European Spine Journal

https://doi.org/10.1007/s00586-020-06446-z

ORIGINAL ARTICLE

Human intervertebral discs harbour a unique microbiome

and dysbiosis determines health and disease
Shanmuganathan Rajasekaran1   · Dilip Chand Raja Soundararajan1 · Chitraa Tangavel2 · Raveendran Muthurajan3 ·
K. S. Sri Vijay Anand1 · Monica Steffi Matchado2 · Sharon Miracle Nayagam2 · Ajoy Prasad Shetty1 ·
Rishi Mugesh Kanna1 · K. Dharmalingam4

Received: 15 October 2019 / Revised: 4 March 2020 / Accepted: 2 May 2020

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Background  To document the role of sub-clinical infections in disc disorders and investigate the existence of microbiome
in intervertebral discs (IVD).
Methods  Genomic DNA from 24 lumbar IVDs [8—MRI normal discs (ND) from brain dead yet alive organ donors, 8—disc
herniation (DH), 8—disc degeneration (DD)] was subjected to 16SrRNA sequencing for profiling the diversity of human
disc microbiome in health and disease. The disc microbiome was further compared to established human gut and skin
microbiomes.
Results  All healthy MRI normal discs from brain dead yet alive organ donors also had a rich bacterial presence. A total of
424 different species (355-ND, 346-DD, and 322-DH) were detected, with 42.75% OTUs being classified at kingdom level,
44% at the phylum level, 22.62% at genus level, and 5.5% at species level. Varying biodiversity and abundance between
healthy and diseased discs were documented with protective bacteria being abundant in normal discs, and putative pathogens
abundant in DD and DH. Propionibacterium acnes had a similar but lower abundance to other pathogens in all three groups
ND (3.07%), DD (3.88%), DH (1.56%). Fifty-eight bacteria were common between gut and IVD microbiomes, 29 between
skin and IVD microbiomes, and six common to gut/skin/IVD.
Conclusion  Our study challenges the hitherto concept of sterility in healthy IVD and documented a microbiome even in MRI
normal healthy discs. The varying abundance of bacteria between ND, DD, and DH documents ‘dysbiosis’ as a possible
etiology of DD. Many known pathogens were identified in greater abundance than Propionibacterium acnes, and there was
evidence for the presence of the gut/skin/spine microbiome axis.

Keywords  Disc degeneration · Sub-clinical infection · Microbiome · Dysbiosis · Inflammaging

Abbreviations
16S rRNA 16 (Svedberg) ribosomal ribonucleic acid
30S Ribosomal subunit contains 16S rRNA
Electronic supplementary material  The online version of this 50S Ribosomal subunit
article (https​://doi.org/10.1007/s0058​6-020-06446​-z) contains DADA2 Data analysis decision and action
supplementary material, which is available to authorized users.
DD Disc degeneration
* Shanmuganathan Rajasekaran DH Disc herniation
rajasekaran.orth@gmail.com DNA Deoxyribose nucleic acid
ESI Electrospray ionization
1
Department of Spine Surgery, Ganga Hospital, 313, GGv138 Green GENES database version 138
Mettupalayam Road, Coimbatore, India
IRB Intuitional Review Board
2
Ganga Research Centre, No 91, Mettupalayam Road, LBP Low back pain
Coimbatore 641030, India
LC Liquid chromatography
3
Department of Plant Biotechnology, Tamil Nadu Agricultural Mascot Software search engine to identify proteins
University, Coimbatore 641003, India
uses MOWSE algorithm
4
Aravind Medical Research Foundation, Madurai 625020, MRI Magnetic resonance imaging
India

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Vol.:(0123456789)

MS/MS Tandem mass spectrometry
MW test Mann–Whitney test
ND Normal disc
OTU Operational taxonomic unit
PICRUSt Phylogenetic investigation of communities
by reconstruction of unobserved states
q2 QIIME2
QIIME Quantitative insights into microbial
ecology
R packages Statistical tool designed by Ross Ihaka and
Robert gentleman
Sequest HT Tandem mass spectrometric data analysis
program used for protein identification
STAMP Statistical analysis of taxonomic and func-
tional profiles
T-test Testing hypotheses on the mean of the
normal distribution
V1–V9 Nine hypervariable regions
TRANSTAN Transplant authority government of Tamil
Nadu, India
HCL Hydrochloric acid
NaCl Sodium chloride
rpm Revolutions per minute
SDS-PAGE Sodium dodecyl sulphate polyacrylamide
gel electrophoresis
fm Femtomole
SPSS Statistical package for the social sciences
PPM Parts per million
Introduction
There is increasing interest and proof of a causative influ-
ence of microbiome in many human diseases. The Nobel
prize-winning discovery of establishing Helicobacter
pylori, a bacteria as the main cause of gastritis and acid
peptic disease, led to a radical shift in the management of
peptic ulcers from surgical treatment to a short regimen of
antibiotics and acid secretion inhibitors [1]. Subsequently,
microbial role in the pathogenesis of many clinical condi-
tions such as obesity, autism, diabetes, inflammatory bowel
diseases, and colorectal cancer has been well established
[2]. Despite accumulating evidence for the presence of low
virulence microorganisms such as Propionibacterium acnes
in diseased intervertebral discs, the role of sub-clinical infec-
tion as an etiology for DDD is still controversial for many
reasons [3].
Firstly, human discs are still considered sterile in health
as they are the largest avascular structures in the body. Most
of the studies have performed their analysis on degenerate
discs and have lacked true controls, to analyse a microbi-
ome of normal discs. Thirdly, most authors have relied on
culture-dependent techniques which are often ineffective
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European Spine Journal
to grow slow growing fastidious organisms [4]. Although
recent studies have documented the presence of diverse
bacteria in degenerated intervertebral discs and discs with
Modic changes, the exact role of the bacterial infection is
still not clear [5]. The concept of disc microbiome is in radi-
cal contrast with the current understanding of disc-related
pathologies. However, its establishment can lead to novel
therapeutic modalities of treatment in low back pain. It
assumes further importance, with the increasing number of
surgical costs and health care expenditure in the manage-
ment of LBP, which is the largest global disabling musculo-
skeletal problem resulting in a huge socioeconomic loss [6].
We present here the first-ever report on the microbiome
of MRI normal human lumbar discs (ND) harvested from
brain dead organ donors with no history of LBP and com-
pare it with herniated (DH) and degenerated (DD) discs,
using high-throughput next-generation sequencing (NGS).
Our results postulate the new and interesting possibility that
‘dysbiosis’ can be an important cause of disc degeneration
(DDD).
Materials and methods
With institutional review board (IRB) approval, we har-
vested eight-disc samples from patients with disc hernia-
tion (DH) and eight from disc degeneration (DD) at L4-L5
level during routine surgical intervention under strict aseptic
precautions. Following disc removal, the tissues were imme-
diately washed with a phosphate buffer solution to remove
blood stains, stored in sterile cryovials, and snap-frozen in
the liquid nitrogen container (− 196 °C). For the control
group, MRI normal discs were harvested under sterile condi-
tions from brain dead yet alive voluntary organ donors with
no history of back pain (ND) and stored similarly (Fig. 1).
Genomic DNA and total proteins extracted from a total of
24 disc tissues (demographic details in Table 1) were sub-
jected to bacterial DNA enrichment, followed by amplicon
sequencing, and in-gel tryptic digestion, followed by mass
spectrometric analysis respectively (Fig. 2; Supplementary
file-1).
Genomic DNA was enriched for bacterial DNA using
­NEB® microbiome kit, amplified using 16S rRNA specific
primers (V1–V9), and sequenced using the Illumina MiSeq
platform. Raw reads were imported into QIIME-2 v-2019.4
for quality control (DADA2-package), taxonomic classifica-
tion, and diversity analysis. Saturation of rarefaction curves
indicated that samples were sequenced to sufficient depth
for capturing the entire spectrum of bacteria. Taxonomic
and functional predictions of the metagenomic profiles were
carried out using PICRUST, STAMP, and R packages.
The presence of bacteria in the discs was identified by
the insilico analysis of the IVD proteome data. The raw files
European Spine Journal

