“Are we barking up the wrong tree? Too much emphasis on Cutibacterium acnes and ignoring other pathogens”— a study based on next-generation sequencing of normal and diseased discs

• Published in: The spine journal 2023-10, Vol.23 (10), p.1414-1426
• Main Authors: Rajasekaran, Shanmuganathan, Vasudevan, Gowdaman, Easwaran, Murugesh, Devi Ps, Narmatha, Anand K S, Sri Vijay, Muthurajan, Raveendran, Tangavel, Chitraa, Murugan, Chandhan, B T, Pushpa, Shetty, Ajoy Prasad, Kanna, Rishi Mugesh
• Format: Article
• Language: English
• Subjects: Bacterial metabolites; C. acnes; Disc degeneration; Dysbiosis; Metabolomics; Microbiome; Modic changes; Next-generation sequencing; Quorum sensing
• ISSN: 1529-9430; 1878-1632
• DOI: 10.1016/j.spinee.2023.06.396

The majority of literature on bacterial flora in the disc stands disadvantaged in utilizing traditional culture methods and targeting a single bacterium, Cutibacterium acnes. Our objective was to document the diversity in the bacterial flora between normal and degenerated discs for shortlisting potential pathogens using next-generation genomic tools. Experimental case-control study. Researchers employed 16S metagenome sequencing to profile bacterial diversity in magnetic resonance imaging normal healthy discs from brain-dead organ voluntary donors (n=20) and 40 degenerated disc samples harvested during surgery (Modic [MC]=20 and non-Modic [NMC]=20). The V3-V4 region was amplified using universal bacterial primers 341F and 806R, and the libraries were sequenced using Illumina NovoSeq 6000 platform. Statistical significance was set at bacteria with a minimum of 100 operational taxonomic unit (OTU) and present in at least 70% of the samples. The quality check-filtered reads were processed using the QIIME-2 pipeline. The OTU clustering and taxonomic classification were carried out for the merged reads using the Greengenes/SILVA reference database. Validation was done by identification of bacterial metabolites in samples using the liquid chromatography–mass spectrometry approach. Abundant bacteria differing widely in diversity, as evidenced by Alfa and Beta diversity analysis, were present in all control and degenerative samples. The number of bacterial genera was 27 (14-gram-positive: 13-gram-negative) in the control group, 23 (10-gram-positive: 11-gram-negative) in the Modic group, and 16 (11-gram-positive: 5-gram-negative) in the non-Modic group. In the Modic group, gram-negative bacteria OTUs were found to be predominant (more than 50% of the total bacteria identified), whereas in control and non-Modic groups the OTUs of gram-positive bacteria were predominant. Species-level analysis revealed an abundance of opportunistic gram-negative pathogens like Pseudomonas aeruginosa, Sphingomonos paucibacillus, and Ochrobactrum quorumnocens in the discs with Modic changes, more than in non-Modic discs. The presence of bacterial metabolites and quorum-sensing molecules like N-decanoyl-L-homoserine lactone, 6-hydroxynicotinic acid, 2-aminoacetophenone, 4-hydroxy-3-polyprenylbenzoate, PE (16:1(9Z)/18:0) and phthalic acid validated the colonization and cell-cell communication of bacteria in disc ruling out contamination theory. Cutibacterium acnes was not the predominant