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Infection and Immunity, October 2000, p. 6012-6026, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Reverse Transcriptase-PCR Analysis of Bacterial
rRNA for Detection and Characterization of Bacterial Species in
Arthritis Synovial Tissue
Karen E.
Kempsell,1,*
Charles J.
Cox,2
Michael
Hurle,1
Anthony
Wong,1
Scott
Wilkie,1,
Edward D.
Zanders,1
J. S. Hill
Gaston,2 and
J. Scott
Crowe1
Glaxo Wellcome Medicines Research Centre,
Stevenage SG2 1NY,1 and Department of
Medicine, University of Cambridge School of Clinical Medicine,
Addenbrookes Hospital, Cambridge CB2 2QQ,2
United Kingdom
Received 13 December 1999/Returned for modification 15 March
2000/Accepted 2 May 2000
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ABSTRACT |
Onset of rheumatoid arthritis (RA) is widely believed to be
preceded by exposure to some environmental trigger such as bacterial infectious agents. The influence of bacteria on RA disease onset or
pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA
disease tissue. Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue
of late-stage RA and non-RA arthritis controls. This may be suggestive
of the presence of live bacteria. Sequencing of cloned complementary
rDNA (crDNA) products revealed a number of bacterial sequences in joint
tissue from each patient, and from these analyses a comprehensive
profile of the organisms present was compiled. This revealed a number
of different organisms in each patient, some of which are common to
both RA and non-RA controls and are probably opportunistic colonizers
of previously diseased tissue and others which are unique species.
These latter organisms may be candidates for a specific role in disease
pathology and require further investigation to exclude them as
causative agents in the complex bacterial millieu. In addition, many of
the detected bacterial species have not been identified previously from
synovial tissue or fluid from arthritis patients. These may not be
easily cultivable, since they were not revealed in previous studies
using conventional in vitro bacterial culture methods. In situ
hybridization analyses have revealed the joint-associated bacterial
rRNA to be both intra- and extracellular. The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in
either RA or non-RA arthritis controls is unclear and requires further investigation.
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INTRODUCTION |
In addition to the influence of
innate susceptibility factors, most notably certain HLA class II
alleles, onset of rheumatoid arthritis (RA) is widely believed to be
preceded by exposure to some environmental trigger. The precise nature
of this initiating factor has not yet been elucidated despite much
study. There has been considerable interest in a possible role for
bacterial infectious agents in disease onset (20, 35, 38),
since there are certain similarities between RA and other inflammatory
arthritides, e.g., reactive arthritis (ReA) and Lyme arthritis (LyA).
These latter conditions are known to be preceded by bacterial infection
at a site distant from the involved joint and also show an association with HLA alleles, ReA with HLA-B27 (40) and LyA more weakly with HLA-DR4 (30). ReA can be triggered by gastrointestinal or genitourinary infection with a number of different bacterial species
including Yersinia, Salmonella,
Campylobacter, and Chlamydia (12), and
LyA is triggered by infection with the tick-borne spirochaete
Borrelia burgdorferi (12).
There is an accumulating body of evidence suggesting that these
conditions, previously thought to be sterile arthropathies, may be
perpetuated by small numbers of persistent organisms which have
trafficked to the affected joint. Spirochetes can occasionally be
recovered by culture of synovial fluid from individuals with LyA but
are only detected by PCR or electron microscopy in synovial tissue
(12, 15, 28, 48). Live organisms have not reproducibly been
recovered by culture from ReA-affected joints; however, DNA from
Yersinia and Chlamydia species has been detected
by PCR in the synovial fluid of some patients with ReA (12, 62,
63). Bacterial rRNA has also been detected (18, 23),
which is suggestive of the presence of live replicating organisms due
to the relative lability of rRNA compared to DNA in nonviable organisms
(59). No such evidence of specific infection as a trigger
for arthritis onset has been uncovered in RA. Early culture studies of
synovial fluid yielded a variety of different bacterial species
(26, 58). The organisms identified varied substantially
between different investigative groups, suggesting the absence of
common culturable etiological agents involved in disease pathology. In
addition, contamination during sample handling and culture procedures
often could not be ruled out, and the organisms could not be associated directly with the disease process.