Fig. 1  Intervertebral disc samples harvested from ‘true controls’—
normal discs (ND). We had the unique opportunity of having ‘pure
controls’ from spine segments showing MRI normal discs from brain
dead voluntary organ donors with no history of back pain. The spine
segments were harvested with the approval of TRANSTAN (Trans-
plant Authority of Government of Tamil Nadu) holding registration
number-214/2016. Three to four motion segments were harvested fol-
lowing retrieval of other vital organs and were immediately snap-fro-
zen at − 160 °C. Only MRI normal discs belonging to donors with no
history of back pain were dissected out from the motion segments and
included in the study to represent true normal discs (ND)

(.msf/.raw files) of the IVD proteome was blasted against top Table 1  Demographic details of the samples considered for this study
ten bacterial species identified from metagenome analysis Sample Group Age Sex Disc level Pfir-
of the study with the help of proteome discoverer 2.3 using rmann
mascot and sequest search engines. grade
Contamination was avoided by adopting strict aseptic
1 ND 13 M L4L5 1
precautions during harvest and sample-processing. Besides,
2 ND 22 M L4L5 2
internal controls were used to rule out contamination. We
3 ND 28 M L4L5 1
performed the same genomic analysis on the buffer solution
4 ND 32 F L4L5 2
used in the operating room to rule out contamination from
5 ND 43 M L4L5 2
the solution. The reagent kits used for DNA extraction were
6 ND 45 M L4L5 2
also sequenced. All the internal controls were devoid of bac-
7 ND 55 F L4L5 2
teria, confirming the absence of contamination.
8 ND 63 M L4L5 2
The raw data have been uploaded to the NCBI SRA data-
9 DH 21 M L5S1 3
base (SUB6416370). For comparing the disc microbiome
10 DH 37 M L3L4 2
with the existing human gut and skin microbiome, the cited
11 DH 37 M L4L5 3
databases of previous microbiome studies were used [7–13].
12 DH 38 F L4L5 5
13 DH 38 M L4L5 4
14 DH 43 M L5S1 4
Results 15 DH 43 F L4L5 4
16 DH 44 F L4L5 4
Presence of bacteria in the IVD and its diversity 17 DD 42 M L5S1 4
18 DD 46 F L5S1 4
A rich bacterial presence was noted in all the 24 specimens,
19 DD 42 M L3L4 5
including MRI normal healthy discs (ND); however, their
20 DD 45 F L4L5 4
microbiome differed from that of degenerated (DD) and
21 DD 60 M L4L5 4
herniated discs (DH) both in biodiversity and abundance.
22 DD 69 F L5S1 4
About 42.75% operational taxonomic units (OTUs) were
23 DD 69 F L4L5 5
classified at the kingdom level, 44% at the phylum level,
24 DD 78 M L2L3 4
22.62% at the genus level, and 5.5% at species level. Anno-
tation of OTUs against the GG v138 database identified ND MRI normal discs harvested from brain dead alive organ donors
11 different phyla common to all three groups (Table 2). without low back pain
Five highly abundant phyla were Proteobacteria, Parcu- DH discs harvested from patients undergoing microdiscectomy for
disc herniation
bacteria, Firmicutes, Cyanobacteria, and Actinobacteria.
DD discs harvested from patients undergoing lumbar fusion for
degenerative disc disease

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Fig. 2  Workflow with the

descriptive methodology fol-
lowed in this study

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 Table 2  Relative abundance of the top five phyla across the three groups and their significance
Phylum Significant properties Clinical significance in Humans

Proteobacteria 1. Largest Phylum within bacterial domain 1. Earliest colonizers of neonatal gut
2. Nitrogen fixation (essential for protein turnover) 2. Facilitate the colonization of beneficial Firmicutes and Bacteroi-
European Spine Journal

Gram-negative
3. Most of them are motile detes in adult gut
Facultative and obligate Anaerobes
3. Outer cell wall has Lipopolysaccharides which can act as endo- 3. Most dominant phyla of breast tissue and increased abundance in
DH DD ND toxins female gut during late pregnancy
36.60 43.02 46.40 4. Significant contributor for protein and lipid metabolism in 4. Present in normal adult human gut but in relatively lower abun-
animal gut dance
5. Known pathogens include Pseudomonas, Brucella, Bordetella,
Rickettsia, Shigella, Salmonella, Yersinia, Escherichia, and Heli-
cobacter
OD1 (Parcubacteria) 1. Non-cultured species of microorganisms 1. Mainly isolated from marine environments
Gram-positive 2. Have lesser ability to survive on their own, due to lack of bio- 2. Increased concentrations in gut of green turtles following hospi-
synthetic abilities talization
Anaerobes
3. Most of them are ectosymbionts as they have potential for 3. Role in humans is unknown
DH DD ND attachment and also possess cell to cell attachment proteins
39.38 30.84 12.75 4. However have nitrogen and fatty acid metabolic properties
Firmicutes 1. Second most abundant phylum of bacteria 1. Major constituent of normal human gut
Gram-positive 2. Ferment resistant starches and plant polysaccharides and oligo- 2. Higher Bacteroidetes/Firmicutes ration is implicated in Type 1
saccharides DM and lower ratio in Type 2 DM
Clostridia- Anaerobes
3. Produce short-chain fatty acids (SCFA) in human gut 3. Decrease in Firmicutes in gut indicates ageing process
Bacilli-Obligate or Facultative aerobes
3. Have the ability to produce resistant endospores 4. Lactobacillus is a well-established probiotic
DH DD ND 4. Sulphate reduction and methanogenesis are other significant 5. Useful in diarrhoea, irritable bowel syndrome, vaginal infections,
6.99 5.50 18.03 properties skin eczema, flu, allergy and also promotes weight loss
6. Known pathogens include Staphylococcus, Streptococcus, Lis-
teria and Clostridia
Actinobacteria 1. Produce 15-25 secondary metabolites with antibiotic properties 1. Essential for normal human gut function for fermentation of
Gram-positive and gram-negative 2. Nitrogen fixation in plants complex carbohydrates
3. Produce short-chain fatty acids (SCFA) such as such as acetate, 2. First line of defence against other pathogens
Aerobe and Anaerobe
propionate and butyrate, from carbohydrate fermentation 3. Micromonospora, Salinispora, Streptomyces, and Bifidobacte-
DH DD ND 4. Have high guanine and cytosine content in their DNA rium are medically important bacteria from which many anti-can-
0.58 2.09 7.71 5. Stimulate intraepithelial lymphocytes, and production of cer drugs, probiotics and antibiotics are being formulated
mucosal immunoglobulins, promoting immune response in 4. Propionibacterium and Gardnerella are few known pathogens.
human gut 5. Current studies indicate beneficial roles of Propionibacterium
Cyanobacteria 1. They are the only photosynthetic prokaryotes to synthesize 1. Responsible for oxygenation of the entire world
Gram-negative oxygen 2. Have been reported in gut microbiome
2. Believed to be ancestors of plants 3. Melainabacteria a sibling of Cyanobacteria is essential for gut
Anaerobe
3. Also have nitrogen fixation ability homeostasis
DH DD ND
0.91 3.70 6.62

