Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction. The technological advancements have facilitated an untargeted approach to explore the microbiome in low-biomass samples with the use of next-generation sequencing. Specifically in tissue that was long thought to be sterile, such as the intervertebral disc, this has led to a shift in perspective and has sparked intense debates within the research community. However, a study on the MC microbiome recently published by Rajasekaran et al. challenged this with the hypothesis that the resident IVD microbiome is in a state of dysbiosis in MC11. Therefore, it is crucial to ensure that such a paradigm shift changing finding is reproducible and is built upon a methodology that is robust, replicable, and accurate. Without a consensus pipeline, the results of different studies cannot be compared, and the groundbreaking potential is lost. The purpose of this study was two-fold: (i) to analyze the microbiome of the Modic type 1 (MC1), MC2, and non-Modic (nonMC) discs and (ii) to emphasize the importance of reaching a methodological consensus including decontamination strategies for low-biomass samples.

Methods. A total of 70 discs (24 nonMC, 25 MC1 and 21 MC2) were collected from patients undergoing spinal fusion surgery. To control for contamination, 10 samples with buffer only were included throughout all steps. DNA was isolated from all 70 discs, the V3-V4 region was amplified and then sequenced using Illumina NextSeq 2000. Reads mapping to the human reference genome GRCh38p13, resulting from off-target amplification, were filtered out2. The remaining reads were trimmed for adapter content. The data was processed with the QIIME-2 pipeline and the obtained amplicon sequence variants (ASVs) were annotated with SILVA database. Bacterial contaminants were identified and removed with Decontam R package, using the prevalence method comparing the composition of the positive samples to the negative controls.

Results. Disc degeneration measured by Pfirrmann grade was similar in all groups (p = 0.448). Alpha-diversity presented no difference between the groups. Beta-diversity indicated that the nonMC group has a greater diversity compared to MC1 and MC2 (Figure 1). The total number of ASVs detected was 180 of which 80% were found in all samples. On a phyla level, proteobacteria were most prevalent, followed by firmicutes and actinobacteria. Firmicutes significantly decreased their abundance in MC1 and MC2 compared to nonMC discs (Figure 3). MC1 discs had less gram-positive bacteria compared to both nonMC (q = 0.013) and MC2 (q = 0.001), suggesting a favourable environment for gram-positive bacteria. in contrast MC2 discs had significantly more gram-negative bacteria (q < 0.001) (data not shown). The widely debated C. acnes was identified in all three groups, without significant group differences (data not shown). Compared to the disc microbiome studies of Rajasekaran et al. our findings show vast differences in dominant bacterial species1.

Discussion. The direct comparison of our findings to other studies emphasizes the differences in results generated either through methodological or ethnicity-based differences and highlights the importance of a consensus that needs to be agreed upon to allow addressing clinically relevant questions.