In more recent molecular studies, Melief et al. (43) found
intestinal flora-derived peptidoglycan polysaccharides within macrophages and dendritic cells from synovial tissue of RA patients. It
is not clear whether bacterial antigen was derived from material carried by mononuclear cells from other body sites or from live organisms within the joint. Evidence for the presence of live bacteria
in the synovial tissue of RA patients has come from the work of Medrano
and Galbete, who observed cell-associated unidentified bacilli in 33 of
34 synovial membrane explant cultures (41). The bacilli in
this study were not characterized; therefore it is not known whether
these organisms are similar to those identified in early culture
studies. Medrano and Galbete made the observation that the bacteria
appeared to exist in a partially cell wall-deficient (CWD) form and
were difficult to culture. This may suggest the possible involvement of
uncultivable or "difficult-to-grow" organisms in RA. CWD (or
L-form) bacteria are notoriously difficult to culture owing to their
osmotic sensitivity and have been implicated in other diseases of
unknown etiology like Crohn's disease (60) and sarcoidosis
(29). However, owing to the lack of reproducibility by other
workers, these observations are still the subject of some controversy.
The detection of bacteria by conventional culture methods, staining, or
species-specific PCR is not perhaps the most sensitive or comprehensive
means of assessing the range of bacteria that could be present in
disease tissue. With the advent of PCR-based detection techniques based
on bacterial rDNA (49), a number of other conditions of
unknown etiology have been found to be caused by previously
unidentified and uncultivable bacteria, e.g. Tropheryma
whippelii in Whipple's disease (50). It is conceivable that uncultivable or difficult-to-grow bacteria could be involved in
RA. Wilbrink et al. have demonstrated the presence of bacterial DNA in
synovial biopsy specimens from individuals with septic and inflammatory
arthropathies by PCR of rDNA with universal primers (61). By
rDNA sequencing, this group were able to partially characterize the
bacteria found in joints of four individuals with undifferentiated
arthritis (UA), some to the species level. Multiple bacterial species
were observed in each, suggesting colonization with more than one
organism. It is not known from this study whether these tissues
contained any previously unidentified microorganisms.
Here we present data demonstrating the presence of multiple bacterial
species in joint tissue of both late-stage RA patients and non-RA
arthritis controls, using the similar technique of reverse
transcriptase-PCR (RT-PCR) of bacterial rRNA. We have used this
adaptation of established DNA-based techniques because bacteria have
multiple copies of rRNA compared with their rRNA genes (16);
thus, RT-PCR of rRNA may offer a severalfold-increased sensitivity over
rDNA PCR. This technique has been used to detect bacterial rRNA in
arthritis joint tissue, suggesting the presence of viable organisms,
and to carry out detailed characterization of the bacteria present by
sequencing of complementary rRNA (crDNA) products. These analyses have
revealed the presence of both previously characterized and novel
bacterial species. The in situ localization of these microorganisms has
also been investigated by conventional bacteriological staining of
tissue sections and hybridization with digoxigenin-labeled rDNA
oligonucleotides. Microorganisms present in these apparently
subclinically infected joints appear to be both cell associated and extracellular.
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MATERIALS AND METHODS |
Patients.
RA and osteoarthritis (OA) synovial tissue
specimens were collected with patient consent at surgery for joint
replacement, with the exception of the specimens from RA patient 2, which was obtained by needle biopsy, and OA patient 20, which was
collected at the first metatarsal-phalangeal joint MTP surgery. Normal
synovial tissues from patients 22 and 23 were collected by needle
biopsy, and normal synovial tissue from patient 21 was collected at
arthroscopy for unexplained knee pain (clinical details of all patients
are given in Table 1). Normal controls
were not age and sex matched to the RA patient group, but trauma
specimens were unlikely to have features of joint disease pathology in
common with arthritis patients of many years duration. These tissues
were used as process controls and were run with each study sample. All
RA patients were classified according to the American College of
Rheumatology criteria (5) and had late-stage disease, i.e.,
disease of many years duration with joint destruction requiring
arthroplasty. A classification of UA was made on the basis of mono- or
oligoarthritis where all other diseases had been excluded.
Materials.
All chemicals including Gram stain reagents were
purchased from Sigma-Aldrich Co. Ltd., Poole, England. TB
Carbolfuschein staining reagents were supplied by Difco Laboratories,
West Molesey, England. Faramount aqueous mountant medium and nitroblue
tetrazolium-5-bromo-4-chloro-3-indolyl phosphate-iodonitrotetrazolium
violet (NBT-BCIP-INT) were purchased from Dako, Ely, England. The
Hybaid Ribolyser kit was purchased from Hybaid, Teddington, England.