DH disc herniation group, DD disc degeneration group, ND normal discs from organ donor group, numerical values represent relative abundance of microorganisms

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Fig. 3  Genus-level distribution. Genus level distribution was ana-
lysed with QIIME2 against the GG database. Taxonomic groups with
abundance < 1% were included in the group “others”. Pseudomonas
was found to be the top genus across all the three groups ND, DH,
DD, but relatively more abundant in diseased condition (DH and DD)
when compared to ND. Besides, normal discs were characterized by
All eleven bacterial phyla showed significant differences in
their abundance and distribution between the three groups.
Proteobacteria was the most abundant in ND (46.4%) and
DD (43.01%), whereas Parcubacteria was highly abundant
in DH (39.38%). Essential gut bacteria with symbiotic and
immuno-protective properties like Firmicutes and Actino-
bacteria were relatively more abundant in ND (18.03% and
7.71%) compared to DD (5.50% and 2.09%) and DH (6.99%
and 0.58%).
At the genus level, ND harboured 21 genera (Fig. 3) com-
pared to only 13 in DD and 11 in DH. Pseudomonas was
abundant, the highest in DH (51%), followed by DD (50%)
and only 14.55% in ND. The second most abundant genus in
ND, Sphingomonas (12.28%), was decreased in DD (4.68%),
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European Spine Journal
a larger number of abundant genera that keep the pathogenic genus
in check. In DD and DH, the presence of a lower spectrum of bac-
teria comprising mainly pathogenic organisms indicates dysbiosis.
Herbaspirillum, Lentibacillus, Planomicrobium, Virgibacillus, and
Saccharopolyspora were present only in Normal discs
and DH (4.58%). Beneficial bacteria like Proteobacteria
(Herbaspirillum and Devosia), Firmicutes (Lentibacillus,
Planomicrobium, and Virgibacillus), and Actinobacteria
(Saccharopolyspora) were present only in ND.
Among the 424 species (355-ND, 346-DD, and 322-DH)
identified, the top 25 abundant bacteria with its clinical sig-
nificance are listed in Table 3. Pseudomonas veronii was the
most abundant [24.48% (DH), 9.34% (DD), and only 4.91%
in ND]. In comparison, Propionibacterium acnes had a rela-
tively low abundance in all three groups being ninth in ND
(3.07%), eighth in DD (3.88%), and twelfth in DH (1.56%).
European Spine Journal

Table 3  Clinical significance of the top 25 bacterial species common to all three groups (bacteria unique to each group are excluded)
Bacterial species Order of abun- Relative abundance Clinical implication in humans
dance (%)
ND DH DD ND DH DD

Anoxybacillus kestanbolensis 1 2 1 13.83 16.57 14.61 There are only 3 articles on this organism and its role in humans is
unknown
Acinetobacter lwoffii 2 3 3 13.04 9.55 10.63 Normal gut commensal - Can cause bacteremia in hospitalized
patients
Sphingomonas yabuuchiae 3 9 9 9.76 2.08 2.98 IAA producing species exerts antimicrobial activity against E.coli &
B. subtilis
Stenotrophomonas acidaminiphila 4 11 2 8.11 1.69 10.75 Opportunistic pathogen causing nosocomial infections in immune-
compromised
Pseudomonas veronii 5 1 4 4.92 24.48 9.35 Has been reported to cause intestinal inflammatory pseudotumour
Acinetobacter johnsonii 6 8 10 4.64 2.36 2.85 Rarely cause human infections
Pseudomonas stutzeri 7 4 5 4.47 8.31 8.37 Opportunistic pathogen causes infective endocarditis
Bradyrhizobium elkanii 8 14 14 3.19 1.22 1.28 Its role in humans is unknown
Propionibacterium acnes 9 12 8 3.07 1.56 3.88 Role in acne vulgaris is debated and is implicated in Chronic Inflam-
matory diseases
Stenotrophomonas geniculata 10 23 6 2.65 0.36 4.99 Biofilm formation and nosocomial infections
Pseudomonas fragi 11 5 11 1.95 7.49 2.60 Its role in humans is unknown
Acinetobacter rhizosphaerae 12 7 13 1.86 3.22 2.23 Many of Acinetobacter species are reported in nosocomial infections
Staphylococcus epidermidis 13 18 15 1.84 0.73 1.10 Frequent causative agent of nosocomial infections-
Corynebacterium durum 14 41 30 1.49 0.14 0.33 Its role in humans is unknown
Microbacterium lacticum 16 6 7 1.01 4.05 4.25 Not reported in humans but possess any bio degradative process
Variovorax paradoxus 17 30 34 0.93 0.23 0.24 Its role in humans is unknown
Pseudomonas nitroreducens 18 10 12 0.84 1.93 2.57 Its role in humans is unknown
Prevotella copri 19 17 22 0.83 0.76 0.58 Maintains intestinal health but can cause chronic inflammatory
diseases
Faecalibacterium prausnitzii 20 24 27 0.82 0.35 0.38 Probiotics; protective against inflammatory bowel disease and colo-
rectal cancer
Bacillus flexus 21 43 39 0.81 0.13 0.22 Produces lipase that is majorly used in food & pharmaceutical
industries
Rhodococcus fascians 22 31 26 0.80 0.22 0.43 Has the ability to forms biofilm in similar fashion to other Rhodoc-
occus species
Brevibacillus reuszeri 23 15 19 0.67 1.00 0.69 Its role in humans is unknown
Pseudomonas viridiflava 30 13 18 0.46 1.40 0.71 Its role in humans is unknown
Pseudomonas alcaligenes 31 16 16 0.46 0.94 1.10 Involved in infective endocarditis and blood stream infections
Brevundimonas diminuta 24 49 49 0.67 0.10 0.48 Intrinsically resistant to fluoroquinolones

ND normal discs, DH disc herniation, DD disc degeneration, IAA indole-3-acetic acid

