Poster Presentation: Detection of microbial DNA in gut and intervertebral discs of germ free and specific pathogen free mice

Introduction: Recent evidence suggests that the microbiome may play a key role in intervertebral disc degeneration (IDD). Bacterial infiltration into the disc tissue could contribute to IDD and low back pain, and several studies have identified Cutibacterium acnes in pathological discs. This study evaluates whether mice are a suitable model for examining IVD microbial colonization by analyzing IVD and gut samples from specific pathogen-free (SPF) mice, which lack defined pathogens but retain normal microbiota, and germ-free (GF) mice, which are completely free of microorganisms. Methods: DNA was extracted using the QIAamp UCP Pathogen Mini Kit from IVD and gut tissues of female C57BL6J mice (n = 3) under SPF or GF conditions. DNA concentrations were measured with Qubit. A defined bacterial mixture (ZymoBIOMICS™ Spike-in Control I) was added before 16S rRNA amplification. Sequencing was performed using nanopore technology on a MinION device, followed by base calling with Dorado and filtering with Nanofilt (1400–1600 bp, q-score > 9) Results: No bacterial DNA was detected in any IVD samples from SPF or GF mice, indicating absence of microbial load. This finding matched the results from GF gut samples, confirming sterility. In contrast, bacterial DNA was detected in gut tissue from two of three SPF mice. Analysis of the ZymoBIOMICS™ Microbial Community Standard demonstrated a strong correlation between expected and measured relative abundances, supporting methodological accuracy. Discussion: These findings indicate that SPF mice may be unsuitable for studying a native IVD microbiome, as discs lacked detectable bacterial DNA. The study also confirmed the GF status of GF mice. Future work will include faecal samples to further understand the gut-disc axis and the potential relationship between gut microbes and IVD health. Keywords: Microbiome, low back pain, intervertebral disc, degeneration Acknowledgments: Supported by SNSF Weave Grant (#320030E₂24175) and DFG (#437213841)