St. George's Hospital and Medical School,
Cranmer Terrace, London SW17 0RE, United Kingdom
Received 13 July 1999/Returned for modification 26 August
1999/Accepted 22 September 1999
 |
INTRODUCTION |
Mycoplasmas are proven causes of
arthritis in animals, notably, Mycoplasma bovis and M. capricolum in ruminants, M. hyosynoviae in pigs,
M. synoviae in poultry, and M. pulmonis and
M. arthritidis in rodents (18). Mycoplasmas have
been suspected of being associated with arthritis in humans. This is
particularly the case for M. fermentans, which was first
isolated from rheumatoid arthritis (RA) patients by Williams et al.
(20), and an increase in cell-mediated immunity to this
organism in RA patients was noted (21). However, others
(12) failed to confirm these findings due to the fastidious growth requirements of M. fermentans and poor success with
culture. When PCR methods became available, this organism was detected in the saliva samples from 44% of healthy people (4), in
the throat, urine, or peripheral blood mononuclear cells of 33% of human immunodeficiency virus-seronegative patients attending a venereal
diseases clinic (10), in 11% of the peripheral blood mononuclear cells from human immunodeficiency virus-seronegative subjects (11), and in the synovial fluid of 14% of patients with rheumatoid and other inflammatory arthritides (15, 16), although the organism was present in 40% of the biopsy specimens of
the RA patients' synovial lining cells. It has not been reported from
osteoarthritis (OA) patients, who were included in the present study as
controls. Unfortunately, however, the PCR detection methods for
M. fermentans were based on supposedly specific primers
(19) which have recently been found to detect M. orale also (5), especially if this organism is present
at a high concentration. The work described in this paper was designed
to search for and identify M. fermentans in human synovial
fluid by a very sensitive, fully nested PCR method and to unequivocally
identify the organism by sequencing part of both of its 16S rRNA genes,
which proved to be polymorphic. A similar method has been used to
identify strains of M. mycoides (14).
| |
MATERIALS AND METHODS |
Patients.
Synovial fluid samples were collected from the
knees of 26 patients with RA, all of which fulfilled the criteria for
diagnosis of the American Rheumatism Association (2).
Samples were also collected from 10 patients with OA and from 8 patients with other inflammatory arthritides. The latter included 2 patients suffering from ankylosing spondylitis (AS), 2 patients with
gout, 2 patients with psoriatic arthritis (PsA), and one patient each
with reactive arthritis (ReA) and pauciarticular juvenile chronic
arthritis (JCA). The total number of samples collected from patients
with RA was 34, since on five occasions samples were obtained from both
the patient's knees, and 2 consecutive samples were obtained from
three patients at least 3 months apart. The total number of samples
from the eight non-rheumatoid inflammatory arthritis patients was 11, since samples were obtained from both knees for one AS patient and for
one ReA patient and two consecutive samples 1 year apart were obtained
from the JCA patient. Nonsteroidal anti-inflammatory drugs (NSAIDS)
and/or prednisolone were taken by 23 of 26 RA patients and 6 of 8 of
the non-rheumatoid inflammatory arthritis patients, whereas only 3 of
10 of the OA patients were taking NSAIDS. Patient information is shown
in Table 1.
Synovial fluid collection.
The synovial fluid samples were
obtained by sterile aspiration from the knees of patients attending the
rheumatology clinic when aspiration was indicated as part of routine
clinical practice. Consent for evaluation of the fluid was obtained
from the patients. The samples were handled aseptically and were
centrifuged at 500 × g for 30 min at 4°C to remove
the cells. Aliquots (usually 1 ml) were stored at 
70°C. It was
important that these samples not be thawed and refrozen.
Materials.
The suppliers of the apparatus and chemicals are
listed below. Hyaluronidase and proteinase K were obtained from Roche
Diagnostics Ltd., Lewes, United Kingdom. The primers, including the
biotin-labelled primer with the hexamethylene spacer, came from Oswel
DNA Service, University of Southampton, Boldrewood, United Kingdom. The
Alf Express automatic sequencer, Cy5-labelled sequencing primers, Taq polymerase, and the solid-phase sequencing plastics and
chemicals came from Amersham Pharmacia Biotech Ltd., Little Chalfont,
United Kingdom. The thermal cycler and Taq Gold came from
Perkin-Elmer Applied Biosystems Ltd., Warrington, United Kingdom, and
Takara Ex Taq came from BioWhittaker UK Ltd., Wokingham,
United Kingdom. The Tris-borate sequencing buffer came from National
Diagnostics, Hull, United Kingdom, and the Finnpipettes from Life
Sciences International, Basingstoke, United Kingdom.