Common bacteria to spine, gut, and skin
To explore the common presence of these microbes in skin
and gut, we compared our disc microbiome with exist-
ing human gut and skin microbiome data. We identified
58 common bacteria between IVD and gut microbiome,
and 29 common bacteria with skin microbiome (Table 4;
Fig. 4a). Among the top 25 abundant species present in the
disc microbiome, four species (Acinetobacter johnsonii,
Pseudomonas stutzeri, Staphylococcus epidermidis, and
Corynebacterium durum) were common to IVD/skin, while
two (Prevotella copri and Faecalibacterium prausnitzii)
were common to IVD/gut. Propionibacterium acnes and
Staphylococcus epidermidis were common to skin/gut/IVD
(Table 5).
Diversity of bacteria
Alpha diversity analysis (Fig. 5a) by the OTU index was
significantly higher in DD and DH, compared to ND (ND
vs. DH, p = 0.015; ND vs. DD, p = 0.008) without differ-
ence between DH and DD (p > 0.8). Alpha diversity by
Shannon diversity index (microbial distribution) revealed

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Table 4  Bacterial species common to gut, spine* and skin

Common between gut and spine (n = 52) Common to gut, spine and skin (n = 6) Common between skin and spine
(n = 23)

Bacteroides uniformis Bifidobacterium breve Haemophilus parainfluenzae Corynebacterium kroppenstedtii

Microvirgula aerodenitrificans
Blautia producta
Ruminococcus bromii
Bacteroides coprophilus
Eggerthella lenta
Megamonas hypermegale
Lactobacillus delbrueckii
Bifidobacterium pseudolongum
Sphingobacterium faecium
Lactobacillus brevis
Collinsella stercoris
Bacteroides fragilis
Clostridium butyricum
Providencia stuartii
Bacteroides ovatus
Parabacteroides distasonis
Akkermansia muciniphila
Prevotella intermedia
Bacteroides plebeius
Lactobacillus helveticus
Clostridium hiranonis
Blautia obeum
Streptococcus sobrinus
Bifidobacterium adolescentis
Prevotella ruminicola
Ruminococcus flavefaciens
Bifidobacterium bifidum
Dorea formicigenerans
Faecalibacterium prausnitzii
Streptococcus anginosus
Lactobacillus ruminis
Mitsuokella multacida
Bacteroides caccae
Prevotella copri
Leuconostoc mesenteroides
Collinsella aerofaciens
Lactobacillus salivarius
Lactobacillus reuteri
Bifidobacterium longum
Lactococcus garvieae
Roseburia faecis
Bacteroides eggerthii
Pediococcus acidilactici
Bifidobacterium animalis
Coprococcus eutactus
Bacillus coagulans
Clostridium perfringens
Prevotella stercorea
Oxalobacter formigenes
Catenibacterium mitsuokai
Propionibacterium acnes
Veillonella parvula
Prevotella melaninogenica
Staphylococcus epidermidis
Veillonella dispar
Kocuria rhizophila
Brevibacterium casei
Rothia dentocariosa
Rhodobacter sphaeroides
Brevibacterium paucivorans
Pseudomonas stutzeri
Acinetobacter johnsonii
Enhydrobacter aerosaccus
Pseudomonas nitroreducens
Pedomicrobium australicum
Corynebacterium simulans
Peptostreptococcus anaerobius
Rothia mucilaginosa
Helicobacter pylori
Lactobacillus iners
Kocuria palustris
Methylobacterium mesophilicum
Rothia aeria
Staphylococcus haemolyticus
Propionibacterium granulosum
Corynebacterium durum
Micrococcus luteus

Spine* represents the total IVD microbiome (n = 424) of all the three groups normal discs (ND) and diseased (DH, DD) identified from this
study

a higher diversity in DD compared to ND (Kruskal–Wallis
test p = 0.006) and DH (p = 0.011).
Species biodiversity between the three groups (beta diver-
sity) showed that the UniFrac distance of ND was different
from DH and DD with no significant difference between DH
and DD (Fig. 5b).
Altered microbiome in health and disease
The relative abundance of bacteria varied significantly
between ND against DD (Fig. 6a) and DH (Fig. 6b). Clini-
cally relevant species, viz. Lactobacillus mucosae (beneficial
probiotic), Propionibacterium granulosum (skin commen-
sal), Sphingomonas yabuuchiae (anti-tumoural), and Staphy-
lococcus epidermidis (oral commensal preventing pathogen
invasion), were found to be abundant in ND compared to
DH. Blautia producta (gut-microbe), Propionibacterium
granulosum (skin-commensal), and Sphingomonas yabu-
uchiae (anti-tumoural) were found to be abundant in ND
when compared to DD. Pseudomonas fragi (proteolytic)
were more abundant in DH (Fig. 7).
Bacteria unique to the health and diseased status of IVD
are listed in Table 6. It was interesting to observe 32 unique
bacteria in ND, 5 in DH, and 10 in DD (Fig. 4b)). In ND,
known probiotics (Bacillus coagulans and Bacillus clausii)
and bacteria with antimicrobial properties against Pseu-
domonas aeruginosa and Staphylococcus aureus (Bdello-
vibrio bacteriovorus) were uniquely present. Interestingly,
three bacteria (Prevotella tannerae, Halomonas nitritophi-
lus, and Streptococcus alactolyticus) occurring in both DD
and DH but not in ND are known human pathogens. Few
other known pathogens, Streptococcus anginosus, Prevo-
tella pallens, Avibacterium gallinarum, and Enterobacter
cowanii, were found only in DD.

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Confirmation of bacterial presence by tandem mass

spectrometry

ESI-LC–MS/MS data of IVD proteome, analysed by MAS-

COT & SEQUEST search engines, identified many con-
served bacterial ribosomal proteins confirming the bacte-
rial presence and numerous metabolic proteins indicative of
various biochemical processes in vivo ruling out contamina-
tion (Supplementary File-2). Metagenomic pathways were
generated by using PICRUST, and their abundance was cal-
culated using STAMP v2.1.3 tool 5 (Supplementary File-3).

