Comparative metagenomic analysis of human intervertebral disc nucleus pulposus and cartilaginous end plates

Abstract

Study designThe diversity of microflora inhabiting endplate (EP) and nucleus pulposus (NP) tissues of human intervertebral disc (IVD) was profiled through NGS-supported 16S rRNA amplicon sequencing. Sixteen EP and their corresponding NP were excised from the brain-dead voluntary organ donors with no clinical history of low back pain, and 12 herniated and 8 degenerated NP tissues isolated from the patients undergoing spinal surgery were subjected to study the alteration in the microbial diversity.Objective(s)To understand in normal IVD, whether the colonization of bacteria to the NP is through the EP in discs with intact annulus fibrosus. To identify significantly differing microbial population(s) between normal and diseased IVD (NP).Background of the studyThere is increasing evidence for subclinical infection by fastidious low, growing bacteria to be a cause of disc degeneration. Although the presence of bacteria in NP has been reported well in literature, the source of bacteria is not clearly proved as the disc is avascular in healthy condition. Documentation of similar bacterial populations in the EP and NP may add proof that bacterial inoculation of NP occurs via the EP.Materials and methodsSixteen EP and their corresponding NP excised from brain-dead voluntary organ donors with no history of back pain and 20 diseased discs collected from patients undergoing microdiscectomy/fusion surgery were used for profiling microbiome through 16S rRNA amplicon sequencing using primers specific for V1-V9 hypervariable regions. Changes in bacterial diversity and abundance were analysed to identify the key microbial populations in normal IVD NP and EP tissues and those significantly altered in diseased IVD (NP).ResultsNP and EP shared a similar spectrum of microbiome but with varying abundance. The five dominant phyla identified were Proteobacteria, Firmicutes, Actinobacteria, OD1, and Bacteroidetes. Proteobacteria was found to be the most abundant phyla in both NP (62%) and EP (53%) of the normal IVD. This was followed by Firmicutes (16%), Actinobacteriota (11%), OD1 (Parcubacteria) (7.6%), and Bacteroidetes (2%) in NP and Firmicutes (23.4%), OD1 (Parcubacteria) (17.6%), Actinobacteriota (2.8%), and Bacteroidetes (2.6%) in EP, respectively. Under diseased conditions, Proteobacteria (68%) was dominant when compared with other phyla. However, there was no significant difference in the abundance of Proteobacteria between the normal and diseased discs. Interestingly, the other dominant phyla such as Firmicutes (Normal-NP: 16.2%; Diseased-NP: 4.02%) and Actinobacteria (Normal-NP: 11%; Diseased-NP: 0.99%) showed a significant reduction in degenerated discs. To understand the key microbial populations that are significantly altered during disease, correlation analysis was performed among the three phyla, which revealed a negative correlation in the ratio of Actinobacteria + Firmicutes vs. Proteobacteria (p = 0.001) in DD.ConclusionResults of our study clearly demonstrated a similar bacterial diversity but with varying abundance between the EP and NP, suggesting the existence of the endplate–nucleus pulposus axis in the normal IVD microbiome. Further, our results have indicated that the changes in the abundance of Actinobacteria + Firmicutes vs. Proteobacteria during DDD need further investigation.

Insight into the Causal Relationship between Gut Microbiota and Back Pain: A Two Sample Bidirectional Mendelian Randomization Study

“…Lachnospiraceae and Ruminococcaceae belongs to Firmicutes . A metagenomic [ 40 ] study demonstrated a significant decrease in the abundance of Firmicutes , Actinobacteria , and Bacteroidetes in diseased discs. An animal study [ 41 ] found higher relative abundance of Lachnospiraceae and Ruminococcaceae and lower relative abundance of Lactobacillus in lumbar disc herniation (LDH) mice treated with Lactobacillus s16 .…”

The role of the complement system in disc degeneration and Modic changes

“…14,16,28,91,92 Disc herniations in most cases also disrupt the CEP because most herniations occur as avulsion type between the CEPs and bony endplate. 91,92 Consequently, endplate damage and herniations are seen as gate for bacterial entry to the disc (i.e., C. acnes), 93,94 which might lead to complement activation via the classical and alternative pathway in the disc 95 C. acnes are found in disc tissue, specifically in MC discs, but not in the BM. 37,96 Hence, it remains to be elucidated if (i) the complement system can be locally activated in disc, (ii) which complement factors are present in disc tissue, either through local production by disc cells or through influx from the BM, and (iii) if and how bacterialmediated complement activation in the disc also affects the MC marrow (Figure 3, Nr.…”

Mitochondria: It is all about energy

“…The persistence of pathological microbes harms the integrity of the intestinal barrier, mediating immune and mitochondrial failure. Even biomechanical disorders, such as intervertebral discopathies, seem to be associated with disc infection caused by oral and skin-derived pathogens ( Rajasekaran et al, 2020 ; Li et al, 2022b ; Shanmuganathan et al, 2022 ). Besides the aforementioned mechanisms, dysbiosis reduces the supply of calcium to the bone and impairs the ability to manufacture vitamins K2 and D and other important immuno-metabolic substances.…”