Preparation of samples for PCR.
Synovial fluid samples were
prepared in batches of 16 or 32 and normally contained two blanks with
1 ml of Tris-buffered saline. A 10-mg/ml solution of hyaluronidase in
pyrogen-free water was freshly prepared and was sterilized by
filtration. The viscosity of the 1-ml synovial fluid samples was
lowered by adding 20 µl of hyaluronidase solution per ml and allowing
the mixture to stand for 0.5 h at room temperature. The samples
were then centrifuged at 18,000 × g for 0.5 h at
20°C, and the small pellet that was obtained was washed twice with
sterile pyrogen-free saline that had been buffered at pH 8.0 with 10 mM
Tris-HCl (TBS). For the blanks, hyaluronidase was added to 1 ml of TBS,
and the tubes were treated as described above for the other samples.
A fresh 10-mg/ml solution of DNA-free proteinase K was prepared. The
sample buffer contained 10 µl of 1 M Tris-HCl (pH 8.0), 12 µl of
proteinase K solution, and 0.978 ml of pyrogen-free water. Then, 50 µl of filter-sterilized 9% Tween 20 was added, and 0.1 ml of this
mixture was added to the samples and the blanks. These were heated at
37°C for 30 min and 60°C for 1 h, and then the proteinase K
activity was destroyed by heating the samples at 96°C for 10 min.
These samples were stored frozen at 20°C.
Primers.
The PCR primers were based on those described by
Hopert et al. (7), but only those which fit the published
16S rRNA gene sequence for M. fermentans exactly were used.
The primers were purified by high-performance liquid chromatography,
and their sequences are shown in Table 2.
PCR.
The PCR mixture (50 µl) contained 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% (wt/vol) gelatin
(Perkin-Elmer Taq Gold buffer diluted 10 times), the
nucleotides dATP, dCTP, dGTP, and dTTP, each at a concentration of 200 µM, each primer at a concentration of 200 nM, 1 U of Taq
polymerase, and 5 µl of sample. When a nested PCR was to be
performed, the first round used Taq polymerase from
Pharmacia or, in some cases, TaKaRa Ex Taq, which contains a
proofreading enzyme which increases the sensitivity of the reaction
two- to threefold. In this case the buffer and deoxynucleoside
triphosphate solutions supplied by the manufacturer were used. It was
important to keep the pH at 8.3 for the biotinylated primer used in the
second round, so highly specific Taq Gold was used. The
primer 8A recommended by Hopert et al. (7) caused problems
when it was biotinylated, probably because the primer binds close to a
loop on the DNA. However, primer 8A worked well when the biotin was
attached with a hexaethylene glycol spacer. The PCR was carried out in
0.2-ml tubes, and the reaction mixture was overlaid with 30 µl of
sterile mineral oil. The thermal cycler was a Perkin-Elmer GeneAMP PCR
system 9600.
The sample was initially dissociated by heating at 95°C for 3 min
(Taq Gold for 15 min) and was then thermally cycled 50 times at 95°C for 35 s, 58°C for 60 s, and 72°C for 35 s, finally being left to extend at 72°C for 7 min before being cooled
to 4°C. The cycling conditions were the same for both sets of
primers, and 50 rather than 30 cycles were used to obtain as strong a
PCR band as possible for sequencing. The relatively long annealing
period was found to produce the best results. An 8-µl sample of the
final reaction products was run on a 3% agarose gel containing 0.5 µg of ethidium bromide ml
1, and the gel was
photographed under a UV light. Those samples showing a clear PCR band
were retained for sequencing. Initially, primers *8A and 3A were used
with Taq Gold under the same PCR conditions described above.