Fig. 4  Comparison of IVD metagenome with reported gut and skin

microbiome. Taxonomic classification at the species level was per-
formed with Kraken2 against the GG database. A total of 424 bacte-
rial species were identified across all three groups without any cut-
off. a Comparison of the total IVD 424 bacterial species (ND, DH,
DD) without any cut-off with gut and skin microbiome revealed 58
common bacteria between IVD and gut microbiome and 29 common
bacteria with skin microbiome. The much-discussed Propionibacte-
rium Acnes had almost an equal presence in all three groups as well
as skin and gut. b Bacterial species present in at least 50% of the
samples were taken, which accounted for 216 species for comparison
between the three groups, and showed 32 unique protective bacteria
in ND compared to only 10 in DD and 5 in DH
Discussion
In this study, for the first time, we have performed next-
generation sequencing and proteomic analysis in what can
be considered as pure controls—discs harvested in live con-
dition without contamination in sterile surgical conditions
from brain dead voluntary organ donors with no history of
LBP and MRI normal discs. The results have documented
the presence of a ‘disc microbiome’ similar to other tissues
and show disc dysbiosis as an important cause of inflammag-
ing and disc degeneration. These results will considerably

Table 5  Clinical significance of the abundant IVD bacterial species found in common between gut and skin

Common to Number Bacterial species Function Clinical significance in humans

Gut and spine 2 Prevotella copri Commensal of human gut & intestines
Faecalibacterium prausnitzii Gut commensal and important
producer of butyrate to the colonic
epithelium
Skin and Spine 4 Acinetobacter johnsonii Skin commensal
Pseudomonas stutzeri Denitrifying bacterium degrades car-
bon tetrachloride
Pseudomonas nitroreducens Synthesizes polyhydroxybutyrate
homopolymer (a polyester) from
medium chain length fatty acids
Corynebacterium durum Skin commensal and part of orophar-
ynx microbiome
Gut, Spine and Skin 2 Propionibacterium acnes Most essential common colonizer of
normal human skin and also nor-
mally found in gut
Secretes propionic acid.
Role in Skin immunomodulation.
Antitumour properties.
Staphylococcus epidermidis Skin & mucous commensal that forms
biofilms on indwelling medical
devices
Maintains intestinal health but currently
linked to chronic inflammatory condi-
tions, such as arthritis and mucosal
and systemic T-cell activation
It is a Probiotics and negatively corre-
lates with inflammatory bowel disease
and colorectal cancer
Rarely causes infection
Opportunistic pathogen causes infective
endocarditis
Soil bacterium reported to be used in
bioremediation process
Potential role in respiratory tract infec-
tions
Wrongly claimed to have role in Acne
vulgaris.
Role in Chronic Inflammatory diseases.
Most associated organism with disc
degenerative disease and Modic
changes.
Protects the skin from more pathogenic
Staphylococcus aureus
Opportunistic pathogen apart from
native-valve endocarditis
Known causative agent of nosocomial
infections especially in patients with
implants

13

impact on how we look at the etiopathology of disc degen-
eration, its prevention, and management.
Research in sub-clinical infection as an initiating factor
of DDD has not seen much progress despite rising reports
of bacterial isolation from discs harvested during routine
13
European Spine Journal
spine surgery. One of the significant challenges is the isola-
tion of these fastidious organisms by usual culture methods,
which has resulted in variable literature. It is well known
that even in well-established sepsis of discs, routine cul-
tures are successful in isolating the specific microorganism
 European Spine Journal
◂Fig. 5  Alpha and beta diversity of bacteria across normal (ND),
degenerate (DD) and herniated (DH) discs. a The alpha diversity
(sample evenness within a group) analysis revealed that the observed
OTU index was significantly higher in diseased groups when com-
pared to normal disc (ND vs. DH, p = 0.015; ND vs. DD, p = 0.008).
There was no significant difference between DH and DD (p > 0.8).
Alpha diversity based on the Shannon diversity index (microbial
distribution) revealed that the index was significantly higher in DD
compared to ND (Kruskal–Wallis test p = 0.006) and DH (p = 0.011).
Though normal discs have more bacterial types, each sample belong-
ing to this group had almost no variation among themselves, unlike
DD and DH, where sample unevenness was observed, as evidenced
by a higher alpha diversity. Results indicate that normal discs had
more sample evenness, while degenerate disc samples differed from
each other. b Variation of the IVD microbial data was assessed by
beta diversity metrics. a, b Principal coordinate analysis based on
unweighted and weighted UniFrac distances with a p value ≤ 0.05.
Healthy normal discs are represented in green and diseased DH in
orange and DD in blue. The clustering of normal disc samples (green
dots) indicates that the microbial community of each sample is almost
the same as the other in ND, and their far distance from DD and DH
samples suggests that the microbial composition of discs is very dif-
ferent between healthy and diseased states. On the other hand, Uni-
Frac distances between DD and DH groups are much less, represent-
ing a similar pathogenic microbial profile in both
in only around 50% of individuals [14]. Broad-range 16S
ribosomal RNA (rRNA) gene analysis has been proven to be
efficacious in identifying organisms, even in these culture-
negative specimens [15]. Our ability to review the breadth
of the microbiota has vastly improved in the last decade due
to the advent of culture-independent approaches such as 16 s
rRNA sequencing and shotgun metagenomics.
Is there a ‘normal disc biome’?
Traditionally, the central nervous system, eye, and IVD
were considered sterile with a unique immune privilege,
protected from the uncontrolled inflammatory response
[16]. This concept is becoming fast outdated as culture-inde-
pendent approaches like 16S rRNA sequencing and shotgun
metagenomics have expanded our ability to identify all bac-
teria [17]. The enormity of the human microbiome and its
importance in health and disease are now baffling [18].
Our study is the first to demonstrate a rich bacterial pres-
ence in ND, DH, and DD, thus establishing the presence of
a ‘disc microbiome’. 355 bacterial species were identified
in ND with 32 unique bacteria compared to 346 in DD (10
unique bacteria) and 322 in DH (5 unique bacteria). Tandem
mass spectrometry identified conserved bacterial proteins
and also many vital enzymes (Table 7) confirming various
active biochemical processes, thus ruling out contamination
as the cause of bacterial presence. It should be noted that
contamination was doubly ruled out by the absence of bacte-
rial reads, in internal controls.
Mapping allowed 42.75% OTUs to be classified at king-
dom level, 44% at phylum level, 22.62% at the genus level,
and 5.5% at species level. Many bacterial OTUs were still
left unidentified, pointing to the presence of many unclassi-
fied bacteria, indicating that we are still far away from fully
comprehending the magnitude and implications of the disc
biome. The influence of a biome in diseases like Parkin-
son’s disease, sarcoidosis, spondylo-arthritis, inflammatory
bowel disease (IBD), prostrate disorders, and type 2 diabetes
is well established, and our results raise the possibility of
establishing a similar effect of the microbiome on spinal
disorders [19].
Biodiversity in health and disease
Rather than just abundance, it is the relative ratio of bacteria
that determines the biological state. A higher Firmicutes/
Bacteroidetes ratio influences obesity, and a higher Bac-
teroidetes/Prevotella ratio causes type 2 DM [20, 21]. We
have documented differing abundance at phylum, genus, and
species levels in all three groups. Firmicutes and Actino-
bacteria were abundant in ND; both are known to produce
short-chain fatty acids and also constitute the first line of
defence in the gut wall [22]. They inhibit Proteobacteria
(entero-haemorrhagic Escherichia coli and Shigella) also
stimulating lymphocytes and immunoglobulin production.
Also uniquely present in ND samples was Saccharopolys-
pora, which produces macrolide antibiotics effective against
most gram-positive bacteria and some gram-negative bac-
teria [23].
In contrast, many known human pathogens such as Pseu-
domonas veronii, Pseudomonas stutzeri, Streptococcus
anginosus, Prevotella pallens, Avibacterium gallinarum,
Enterobacter cowanii, Prevotella tannerae, Halomonas
nitritophilus, and Streptococcus alactolyticus were either
found in higher abundance or uniquely in DD and DH. Many
of these have been implicated in spondylodiscitis, fracture,
and joint infections, meningitis, and endocarditis, and it will
not be a surprise if they are a cause of sub-clinical infection
and inflammatory response leading to DD [24].
Dysbiosis, inflammaging, and disease
‘Dysbiosis’, a phenomenon implicated in various diseases, is
defined as any perturbation of the normal microbiome con-
tent that could disrupt the symbiotic relationship between
the host and associated microbes resulting in disease [25].
Although all three groups had bacteria related to the same
11 phyla, protective bacteria were more abundant in ND
and known pathogens more abundant in DH and DD. At the
species level, although Pseudomonas veronii and stutzeri
were common to the three groups, it was comparatively
lower in ND (Fig. 7). Besides, when considered as a ratio to
other protective bacteria, we also find that their activity has
been masked by the presence of protective and beneficial
13
 European Spine Journal

