealed no significant differences in overall community composition between groups across all distance measures, and dispersion was comparable between groups. Differential abundance analysis did not identify any bacterial taxa that differed significantly between patients and controls after correction for multiple testing. Conclusions: In this cohort, chronic low back pain was not associated with detectable alterations in the urinary microbiome. These findings suggest that, unlike the gut microbiome, urinary microbial communities may be relatively stable in the context of non-urological chronic pain, highlighting the importance of phenotype specificity and multidimensional approaches in microbiome-based pain research.