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Updated: Jun 22, 2025

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Does the gut microbiome influence disc health and disease? The interplay between dysbiosis, pathobionts, and disc
Shanmuganathan Rajasekaran1, Gowdaman Vasudevan2, Chitraa Tangavel2
1Department of Spine Surgery, Ganga Hospital, 313, Mettupalayam Rd, Coimbatore, India.
Background Context:
Gut microbiome alterations resulting in inflammatory responses have been implicated in many distant effects on different organs. However, its influence on disc health is still not fully investigated.
Purpose:
Our objective was to document the gut biome in healthy volunteers and patients with disc degeneration and to understand the role of gut dysbiosis on human disc health.
Study Design:
Experimental case-control study.
Patient Sample:
We included 40 patients with disc degeneration (DG) and 20 healthy volunteers (HV). HV comprised of age groups 30 to 60 years with no known record of back pain and no clinical comorbidities, with normal MRI. Diseased group (DG) were patients in the same age group undergoing surgery for disc disease (disc herniation-25; discogenic stenosis-15) and without instability (with Modic-20; and non-Modic-20).
Outcome Measures:
N/A.
Methods:
We analyzed 16S V3-V4 rDNA gut metagenome from 20 healthy volunteers (HV) and compared the top signature genera from 40 patients with disc degeneration (DG) across Modic and non-Modic groups. Norgen Stool DNA Kit was used for DNA extraction from ∼200 mg of each faecal sample collected using the Norgen Stool Collection Kit.16S V3-V4 rDNA amplicons were generated with universal bacterial primers 341F and 806R and amplified with Q5 High-Fidelity DNA Polymerase. Libraries were sequenced with 250×2 PE to an average of 0.1 million raw reads per sample (Illumina Novaseq 6000). Demultiplexed raw data was assessed with FastQC, and adapter trimmed reads >Q30 reads were processed in the QIME2 pipeline. Serum C-reactive protein (CRP) was measured by the immunoturbimetry method and Fatty acid-binding protein 5 (FABP5) was measured in albumin-globulin-depleted plasma through global proteome analysis.
Results:
We observed significant gut dysbiosis between HV and DG and also between the Modic and non-Modic groups. In the Modic group, commensals Bifidobacterium and Ruminococcus were significantly depleted, while pathobionts Streptococcus, Prevotella, and Butryvibrio were enriched. Firmicutes/Bacteroidetes ratio was decreased in DG (Modic-0.62, non-Modic-0.43) compared to HV (0.70). Bacteria-producing beneficial short-chain fatty acids were also depleted in DG. Elevated serum CRP and increased FABP5 were observed in DG.
Conclusion:
The study revealed gut dysbiosis, an altered Firmicutes/Bacteroidetes ratio, reduced SCFA-producing bacteria, and increased systemic and local inflammation in association with disc disease, especially in Modic changes. The findings have considerable importance for our understanding and prevention of disc degeneration.
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