bacteria in ND. Such a shift in the relative ratios of the bac- colorectal cancer, diabetes mellitus, and neurodegenerative
teria points to the presence of dysbiosis, a condition impli- disorders [26]. Dysbiosis has not previously been described
cated in inflammatory bowel disease, rheumatoid arthritis, in DD, but our study raises this interesting possibility.

13
 European Spine Journal
◂Fig. 6  Comparison of significantly abundant bacterial species
between normal disc (ND) and a degenerated (DD) and b herni-
ated (DH). a Species-level taxonomic analysis of the metagenomic
sequences obtained from ND and DD using the STAMP software.
Green bars represent ND and blue bars represent DD. a T-test and
b MW test. Sphingomonas yabuuchiae (protective bacteria) had
a higher relative abundance in ND when compared to DD. Saccha-
ropolyspora hirsuta, an antibiotic complex (sporaricin) producing
bacteria, also had higher abundance in normal discs. b Species-level
taxonomic analysis of the metagenomic sequences obtained from ND
and DH using the STAMP software. Green bars represent ND, and
blue bars represent DH. a T-test and b MW test. Pseudomonas fragi,
a bacterium with many proteases and lipases, was abundant in the
DH group. Similarly, Pseudomonas viridiflava (a necrotic agent) was
abundant in DH. In contrast, normal discs had a higher abundance of
protective bacterial species such as Sphingomonas yabuuchiae, Lac-
tobacillus mucosae, and Bacillus flexus  study shows that this area should be a focus of future studies.
Although DD is multifactorial, ‘inflammaging’, a state
of chronic uncontrolled low-grade inflammation, is increas-
ingly implicated as its etiology [27]. A recent report has
documented inflammaging by the presence of complement
activation cascades and also postulated infection as an eti-
ology due to unsuppressed complement activity [28]. Our
documentation of diverse pathogenic bacteria and a state of
dysbiosis strongly supports this theory.
Is Propionibacterium acnes the only bacterium
associated with degenerative disc disease?
Propionibacterium acnes has enjoyed the focus of attention
of recent literature, but our results question this view by
reporting other known pathogens at a higher abundance.
Pseudomonas veronii and Pseudomonas stutzeri were highly
abundant, and Propionibacterium acnes ranked only 8th in
DD (3.88%), 9th in ND (3.07%), and 12th in DH (1.56%). It
is seen that Propionibacterium acnes was found in all groups
without a significant difference in abundance. Their over-
emphasis in previous studies may be due to a narrow focus
on the single bacteria based on initial reports and a flaw in
the methodology of using bacteria-specific primers in PCR
analysis and not utilizing 16S rRNA primers in metagenome
sequencing which identifies the entire spectrum of bacteria.
Gut/skin/spine microbiome axis
The gut microbiome is now the seat of focused attention as
NGS has increased the estimated bacterial community in
the gastrointestinal tract from 500 to over 15,000 species
and is an essential component of the human microbiome.
The human gut with 13 trillion bacterial cells outnumbers
total human cells by 13 times and plays a significant role
in modulating host metabolism, vitamin synthesis, coloni-
zation resistance, and immune homeostasis. Its causative
influence in many non-communicable diseases such as IBD,
colitis, diabetes, and obesity, etc., is well documented [29].
A correlation between the gut microbiome and disc diseases
has not been probed, but our study documents 58 bacteria
common between the IVD and gut and 29 common between
IVD and skin (Table 4). Among the top 25 abundant species,
Acinetobacter johnsonii, Pseudomonas stutzeri, Staphylo-
coccus epidermidis, and Corynebacterium durum were com-
mon to IVD/skin, Prevotella copri and Faecalibacterium
prausnitzii were common to IVD/gut, and only Propionibac-
terium acnes and Staphylococcus epidermidis were common
to skin/gut/IVD. A clinical correlation between UTI and
gastroenteritis with LBP and exacerbation of spondyloar-
thropathy has been reported, but a formal link between the
gut microbiome and LBP has not been established [30]. Our
Role of the microbiome within the spine
The traditional belief that human intervertebral discs are
sterile structures has been proved wrong by this study, where
normal discs have been documented to have a unique micro-
biome very distinct from that of degenerated and herniated
discs. The role of sub-clinical infection in disc degenera-
tion is well established, as many authors have documented
the presence of bacteria mainly Propionibacterium acnes
in degenerated human IVDs using several techniques such
as traditional culture methods, DNA-based analysis, bio-
film demonstration, and electron microscopy of IVD cells.
Similar to our finding, previous sites which were deemed
to be sterile such as reproductive tract, foetus, breast tis-
sue, seminal fluid, and even eye have been investigated and
found to have a distinct microbiome in health and disease.
It was often thought that nutrition to the IVD was mainly
through the end-plates as the adult discs were considered
avascular. With the established role of the microbiome in
providing nutrition to other human cells, analysing the ben-
eficial effects of the microbiome in IVD is a valuable domain
for future research. Albert et al., in his study, documented
improvement in the treatment of low back pain with antibi-
otic therapy for 100 days. More recently, trials with probiot-
ics are also underway in patients with low back pain [31].
With growing evidence of bacterial etiology in DDD, this
study has documented a normal microbiome, necessitating
a further focus on investigating the wide spectrum of micro-
organisms and their role in health and disease.
Implications for future
Increasing understanding of the effect of the microbiome
on human health and diseases has led to newer manage-
ment strategies focusing on alterations of the microbiome
by dietary modifications, use of probiotics, antibiotics,
and even gut microbiome transplants in diseases like pso-
riatic arthritis, atopic dermatitis, IBD, necrotizing colitis,
13