Amplitaq Taq polymerase and buffers were supplied by
Perkin-Elmer, Warrington, England. Oligonucleotide primers for RT-PCR
were purchased from GibcoBRL Life Technologies, Paisley, Scotland. Dual
5' and 3' digoxygenin-end-labeled oligonucleotides were purchased from
Sigma-Genosys Ltd., Pampisford, England. Deoxyribonucleotides and
anti-digoxigenin-coupled alkaline phosphatase were purchased from Roche
Diagnostics, Lewes, England. The Novagen pT7-blue PCR cloning kit was
purchased from Cambridge Bioscience, Cambridge, England.
Tissue handling and RNA isolation.
Resected synovial tissue
samples collected at surgery were immediately frozen in hexane on dry
ice and then stored at 
80°C prior to use. Synovial biopsy specimens
were placed immediately into 500 µl of guanidinium isothiocyanate
extraction buffer (GIEB). Total RNA was isolated from late-stage RA, OA
and UA synovial tissue and normal control tissue using a modification
of the Hybaid RiboLyser guanidinium isothiocyanate-acid phenol
extraction method, in which buffer A was replaced with fresh GIEB
(39). In short, approximately 0.1 g of resected
synovial tissue was thawed in 500 µl of GIEB, chopped finely and
extracted using shear lysis in the presence of 500 µl of phenol (pH
4.0) and 100 µl of chloroform-isoamyl alcohol in a Hybaid RiboLyser,
as specified by the manufacturer. Total RNA was recovered by
precipitation with propan-2-ol, dried under appropriate sterile
conditions, and dissolved in 50 µl of diethylpyrocarbonate-treated
water containing 0.1 mM EDTA.
RT-PCR amplification of bacterial crDNA from bacterial and
synovial tissue RNAs.
To eliminate the risk of contamination with
bacterial nucleic acids from external sources, all reagents were
prepared using distilled water irradiated with UV at 254 nm for 2 min.
Rigorous controls were instigated at each stage of the RT-PCR procedure to ensure no contamination of samples during protocol implementation. Samples of control bacterial rRNAs (25 ng) or total tissue RNA (100 ng)
were reverse transcribed by the same method, in 4 µl of buffer
containing 50 mM Tris-Cl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 40 µM deoxynucleoside triphosphates, and 20 µM primer R2 (all primer sequences are given in Table 2).
This mixture was heated to 65°C for 1 min and then cooled to room
temperature for 3 min. Superscript RT (200 U) was added, and the
mixture was incubated at 37°C for 1 h. The reaction was stopped
by incubation at 65°C for 10 min.
Bacterial rDNA fragments were amplified from total cDNA and bacterial
genomic DNA by PCR amplification using universal bacterial
rRNA-specific oligonucleotide primers R1 and R2. RT mix (1 µl)
or
genomic DNA (5 ng) was used as template in a PCR mixture containing
1×
Amplitaq PCR buffer, 0.2 µM deoxynucleoside triphosphates,
0.2 µM
PCR primers R1 and R2, 1.5 mM MgCl
2, and 2.5 U of Amplitaq
Taq polymerase. These were amplified at 94°C for 4 min and
then
for 30 cycles of 58°C for 1 min, 72°C for 3 min, and 94°C
for
1 min. PCR products were visualized by electrophoresis on a 2%
agarose
gel.
In samples from many patients, bacterial 16S crDNA bands could be seen
(Fig.
1); some were diffuse, suggesting
mixed bacterial
crDNA products and the possible presence of more than
one organism.
PCR products were therefore cloned into the PCR product
cloning
vector pT7-blue (Novagen), as specified by the manufacturer,
for
isolation and sequencing. Individual clones were inoculated into
96-well plates and grown overnight, and then a small amount of
bacterial suspension was transferred into 96-well PCR plates containing
25 µl of PCR mixture as above but containing 3 mM MgCl
2
and PCR
primers which amplify the cloned fragment using flanking
plasmid
primer binding sites (primers B40F and B40R). These were
amplified
at 94°C for 4 min and then for 25 cycles of 60°C for 1 min, 72°C
for 3 min, and 94°C for 1 min. Individual PCR products
were diluted,
sequenced with primer B40F on an ABI automated sequencer,
and
analyzed using the search algorithm BLAST (
3) on
database sequences
and compared horizontally using the Genetics
Computer Group (GCG)
algorithm PileUp (
18a).
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FIG. 1.
Results of RT-PCR of bacterial 16S rRNA from patient RNA
samples, amplification products visualized by agarose gel
electrophoresis, and ethidium bromide staining.
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Detection of tissue-localized bacteria by conventional staining
and in situ hybridization.
