Description of Sporanaerobium hydrogeniformans gen. nov., sp. nov., an obligately anaerobic, hydrogen-producing bacterium isolated from Aravali hot spring in India.

An obligately anaerobic bacterium XHS1971T, capable of degrading cellulose and xylan, was isolated from a sediment sample of Aravali hot spring, Ratnagiri, India. Cells of strain XHS1971T were Gram-stain-negative, spore-forming, motile, long-rods. Growth was observed at temperatures 30-50 °C (optimum 40-45 °C), pH 5.0-10.0 (optimum pH 8.0) and NaCl concentrations 0-0.5% (optimum 0%). Generation time of strain XHS1971T was 5 h under optimised growth conditions. Strain XHS1971T showed the ability to metabolise different complex and simple sugars constituting lignocellulosic biomass. Glucose was fermented majorly into hydrogen, formic acid, acetic acid, and ethanol, whereas carbon dioxide, butyric acid, lactic acid and succinic acid were produced in traces. 16S rRNA gene analysis of strain XHS1971T revealed < 94.5% homology with Cellulosilyticum lentocellum DSM5427T followed by Cellulosilyticum ruminicola JCM14822T, identifying strain as a distinct member of family Lachnospiraceae. The major cellular fatty acids (> 5%) were C14:0, C16:0, C18:0, and C16:1 ω7c. The genome size of the strain was 3.74 Mb with 35.3 mol% G + C content, and genes were annotated to carbohydrate metabolism, including genes involved in the degradation of cellulose and xylan and the production of hydrogen, ethanol and acetate. The uniqueness of strain was further validated by digital DNA-DNA hybridisation (dDDH), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) values of 22%, 80%, and 63%, respectively, with nearest phylogenetic affiliates. Based on the detailed analyses, we propose a new genus and species, Sporanaerobium hydrogeniformans gen. nov., sp. nov., for strain XHS1971T (= MCC3498T = KCTC15729T = JCM32657T) within family Lachnospiraceae.

"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.

BACKGROUND: The majority of literature on bacterial flora in the disc stands disadvantaged in utilizing traditional culture methods and targeting a single bacterium, Cutibacterium acnes. PURPOSE: 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. STUDY DESIGN: Experimental case-control study. METHODS: 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. RESULTS: 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. CONCLUSION: 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. CLINICAL SIGNIFICANCE: These findings invite further investigation into causal relationships of bacterial profile with disc degeneration phenotypes as well as phenotype-driven clinical treatment protocols.

Bacteria in human lumbar discs - subclinical infection or contamination? Metabolomic evidence for colonization, multiplication, and cell-cell cross-talk of bacteria.

BACKGROUND CONTEXT: 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. PURPOSE: 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). STUDY DESIGN: An observational case-control study. PATIENT SAMPLE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. CLINICAL SIGNIFICANCE: 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.

Actinomyces ruminis sp. nov., an obligately anaerobic bacterium isolated from the rumen of cattle.

An obligately anaerobic, rod-shaped, Gram-stain-positive, non-spore-forming, non-motile bacterial strain; designated as CtC72T was isolated from the rumen of cattle. The 16S rRNA gene sequence similarity of less than 98.65% revealed the strain as a member of the genus Actinomyces, nearest to but distinct from Actinomyces qiguomingii DSM 106201T, Actinomyces ruminicola DSM 27982T, Actinomyces procaprae JCM 33484T, Actinomyces succiniciruminis TISTR 2317, Actinomyces glycerinitolerans TISTR 2318. The low values of digital DNA-DNA hybridization (< 70%) and average nucleotide identity (< 95%) further highlighted the distinctive nature of strain CtC72T from its closest relatives. The strain CtC72T could grow at temperatures between 30 and 50 °C (optimum 40 °C), pH between 6.0 and 9.0 (optimum 7.5-8.0), and NaCl between 0 and 1.5% (optimum 0%). The strain hydrolysed cellulose and xylan and utilised a range of mono-, di-, and oligo-saccharides as a source of carbon and energy. Glucose fermentation resulted in acetic acid and formic acid as major metabolic products, while propionic acid, lactic acid, and ethanol as minor products along with CO2 production. The DNA G + C content of strain CtC72T was 68.40 (mol%, Tm) and 68.05 (%, digital). Major cellular fatty acids (> 10%) were C16:0, C18:1 ω9c, and C18:1 ω9c DMA. Based on these data, we propose that strain CtC72T be classified as a novel species, Actinomyces ruminis sp. nov., under the genus Actinomyces. The type strain is CtC72T (= KCTC 15726T = JCM 32641T = MCC 3500T).