Fig. 7  Distribution of Pseudomonas species across normal (ND),
degenerate (DD), and herniated (DH) discs. Pseudomonas genus was
the most abundant genera in all the three groups. However, the rela-
tive abundance of Pseudomonas was the least in the ND group, indi-
cating increased colonization in the diseased state. Nine different spe-
cies were identified, and the relative abundance of all these species
was lowest in the ND group again indicating dysbiosis in DD and DH
and drug-induced diarrhoea. Our findings, which strongly
suggest a sub-clinical infection cause of inflammaging, by
documenting dysbiosis in degenerate discs, indicate that
future studies must aim at identifying clinico-radiological
and biochemical markers that can segregate the groups of
patients with back pain who have a sub-clinical infection so
that appropriate therapies can be developed.
Limitations
The study has analysed 24 samples and has established the
presence of a disc microbiome and postulated ‘dysbiosis’ as
a cause of disc disorders. However, this has to be validated
in a more extensive set of samples and probably in various
ethnic and geographical populations also. The presence of
numerous bacteria, both protective and pathogenic, has been
documented, but the actual relevance between them should
be further studied using metatranscriptomics and metabo-
lomics. We have concentrated mainly on bacteria; however,
13
European Spine Journal
samples. Certain species of Pseudomonas (veronii, viridiflava, fragi,
umsongensis, and citronellolis) were most abundant in herniated discs
(DH) and degenerated discs (DD) had a marginally higher abundance
of (stutzeri, nitroreducens, balearica and alcaligenes). While Pseu-
domonas Veronii (inflammatory pathogen) was most abundant in DH,
Pseudomonas stutzeri (well-known infective pathogen) had a higher
relative abundance in DD
the presence of viruses and fungus also needs investigation
by shotgun metagenomics.
Conclusion
Our study, which is the first comprehensive metagenomic
analysis of the lumbar discs, challenges the concept of steril-
ity of human IVD in health and has established the presence
of a human disc microbiome. We have also documented the
presence of ‘dysbiosis’ evidenced by differing ratios of bac-
terial abundance across the three groups, which points to
the possibilities of newer non-surgical therapeutic avenues.
By identifying 58 bacteria common to IVD and gut and 29
bacteria common to IVD and skin, we also propose the pres-
ence of a gut/skin/spine Microbiome axis, which needs to be
probed further. The findings of our study also reinforce the
importance of sub-clinical infection in the etiopathogenesis
of disc degeneration and low back pain and suggest concen-
trated research on this crucial topic.
 Table 6  List of bacteria specific to normal discs (ND), disc herniation (DH), and disc degeneration (DD) and their functional characteristics
S. no. Phylum Bacteria name Gram stain Respiration Function/characteristics Clinical implication

Unique in ND (normal discs)

1 Firmicutes Oceanobacillus oncorhynchi Positive Obligate aerobe; fac- Non-pathogenic inhabitant of alkaline Believed to be Human gut symbiont simi-
ultative alkaliphilic aquatic environment lar to Oceanobacillus faecalis
European Spine Journal

2 Staphylococcus succinus Positive Anaerobe Bacterial genes were found resistant to In normal human gut, through the activity
heavy metals & toxin production and of staphylococcal aromatic amino acid
they equally participate in Bacteriocin decarboxylase (SadA), they are believed
Production to produce trace amines which act as
neuromodulators
3 Bacillus coagulans Positive Facultative anaerobe Promotes host health by improving Probiotics; Beneficial bacteria for infec-
bowel function and produces lactic acid tions caused by rotaviral diarrhoea in
children
4 Bacillus longiquaesitum Positive Facultative anaerobe Soil bacterium Has not been isolated from human speci-
mens
5 Bacillus ginsengihumi Positive Facultative anaerobe Possess catalase and oxidase activity. Has not been isolated from human speci-
Never degrades macromolecules such mens
as casein, collagen, starch
6 Bacillus foraminis Positive Facultative anaerobe Isolated from alkaline groundwater Has not been isolated from human speci-
mens
7 Bulleidia p-1630-c5 Positive Anaerobe Bio mechanisms of this bacteria is Increased colonization in human gut after
unknown Obesity treatment and associated with
Type 2 DM
8 Bacillus endophyticus Positive Aerobe Disrupts the cell wall and peptidoglycan Has not been isolated from human speci-
synthesis of the gram-positive and mens
gram-negative bacteria
9 Bacillus clausii Positive Aerobe Probiotics; Helps in the maturation of Beneficiary bacteria that helps in treatment
immune system of irritable bowel syndrome, respiratory
and gastrointestinal infections.
10 Bacillus humi Positive Aerobe Endospore-forming bacteria that pro- Some species of Bacillus reported to have
motes plant growth both beneficial and harmful roles
11 Lactobacillus iners Positive Facultative anaerobe Encodes inerolysin, a pore-forming toxin Can be detected in normal conditions as
related to vaginolysin of Gardnerella well as during vaginal dysbiosis
vaginalis

13


Table 6  (continued)
S. no. Phylum Bacteria name Gram stain Respiration Function/characteristics Clinical implication

13
12 Proteobacteria Bdellovibrio bacteriovorus Negative Obligate aerobe Bacterial predator. Found in environment Future Antimicrobial agent. Reported in
and within human gut attacks gram- the studies to destroy isolates such as
negative prey. Pseudomonas aeruginosa and Staphylo-
coccus aureus that cause Cystic fibrosis
13 Aggregatibacter segnis Negative Aerobe Characterized by slow growth and weak Have been isolated from oral dental
carbohydrate fermentation plaques and also patients with infective
endocarditis
14 Enhydrobacter aerosaccus Negative Facultative anaerobe A common constituent of oral microbiota Has Probiotic properties and is the most
in children abundant organism in human milk
samples
15 Thermomonas fusca Negative Aerobe For bioremediation in industries Has not been isolated from human speci-
mens
16 Limnohabitans curvus Negative Aerobe Helps ingrowth of algal-derived sub- Has not been isolated from human speci-
strates mens
17 Shinella granuli Negative Aerobe Found to high oxidase and catalase activ- Has not been isolated from human speci-
ity used for sewage treatment systems mens
18 Candidatus Portiera aleyrodidarum Negative Obligate aerobe Obligate primary endosymbiotic bacte- Has not been isolated from human speci-
rium in white flies mens
19 Hyphomicrobium zavarzinii Negative Aerobe Produces dimethyl sulphone, a energy Has not been isolated from human speci-
and carbon substrate used for microbio- mens
logical degradation
20 Caulobacter henricii Negative Aerobe Important model organism for studying Some of its species are reported to be a
cell division and differentiation cause of post neurosurgical bacterial
meningitis in paediatric patients
21 Actinobacteria Prauserella ugose Positive Aerobe Plant growth promoter and producers of Has not been isolated from human speci-
natural energy substrates mens
22 Cryptosporangium japonicum Positive Aerobe Plays a major role in Cryptosporidium- Some of its species are reported to be
host cell interactions involved in asymptomatic infections
23 Rhodococcus ruber Positive Aerobe Helps in bioremediation and detoxifica- Closely related to Rhodococcus equi, a
tion of contaminated soils known human opportunistic pathogen
24 Brevibacterium paucivorans Positive Obligate aerobe Exists in humans either as commensals Reported to be rare cause of catheter
or opportunistic pathogens related blood steam infections
25 Streptomyces reticuliscabiei Positive Aerobe Associated with netted scab in potatoes Non-pathogenic
26 Kocuria palustris Positive Aerobe Inhabitant of normal skin and mucous Non-pathogenic bacteria which are rarely
membranes associated with human infections
27 Williamsia serinedens Positive Aerobe Effectively helps in biodegradation Reported to be causative agent of perinatal
process sepsis
28 Mycobacterium celatum Positive Aerobe Opportunistic pathogen Causative agent of fatal Pulmonary Infec-
tion in patients with suppressed cell-
mediated immunity
European Spine Journal
 Table 6  (continued)
S. no. Phylum Bacteria name Gram stain Respiration Function/characteristics Clinical implication