Frozen arthritis and control tissues
were embedded in OCT (Agar Scientific), and 0.3-µm sections were
produced on a Shandon Cryotome cryostat. Conventional bacterial
staining was performed on selected sections using Gram stain and
Carbolfuschein reagents as specified by the manufacturers. For in situ
hybridization, sections were rinsed in phosphate-buffered saline (PBS),
rehydrated by immersion in 0.2% Triton X-100-PBS for 15 min, and
washed twice in PBS. Half the slides were treated with 200 µl of
RNase solution (10 mg/ml in 2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) for 30 min at 37°C as a control. RNase-treated
slides were washed twice in 2× SSC and then digested at 37°C for 17 min with a solution containing 0.1 M Tris-Cl, 50 mM EDTA (pH 8.0), and
5 µg of proteinase K per ml. These were then immersed in 0.1 M
glycine in PBS and fixed with 4% paraformaldehyde in PBS. After being
rinsed with PBS and treated in 0.25% acetic anhydride and 0.1 M
triethanolamine solution (pH 8.0), all slides were incubated in 20%
acetic acid at 4°C, washed three times in PBS, dehydrated through
alcohol, and dried.
For oligonucleotide annealing, sections were prehybridized at 37°C
for 30 min in 50 µl of buffer containing 36% formamide,
5× SSC,
10% dextran Sulfate, 5% Denhardt's solution, 0.5% sodium
dodecyl
sulfate (SDS), 100 µg of sheared herring sperm DNA per
ml. A 40-µl
volume of fresh buffer was then added containing a
mix of 5 ng of
digoxigenin-labeled probes ISH1 to ISH3 per µl.
The sections were
incubated at 37°C overnight and then washed
four times in 2×
SSC-0.1% SDS at 45°C, twice in 0.1× SSC-0.1%
SDS at 45°C, and
twice in 2× SSC at room temperature. They were
then treated with 10 µg of RNase per ml in 2× SSC at 37°C for
15 min. For visualization
of the digoxigenin-labeled probe, the
slides were rinsed with
Tris-buffered saline (TBS) and then incubated
for 30 min at room
temperature in TBS buffer containing 10% bovine
serum albumin and
0.5% Triton X-100 and for 5 min with TBS containing
2% normal sheep
serum and 0.5% Triton X-100. The TBS was removed,
100 µl of buffer
was added containing antidigoxigenin immunoglobulin
G conjugated to
alkaline phosphatase diluted 1:100 in TBS-2% normal
sheep
serum-0.5% Triton X-100, and the sections were incubated
for 2 h. The slides were washed three times in TBS, and bound
alkaline
phosphatase was visualized by incubation for 16 h in
NBT-BCIP-INT
solution. The slides were rinsed in water, counterstained
in Mayers
hematoxylin solution (Pioneer Research Chemicals Ltd.),
and mounted in
Mountant. All the sections were visualized by light
microscopy, and
images were captured either using electronic imaging
or on 35-mm Kodak
Ektachrome 64T
film.
| |
RESULTS |
RT-PCR amplification and sequencing of bacterial crDNA fragments
from total synovial RNA.
crDNA amplification products from total
synovial tissue RNAs were observed in 8 of 9 RA specimens, 6 of 11 non-RA arthritis controls (i.e., 4 of 7 OA specimens and 2 of 4 UA
specimens), and 0 of 3 normal specimens (Fig. 1). Due to the high risk
of contamination, great care was taken in sample handling, buffer generation, and RT-PCR analysis with the implementation of appropriate RT and PCR negative controls; these were consistently negative. Since
the normal control samples were also consistently negative, it was
concluded that the RT-PCR signal in positive tissues was derived from
tissue-associated bacterial rRNA and not contamination from skin or
other environmental sources, e.g., introduced during surgical removal
of tissue.
DNase treatment was not conducted on all samples, to preserve the total
signal obtained from both RNA and DNA. However, when
tested on a larger
cohort of patient samples than presented in
this study, DNase treatment
of total synovial RNA prior to RT-PCR
did not abolish the signal,
suggesting the presence of live bacteria
in these tissues (K. Kempsell
and C. Cox, unpublished data). The
intensity of the PCR signals varied
between samples; this cannot
be related directly to total bacterial
numbers since the number
of rRNA transcripts can vary enormously in
bacterial cells according
to rates of growth (
16).
The bacterial crDNA fragments from each positive sample were cloned
into the vector pT7-Blue, and the clones were sequenced.