This gave a single-stage PCR and, with the Cy5-labelled sequencing
primer SEQ1, allowed the type strain PG18 to be partially sequenced and
identified a missing base (N = A), which was used in SEQ2. Samples
from 12 of 32 of the inflammatory arthritis patients gave bands which
could be sequenced by the single-stage PCR. However, some bands which
were too weak to sequence were found, so the nested PCR was used,
together with the Cy5-labelled primer SEQ2. The single-stage PCR and
sequencing primer SEQ1 were aimed at human mycoplasmas in general and
were not specific for M. fermentans, although this was the
only organism detected by the single-stage PCR. The nested PCR used
primers 9A and 3A as the outer primer pair and primers *8A and 5B as
the inner primer pair, with SEQ2 as the sequencing primer. These were specific only for M. fermentans.
Sequencing.
An ALF Express automated sequencer was used. It
was developed particularly for the sequencing of human DNA and readily
detects gene polymorphisms. It was used with the solid-phase sequencing kit supplied by the manufacturer. The biotinylated PCR product was
removed from the reaction mixture by allowing it to adhere to four
streptavidin-coated plastic combs. The Cy5-labelled sequencing primer
was allowed to bind to the PCR product, and the sequencing reaction was
run with T7 polymerase and the dideoxynucleoside triphosphate mixtures
provided by the manufacturer. The reaction products were dissociated
from the combs with the iodoacetamide mixture provided by the
manufacturer and were electrophoresed down a 6% polyacrylamide gel
with 0.089 M Tris-borate buffer (pH 8.3) with 2 mM EDTA, obtained as a
10× concentrate. The dye-labelled products were visualized with a
laser, and the sequence was shown on a computer screen. This sequence,
when reversed and complemented, was compared to the prokaryotic DNA
sequences on the PCGENE or EMBL databases to identify the organism.
Precautions against contamination.
As the mycoplasmal
contents of the synovial fluid samples were extremely small, they were
both difficult to measure and easy to contaminate. It was essential to
do the DNA extraction and PCR procedures under sterile conditions since
the primers used might react to some extent with other bacteria and
contaminate the PCR band. If this happened, the sequencing primer was
normally blocked so no sequence was obtained, even if a band had been observed.
Pyrogen-free saline and water for injection were used in the isolation
of the sample. The Eppendorf tubes and 0.2-ml PCR tubes were sterilized
in an autoclave, and the other plastics and pipette filter tips were
purchased in radiation-sterilized condition. Given that the PCR bands
were sequenced, there was no danger that a band due to a bacterial
contaminant would be identified as a mycoplasma. New Gilson and
Finnpipettes were acquired and were used solely with the filter tips
for PCR. Disposable plastic trays were used to prepare PCRs.
The batch preparation of mixed samples and blanks and the preparation
of more than one sample from each individual in different batches were
again precautions against contamination, although sometimes not enough
material was available for more than one test with a sample. The
sequencing of the PCR band identified the organism, and since
identifiably different M. fermentans strains were
discovered, it was often possible to check for contamination by sequencing.
Nucleotide sequence accession number.
The partial sequence
of the second gene in strain PG18 and in most of the clinical samples
was deposited in GenBank, under the name MF16SRRNA2 and with accession
no. AF031374.
| |
RESULTS |
M. fermentans was found in 31 of 34 samples from the RA
patients, 9 of 11 samples from the non-rheumatoid inflammatory
arthritis patients, and in none of 10 samples from the OA patients. If
more than one sample was obtained from one patient the same strain was
always found in both knees or in two consecutive samples. An exception
was for the first JCA patient sample, which was negative and which may
have been affected by a bacterial contaminant, as only a poor-quality
PCR band which could not be sequenced was obtained. The second sample
from this patient was positive. Table 3
shows the distribution of M. fermentans among the patient
groups. It was confined to patients with inflammatory arthritis but not to patients with RA alone and was not found in the 10 patients with OA.
Figure 1 is a photograph of an agarose
gel stained with ethidium bromide. It shows the second-round PCR
product obtained from some RA, AS, gout, and OA patients with the
primer pair *8A-5B. Bands can be seen only for samples from the
patients with inflammatory arthritis and not for samples from the
patients with OA.
View larger version (73K):
[in this window]
[in a new window]
|
FIG. 1.