29 Rhodococcus globerulus Positive Aerobe Cause opportunistic infections in immu- Reported to cause sub-acute systemic ill-
nocompromised hosts ness in recipient of stem-cell transplant
patient
30 Candidatus Aquiluna rubra Positive Aerobe Isolated from eutrophic pond Has not been isolated from human speci-
European Spine Journal

mens
31 Bacteroidetes Sphingobacterium faecium Negative Aerobe Isolated from the faeces of the cattle Rarely reported as opportunistic pathogens
32 Deinococcus-Thermus Deinococcus geothermalis Positive Aerobe Bioremediation of radioactive waste Believed to protect epithelial cell from
radiation
Unique in DH (disc herniation)
1 Actinobacteria Microbacterium aurum Positive Aerobe Have fibronectin type III (FNIII) Human pathogen causing endophthalmitis
domains that functions as substrate
disrupter
2 Firmicutes Clostridium neonatale Positive Obligate Anaerobe Saccharolytic, sub-terminal spore form- Causes necrotizing enterocolitis, a gastro-
ing bacteria intestinal disease in neonates
3 Paenibacillus barengoltzii Positive Aerobe Catalase and oxidase Reported to be identified in ascitic and
tests are positive synovial fluid. Also known to cause
Catalase and oxidase intravascular catheter-associated infec-
tests are positive tion
High oxidase and catalase activity
4 Eubacterium dolichum Positive Anaerobe High catalase activity Abundant levels of this bacterium are
known to cause high visceral fat induced
cardio-metabolic diseases.
5 Proteobacteria Vibrio rumoiensis Negative Facultative Anaerobe Facultative psychrophilic bacteria exhibit Naturally occurring Vibrio species in
high catalase activity use ­H2O2 as a aquatic environments were found with
bleaching and microbicidal agent virulent factors to elicit disease in humans
Unique in DD (Disc Degeneration)
1 Actinobacteria Bifidobacterium pseudolongum Positive Anaerobe Prevents intestinal colonization of patho- Gut bacteria promotes host health in
genic bacteria exerting competition with exogenous
pathogenic bacteria
2 Firmicutes Streptococcus anginosus Positive Facultative Anaerobes Commensal of gut, human oral cavity Found to cause brain and liver abscesses
cause purulent infections
3 Geobacillus vulcani Positive Aerobe Produce acids from glycerol, lactose and Role in Humans is unknown
ribose
4 Bacteroidetes Prevotella pallens Negative Anaerobe β-Lactamase production Found to be isolated first from oral cav-
ity can be considered as opportunistic
pathogens and several dysbiosis-related
diseases

13


Table 6  (continued)
S. no. Phylum Bacteria name Gram stain Respiration Function/characteristics Clinical implication

13
5 Proteobacteria Avibacterium gallinarum Negative Facultative Anaerobes Produces acid from ingested galactose Etiologic agent of infectious Coryza of
chickens
6 Methylibium petroleiphilum Negative Facultative Aerobes Methyl tertiary-butyl etherdegrading Non-pathogenic
methylotroph used in bioremediation
that destroys numerous toxic com-
pounds
7 Enterobacter cowanii Negative Facultative Anaerobes Functions as both plant & human Reported as food-contaminant, seen in
pathogen patients at intensive care unit or immu-
nocompromised individuals
8 Halomonas campisalis Negative Aerobe Nitrous Oxide–Reducing Denitrifier Causes contamination in dialysis centre
due to its biofilm formation
9 Sphingobium xenophagum Negative Aerobe Hydrocarbon degrading microbe Unknown role in Humans
10 Erwinia dispersa Negative Aerobe Yellow pigmented, and ferment lactose Known to cause respiratory infections,
neonatal sepsis, and bloodstream infec-
tions
Common to both DD & DH
1 Proteobacteria Halomonas nitritophilus Negative Aerobe Involved in the production process of Newly discovered human pathogen
3-hydroxybutryic acid
2 Novosphingobium stygium Negative Aerobe Degrade Aromatic compounds Some of its species are reported to cause
autoimmune disorder of liver
3 Firmicutes Streptococcus alactolyticus Positive Facultative Anaerobes Confined to intestinal flora of pigs, Known to cause human infections in an
chickens, pigeons, and canines cause immunocompetent human host
massive inflammation
4 Bacteroidetes Prevotella tannerae Negative Anerobe Ferment carbohydrates and produce suc- Oral commensal can be an opportunistic
cinic and acetic acids pathogen

This table also enlists bacteria common to DD and DH

European Spine Journal
European Spine Journal

Table 7  Examples of conserved Protein class Description

bacterial proteins, metabolic
proteins, and functional proteins
(entire list in supplementary
file-3) confirming the presence
of live and multiplying bacteria
Ribosomal proteins (conserved)
Metabolic proteins
Other functional proteins
30S ribosomal protein S2, S10, S11 and S12
50S ribosomal proteins- L2, L3, L6, L9, L12, L13, L14, L15, L19,
L20, L21, L22, L28, L33
2-octaprenylphenol hydroxylase
4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (flavodoxin)
5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase
Acetyl-CoA carboxylase
Acetylornithine deacetylase
Acetyltransferase component of pyruvate dehydrogenase complex
Acyl-CoA dehydrogenase oxidoreductase
Alpha/beta hydrolase
2-Hacid_dh_C domain-containing protein
ABC transporter, ATP-binding protein
Acyl_transf_3 domain-containing protein
Aromatic amino acid transport protein AroP1
C4-dicarboxylate transport protein
Chaperone protein ClpB
Cold-shock protein
Collagen pro alpha-chain
ComEA family DNA-binding protein
Conjugal transfer protein TrbP

Acknowledgements  The corresponding author has full access to the
data analysed in this study and holds all responsibilities towards the
submission of this article. We acknowledge the receipt of grants from
the Ganga Orthopaedic Research & Education Foundation (GOREF
2018-08) and AO Spine (AOSIN(R) 2017-04). All authors contributed
equally to the preparation of this manuscript. We acknowledge the
efforts taken by all the authors equally in preparing this manuscript
and also thank GOREF for funding the project.
Funding  The project was mainly funded by Ganga Orthopaedic
Research & Education Foundation (GOREF 2018-08) and partially by
the AO Spine (AOSIN(R) 2017-04).
Compliance with ethical standards  terial genome and culture collection for improved metagenomic
Conflict of interest  The authors declare that they have no conflict of
interest.
10. Tsai Y-C, Conlan S, Deming C, Program NCS, Segre JA, Kong
IRB approval  The study was performed only after approval of the IRB
committee.
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