At least 46 individual clones were sequenced, and where bacterial
species
determination proved difficult due to cloning of nonspecific
or partial
crDNA products, additional sequencing was conducted
(Table
3). In general, samples that consistently
gave a very
strong PCR signal yielded good recovery of bacterial
crDNA-containing
clones. Tissues with weaker RT-PCR signals gave less
efficient
recovery of bacterial crDNA-containing clones; therefore more
clones were sequenced to generate an accurate bacterial species
profile. Tissues which consistently gave very weak signals, e.g.,
patient 12 samples c, e, and f, were not cloned, since the recovery
of
bacterial crDNA-containing clones was expected to be very poor.
Clones
were sequenced on one pass only, which was estimated to
be more than
97% accurate. Data analysis using the BLAST algorithm
on database
sequences revealed a number of bacterial species,
most of which could
be identified to near species level. These
are outlined in Table
4, along with the percent
similarity to
the best-fit database sequence. Further comparisons were
made
between the cloned sequences to determine the overall similarity
of bacterial species between patient samples. Figures
2 and
3 and
Table
4 give graphic color-coded depictions of the profile
of bacterial
species found in each patient.
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TABLE 3.
Summary of RT-PCR analysis of patient RNAs, histologic
tests, and in situ hybridization with digoxigenin-labeled bacterial
16S rRNA oligonucleotidesa
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FIG. 2.
Diagrammatic representation of the bacterial species
identified in RA patients by sequencing of cloned 16S crDNA amplicons.
Each genus is represented by colour coding, and species are depicted by
initials derived from abbreviated species names (Table 4). Section
sizes are representative of the total number of sequences for that
species in each patient.
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FIG. 3.
Diagrammatic representation of the bacterial species
identified in the OA and UA patients. The outline for genus and species
representation is given in Fig. 2.
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It can be seen from these analyses that there were a number of
different bacteria in each patient tissue, with almost unique
complements of bacterial species in each. Some species were unique
to
individuals, while others were shared with other patients in
the study.
The profiles were distinct and highly variable, indicating
no bacterial
contamination of the sample either at the clinical
source or during
processing. A number of well-characterized bacterial
species were
found, distributed across both disease groups. Most
of these are of
commensal origin, in particular from the skin
and gastrointestinal
tract. These include most notably
Staphylococcus epidermidis,
Propionibacterium acnes, and
Escherichia coli, as
well as other coliforms. Bacterial
species that may also be derived
from members of the endogenous
microflora include streptococci,
actinomycetes, and neisseriae. Some of
these organisms have opportunistically
infectious or pathogenic
potential, and their presence is of note.
Many of the bacterial
sequences detected are, however, unique
and have not been previously
characterized by sequencing of rRNA
since they show less than 97%
similarity to known database sequences;
these may represent new
species. It is not clear what the source
of these organisms is, but
they could be either unidentified commensal
organisms or environmental
in origin. The only indication of the
presence of a potentially
pathogenic organism is the finding of
Mycobacterium
tuberculosis group (MTG) crDNA sequences in RA patient
6. The rRNA
genes of the MTG, which also contains the vaccine
strain
M. bovis BCG, are more than 99.9% similar (
31), so that
the presence of these sequences may not indicate clinical infection
with pathogenic
M. tuberculosis.
A total of 92 individual species were found in the RA group and 50 were
found in the non-RA control group, implying that RA-affected
joints, in
addition to having a greater bacterial load as indicated
by their
RT-PCR signal, contain a greater number of species. Overall,
the RA and
non-RA control groups shared 21 species; therefore,
these organisms are
probably opportunistic colonizers of diseased
synovium. Seven species
in the RA group were unique (Fig.
4) and
were found in more than one patient. These were
Corynebacterium species 2,
E. coli species 2 and
3,
Streptococcus species 2,
Pseudomonas species
2,
Leptospira species 1, and
Methylobacterium
species
1. A total of 42 other identifiable bacterial species were
unique
to this group but in one individual only. In the non-RA group
(Fig.
4), two species were unique and were found in more than
one
individual (
Corynebacterium species 9 and unidentified
bacterium
species 1); a further 25 were unique and appeared in only one
individual.
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FIG. 4.
Diagrammatic representation of the total numbers of
bacterial species unique to each patient group. (A) RA patients; (B)
non-RA patients. Dark-shaded segments indicated species unique to that
disease and found in more than one patient. Lighter-shaded segments
indicate species unique to that disease and found in one patient only.
Blank segments indicate species common to both patient groups.
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Synovial tissue samples are not uniform with respect to bacterial
species colonization.
