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The accumulating evidence associating sub-clinical infection with disc degeneration (DD) and the controversy of contamination versus infection mandates a further understanding of the microbial activity in the disc and host-microbiome interaction. To utilize a novel approach of metabolomics to probe the presence of bacterial metabolites involved in colonization, survival, and replication in human lumbar intervertebral discs (LIVD). An observational case-control study. Nucleus pulposus from the LIVD of three brain-dead voluntary organ donors (MRI normal and classified as controls) and of three patients undergoing surgery for disc degeneration (DD) (cases) were utilized. Untargeted metabolite profiling was carried out in six discs (3-controls and 3-cases) after extraction using methanol: acetonitrile: water (2:2:1) solvent system and acquired through HPLC-MS/MS platform using C18 reversed-phase column. From the total IVD metabolome, microbial metabolites were filtered by mapping against HMDB, ChEBI, SigMol, Siderophore database, ecdmb database, and PaMet databases. The biological functions of the metabolites were then studied by MSEA pipeline from Metaboanalyst, and the enrichment ratio, p-value, and Variably Importance Projection scores of the metabolites were calculated. Degeneration responsive changes in the abundance of the microbial metabolites were calculated based on the peak intensities between the control and cases. Mass spectrometry identified a total of 17601 and 15003 metabolites, respectively, in the control and degenerated discs. Preliminary mapping of the above metabolites against HMDB indicated the multiple sources, and of these, 64 metabolites were of microbial origin, accounting for 1.6% of the total IVD metabolome. Principle Component Analysis and Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA) showed distinct clustered patterns between control and disc degene'ration, indicating a strong variation in concentration, peak, and spectral values of the 64 metabolites between controls and cases. After the exclusion of metabolites that were also associated with humans, drugs, and food, 39 metabolites specific to bacteria were isolated. Nine were primary metabolites related to bacterial growth and survival, and the remaining 30 were secondary metabolites related to different environmental stress response activities. The three significant pathways (p<.001) which were predominant in the bacterial metabolites were autoinducer-2 biosynthesis, peptidoglycan biosynthesis, and chorismate pathway. In addition, a significant fold change of >1.0 was found for nine metabolites which included (S)-14-Methyilhexadecanoic acid related to P. acnes , 9-OxoODE, and 13-OxoODE related to gut flora, vibriobactin – a siderophore, tuberculosinol and iso-tuberculosinol, virulence factors of M. tuberculosis. There was also upregulation of Autoinducer- 2, an important "Quorum sensing molecule" involved in bacterial cross-talk. We identified several bacterial-specific metabolites participating in bacterial growth, survival, and cross-talk pathways. These were found in both groups but up-regulated in degenerated discs. The presence of Quorum sensing molecules and cell-cell interactions provides firm proof of colonization and growth. These findings indicate that the bacterial presence may not be mere contamination but could be colonization with a possible role in infection-mediated inflammation in DD. Proof of subclinical infection as an initiator of DD and documentation of exact germ and drug sensitivity will change the way millions of patients with non-specific low back pain (NSLBP) are treated across the world. [ABSTRACT FROM AUTHOR]

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. 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. Experimental case-control study. 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). N/A. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Study Design: Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD). Purpose: To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis. Overview of Literature: Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed. Methods: IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LCMS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases. Results: A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins. Conclusions: This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies. [ABSTRACT FROM AUTHOR]

Background Context: The true understanding of aging and disc degeneration (DD) is still elusive. MRI has not helped our attempts to understand the health and disease status of the discs as it reflects mainly the end morphologic changes and not the changes at a molecular level. Understanding degeneration at a molecular level through proteomics might allow differentiation from normal aging and also aid in the development of biomarkers for early diagnosis and preventive therapies.Purpose: To utilize proteomics to understand the molecular basis of healthy, aging, and degenerating discs and conclusively differentiate normal aging and degeneration.Study Design: Proteomic analysis of human intervertebral disc samples.Methods: L4-L5 disc samples from three groups were acquired and subjected to proteomic analysis. Samples from individuals aged in the second, third, and fourth decades were used to represent young healthy discs (Group A). Those from MRI normal donors aged in the fifth, sixth, and seventh decades represented normal aging (Group B). Five degenerated discs obtained from patients at surgery represented degeneration (Group C). The entire proteome map and alteration in protein expressions were further analyzed using bioinformatics analysis. This was a self-funded project.Results: There were 84 common proteins. Specific proteins numbered 225 in A, 315 in B, and 283 in C. By gene ontology biological process identification, Group A predominated with extracellular matrix organization, cytoskeletal structural and normal metabolic proteins. Group B differed in having additional basal expression of immune response, complement inhibitors, and senescence proteins. Group C was different, with upregulation of proteins associated with oxidative stress response, positive regulators of apoptosis, innate immune response, complement activation and defense response to gram positive bacteria indicating ongoing inflammaging.Conclusions: Our study documented diverse proteome signatures between the young, aging and degenerating discs. Inflammaging was the main differentiator between normal biological aging and DD.Clinical Significance: Multiple inflammatory molecules unique to DD were identified, allowing the possibility of developing specific biomarkers for early diagnosis and thereby provide evidence-based metrics for preventive measures rather than surgical intervention and also to monitor progress of the disease. [ABSTRACT FROM AUTHOR]

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 bacteria in any of the three groups of discs and in fact was in the 16th position in the order of abundance in the control discs (0.72%), seventh position in the Modic discs (1.41%), and 12th position (0.53%) in the non-Modic discs. This study identified a predominance of gram-negative bacteria in degenerated discs and highlights that Cutibacterium acnes may not be the only degeneration-causing bacteria. This may be attributed to the environment, diet, and lifestyle habits of the sample population. Though the study does not reveal the exact pathogen, it may pave the way for future studies on the subject. These findings invite further investigation into causal relationships of bacterial profile with disc degeneration phenotypes as well as phenotype-driven clinical treatment protocols. [ABSTRACT FROM AUTHOR]