In addition to species variation among
patients, intrasample variation was found in tissue samples from the
same patient. There appeared to be signal variation between
individually analyzed samples from the same source. Samples 12 a
to g, taken from the same knee of a patient with OA, were found to be
substantially different in the intensity of their RT-PCR signals (Fig.
1), suggesting that not all parts of the same tissue have the same
bacterial load. In addition to variation in RT-PCR signal intensity
among samples, patient 12 had a different complement of bacterial
species in the three strongly positive tissue samples sequenced,
samples a, d, and g. This suggests that, since not all parts of the
same tissue specimen have a similar bacterial load, they also do not contain the same species, suggesting microcolonization of different tissue areas.
In situ Localization of bacteria in synovial tissue sections.
Since no organisms were found in normal synovial specimens, it is not
thought that the presence of bacterial rRNA sequences in the test
specimens is due to contamination from the surgical procedure or during
subsequent processing and analysis. Staining of bacterial
crDNA-positive tissue sections by conventional Gram stain did reveal
some gram-positive organisms (Fig. 5),
which appeared to be staphylococci, and other bacterium-like bodies, which did not give the expected positive (purple), or negative (pink)
Gram stain result. This implies that bacteria present may not stain
conventionally with these reagents, perhaps due to alterations in cell
morphology. Conventional staining may not be appropriate in tissues
where bacteria may exist in a CWD form.
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FIG. 5.
Cryostat tissue sections from patient 8, stained with
bacterial Gram stain. (A) Extracellular microcolony of staphylococci.
Magnification, ×100. (B) cell-associated bacteria staining
unconventionally brown by this staining technique. Magnification,
×40.
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To validate our observations obtained by RT-PCR and to determine the in
situ localization of the bacteria previously detected,
we conducted in
situ hybridization experiments with digoxigenin-labeled
universal
bacterial rRNA-specific oligonucleotides. These are
complementary to
bacterial rRNA and will bind directly to ribosome-associated
rRNA. This
technique proved to be extremely sensitive in the detection
of bacteria
in infected tissues (Fig.
6).
M. tuberculosis-infected
mouse lung tissue stained by conventional
Ziel-Nielson Carbolfuschein
methods and by in situ hybridization gave
signals of comparable
intensity. Control human and mouse tissues were
negative. A UA
sample that was previously negative by RT-PCR also
proved negative
by in situ hybridization. A weakly positive UA sample
showed sparse
localized intracellular staining around what appeared to
be inflammatory
foci.
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FIG. 6.
Control and undifferentiated arthritis cryostat tissue
sections stained with bacterium-specific stains or by in situ
hybridization using digoxgenin-labeled bacterial 16S rRNA
oligonucleotides ISH1 to ISH3. (A) Control mouse liver stained by in
situ hybridization. (B) Control human kidney stained by in situ
hybridization. (C) M. tuberculosis-infected mouse lung
stained with mycobacterium-specific Ziehl-Nielson Carbolfuschein. (D)
M. tuberculosis-infected mouse was stained by in situ
hybridization. (E) Section from negative undifferentiated arthritis
patient 16 stained by in situ hybridization. (F) Section from
undifferentiated arthritis patient 15 stained by in situ hybridization.
Note the intracellular staining (IC) within a focus of inflammatory
cells. Magnifications, ×20 (A), ×40 (B and F), and ×10 (C to E).
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Human synovial tissues that had previously been found positive by
RT-PCR gave strong hybridization signals with these probes
(Fig.
7). Negative tissues did not, and the
relative intensity
of section staining appeared to correlate with the
result obtained
by RT-PCR (Table
3). This was particularly evident for
samples
from patient 12, where RT-PCR-negative tissue sections b and f
gave weak in situ hybridization signals whereas sections a and
d
stained strongly. In most of the strongly staining sections,
the
staining appeared to be synovial cell associated, implying
an
intracellular location for much of the tissue-associated bacterial
rRNA. Some small regions of extracellular staining may be associated
with bacterial microcolonies, but these are less easy to distinguish
by
light microscopy. Single organisms were not readily detectable
even at
higher magnifications.
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FIG. 7.
RA and OA arthritis cryostat tissue sections stained by
in situ hybridization using digoxigenin-labeled bacterial 16S rRNA
oligonucleotides ISH1 to ISH3. (A) RA patient 7. Note the heavy
staining within a focus of what appear to be inflammatory cells. (B) RA
patient 8. Note the sparse staining in isolated cells. (C) Patient 12 sample a. (D) Patient 12 sample b. (E) Patient 12 sample g. (F) Patient
12 sample e. Signals correlate with those obtained by RT-PCR.
Magnifications, ×20.
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DISCUSSION |
The presence of a wide variety of bacterial species in both RA and
other forms of chronic arthritis was an unexpected and novel discovery
and indicates that arthritic joints are not sterile, as thought
previously. Some of the species we identified are known organisms, but
many are novel and may represent organisms previously uncharacterized
by rRNA sequencing. The source of these organisms is not known, but
they may be derived from environmental sources or from the indigenous
microflora, since it is thought that in the gut at least, only a
proportion of resident commensal microorganisms have been identified
(9). Since many of the bacteria we identified in synovium
have not been characterized previously in other studies of either
environmental or clinical material, it may suggest that they are not
readily cultivable. Certainly the majority of bacterial species
identified in this study have not previously been found in the synovium.
Many species, e.g., P. acnes and S. epidermidis,
were found in both the RA and non-RA patient groups, implying that
their presence in synovium is not disease specific and that they are likely to be opportunistic colonizers of already diseased and compromised tissue. P. acnes is part of the normal skin
microflora and has previously been isolated by culture from RA synovial
fluid (6). Antigen from this organism has also been detected
in synovial fluid leukocytes (7), implying an intracellular
location. S. epidermidis has not previously been isolated
from arthritic joints, other than in overt septic arthritis; however,
since both organisms have pathogenic potential (reviewed in references
17, 25, and 27), particularly
S. epidermidis, their presence may be significant. Since
many of the other species found in both patient groups are also normal
commensal residents of the skin or gastrointestinal tract, the role
that any of these bacteria play in joint pathology must remain uncertain.
The presence of commensal organisms suggests trafficking from sites
such as the gut; it has previously been suggested that gut permeability
and mucosal competence is impaired in RA, although other studies have
implicated nonsteroidal drugs in the causation of these abnormalities
(24, 42), and all of the arthritic patients in this study,
including those with OA, are likely to have been exposed to these
agents. In a previous PCR study, Wilbrink et al. did not report
evidence of bacteria in OA-affected synovium (61). However,
the duration of disease in their OA patients was less than 12 months,
compared to many years in our patients coming to joint replacement
surgery. In this former study it also appears that the synovia of UA
patients with a disease duration of more than 12 months are more often
positive for bacteria by PCR (4 of 4 as opposed to 4 of 16). This would
also suggest increasing colonization of arthritis tissue over time,
irrespective of the cause, and may in part explain differences in
bacterial positivity in OA patients between the two studies.
Many of the OA-affected synovia in our study showed histological
features of late-stage disease and contained a substantial inflammatory
infiltrate. For example, in synovial tissue from OA patient 12, in situ
hybridization analyses showed large numbers of both intracellular and
extracellular bacteria associated with inflammatory cells. Therefore,
we conclude that any chronic synovitis may be colonized by commensal
bacteria, which most probably reached the joint from the gut and skin
within phagocytic cells, particularly macrophages, which are
continuously recruited to the synovium. However, in general a large
number of the RA-affected joint tissues were positive for bacteria and
each contained a larger number of individual species, consistent with
the greater degree of inflammation present in this disease.
Some species of bacteria were found only in RA, and while in many cases
a particular organism was seen in only a single RA patient, some
organisms were seen in more than one. These included corynebacteria and
streptococci, which have been isolated from the synovial fluid of RA
patients previously by culture (26, 58). Whether any of
these organisms could play a role in RA specifically awaits further
investigation, but it is noteworthy that previous studies of ReA have
demonstrated the causative organism in only a proportion of affected
synovia, even when the diagnosis has been firmly established. Thus, a
significant etiologic agent would not necessarily be detected in all
RA-affected synovia. This may be due in part to the limits in
sensitivity of any rDNA amplification and sequencing technique. Studies
such as this are problematical due to inherent sampling errors and the
prohibitive sequencing effort that must be conducted to collate
meaningful results. Low-copy-number sequences in these mixed crDNA
pools are often overlooked; these can be seen by specific nested PCR but are not observed by large-scale sequencing (Kempsell and Cox, unpublished). Thus, large numbers of nonspecific organisms can obscure
any etiological agent in low abundance.
Also conspicuously absent from the list of bacteria identified in RA in
this study are bacterial species which have captured interest in recent
years as possible etiological agents of RA, including mycoplasmas
(52, 53) and Proteus mirabilis (64). M. tuberculosis (10, 51) was also not found, with
the exception of MTG crDNA sequences in patient 6. In addition, we
found no evidence of bacterial species that commonly cause ReA in
RA-affected joint tissue, whereas these organisms can be identified in
ReA-affected synovium (12, 18, 23, 62, 63).
If bacteria are involved in the pathology of RA, the condition might be
expected to respond to antibacterial therapy. Antibiotic trials have
been conducted in RA with different degrees of success (reviewed in
reference 47), but little evidence of a general efficacy of antimicrobials has emerged. Caruso and coworkers have reported striking results using high-dose intra-articular injection of
rifamycin SV (14); however these studies have not been
corroborated by other workers. Some effect of tetracyclines has also
been reported (1, 33, 57), but since these and other
efficacious antibiotics have profound anti-inflammatory properties
(19, 34, 56), the mechanism of any effect seen in RA remains
unclear. However, given the evidence of subclinical bacterial
colonization presented in this paper, part of their mode of action
might well be antibacterial. If this were the case, the results of
treatment might be expected to be variable since not all colonizing
species would be sensitive to the particular antibiotic under trial. In
addition, some of the organisms identified in this study are
notoriously refractory to antibiotic treatment; in S. epidermidis, this is due to the production of protective biofilm
matrices (17). Streptococci can also persist in tissues and
evade killing by sequestration inside host cells (45). Since
some of the bacteria appeared to be intracellular, this would be
another reason to explain the lack of efficacy of some antimicrobials.
ReA (55) and LyA (28, 43) are also relatively
refractory to antimicrobial therapy, particularly in chronic disease,
even though there is no doubt about the causative organism. Again,
intractability to antimicrobials may be due to the persistence of
slow-growing or latent bacteria. Thus, even if bacteria are involved in
the primary pathogenesis of RA, the disease might not respond to
conventional antimicrobial therapy.
Bacteria could cause or influence inflammatory joint disease in a
number of ways including (i) persistent infection (35); (ii)
induction of autoimmune pathology, perhaps through molecular mimicry
(2, 35); (iii) production of bacterial superantigens (46, 54); and (iv) induction of immune dysfunction through other mechanisms (25). Which of these mechanisms is
responsible for ReA or LyA remains obscure, and several may be
involved. In LyA there is evidence of both persistent infection
(27) and induction of immune dysfunction or autoimmune
pathology, the latter arising from the autoreactive potential of T
cells recognizing the B. burgdorferi outer membrane protein
OspA (11, 21, 32, 37). Whether any of the bacterial species
identified in RA could induce disease pathology by similar means
remains unclear.
Other bacterially mediated autoimmune diseases in which persistent
infection and induction of autoimmune pathology have been postulated to
contribute to disease pathology include gastric inflammation
(Helicobacter pylori) (4, 44) and
arteriosclerosis (Chlamydia pneumoniae) (22, 36).
In the former case, the organism is clearly implicated, since the
disease responds to elimination of the organism, whereas in the latter
case, the organism may either contribute to the pathogenesis of the
atherosclerotic plaque or merely act as a colonizer of the diseased and
compromised tissue. The present study raises the same questions with
regard to the role of synovial bacteria in chronic arthritis. While the
general colonization of the synovium which is a feature of all chronic synovitis may exacerbate inflammation irrespective of the cause of the
arthritis, particular organisms may play an initiating role in diseases
such as RA. It will be particularly informative to compare the spectrum
of bacterial species isolated from long-established chronic synovitis
and from acute disease, since in the latter case nonspecific
colonization may not yet have occurred and any organisms isolated at
this early stage would be more likely to be relevant to pathogenesis as
initiators of disease.
| |
ACKNOWLEDGMENTS |
We thank Gabriel Panayi, Dorian Haskard, Peter Sewell, George
Southgate, Gill Pountain, Andrew Hassell, and Sally Roberts for the
kind provision of clinical material for use in this work. We also thank
Alan Lewis for help with sequence analysis and other technical assistance.
We thank Glaxo Wellcome UK for sponsoring this project and for
financial support of Charles Cox.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Glaxo Wellcome
Medicines Research Centre, Department of Immunopathology, Gunnels Wood Rd. Stevenage, Hertfordshire SG1 1NY, United Kingdom. Phone: (44) 1438 768027. Fax: (44) 1438 764818. E-mail:
kek23980{at}GlaxoWellcome.co.uk.
Present address: The Chester Beatty Institute for Cancer Research,
London SW3 6JB, United Kingdom.
Editor:
E. I. Tuomanen
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Infection and Immunity, October 2000, p. 6012-6026, Vol. 68, No. 10
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