Second-round PCR products produced by the synovial fluid
samples which were treated with primer pair *8A-5B. Lanes 1 and 2, samples from OA patients; lane 3, sample from a gout patient infected
with M. fermentans; lane 4, samples from an AS patient
infected with M. fermentans; lanes 5 and 6, samples from RA
patients infected with M. fermentans; lane 7, molecular size
ladder.
|
|
Figure 2 shows the coding strands of part
of the 16S rRNA gene from M. fermentans. It was discovered
that for the type strain of M. fermentans, strain PG18, and
for most of the strains found in the clinical samples, the two 16S rRNA
genes of M. fermentans were not identical. The ambiguities
occurred at positions 253 and 298 in the M. fermentans
sequence shown, where the base was T on one gene and C on the other and
is described by the letter Y in the sequence. For strain PG18 and most
of the strains from patients, position 253 was heterogenous (Y) but the
base at position 298 was T on both genes and was Y for only one strain
from an RA patient. PCR products from strains from two RA patients, the PsA patient, and strain "incognitus" homogeneously showed a T base
at position 253, and strains from four RA patients homogeneously showed
a C base at position 253. Unusual sequences were resequenced for
confirmation. Strains from two RA patients were either C or Y, as the
(repeated) sequences obtained were poor and ambiguous. This could have
been an experimental defect, or perhaps two M. fermentans
strains were present. One other base, described as N on the original
sequence, was identified and is underlined and in boldface type in Fig.
2. It is part of SEQ2.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 2.
Sequence of part of the 16S rRNA gene of M. fermentans showing the positions of the primers and the
ambiguities in the sequence. The positions of the outer and inner
primers are marked with dotted lines and arrows, and the sequencing
primers are underlined. MF, edited part of M. fermentans
sequence MFRRNAF. Y (arrows) is C or T.
|
|
| |
DISCUSSION |
M. fermentans was restricted to the joints of patients
with inflammatory arthritis and was not detected in any of the patients with OA. Two of three of the RA patients negative for M. fermentans were not taking anti-inflammatory drugs and had
clinically quiescent disease. The third patient was taking NSAIDS, and
this patient, as well as one of the other M. fermentans-free
patients, had only recently developed the disease. A recent publication
by O'Dell et al. (13) has shown that treatment of
recent-onset RA patients with minocycline, to which M. fermentans is susceptible (6), produces a long-term
decrease in the severity of their symptoms.
Comparison with previous studies.
The results of tests for the
detection of M. fermentans confirm those of Williams et al.
(20) and Schaeverbecke et al. (15, 16), except
that 88% of the synovial fluid samples from our RA patients contained
M. fermentans, whereas 14% of the samples reported by
Schaeverbeke et al. (15, 16) contained M. fermentans, and both of our patients with gout were infected with
M. fermentans. Four different strains of M. fermentans were found, and different strains have been reported to
have been cultivated from arthritis patients (17).
There are a number of reasons for the greater sensitivity of the PCR
procedures reported from this work. M. fermentans has been
reported to associate with B cells rather than neutrophils in the
peripheral blood (3), so it was possible to remove the irrelevant, largely neutrophilic infiltrate from the synovial fluid,
allowing the small amount of residual precipitate to be treated with
proteinase K, which is a 10-fold more sensitive method of DNA
preparation than ethanol precipitation from phenol-chloroform (1). This was followed by the use of a fully nested PCR,
which is about 100-fold more sensitive than a single or seminested PCR. However, other measures contributed to the result, such as the use of
Taq Gold and the treatment of the sample, particularly the
specific removal of hyaluran with hyaluronidase, which left any
mycoplasmas behind.
The sequencing of the mycoplasmal PCR product not only confirmed the
identity of the mycoplasma but also showed the presence of different
strains. M. fermentans has an unusual rRNA gene
organization, with two sets of rRNA genes and with the two 16S-23S rRNA
gene clusters being arranged in a tail-to-tail orientation
(8). The sequencing method used in this work was originally
developed to sequence the human HLA genes. The whole PCR product is
sequenced, so heterozygotes (or polymorphism) can be detected. The
sequences of both of the two 16S rRNA genes of M. fermentans
were therefore determined, and if a lack of identity occurred they were
usually not identical at only one or rarely two bases.
M. fermentans was found in the synovial fluid of nearly all
the inflammatory arthritis patients. It has not previously been detected in gout patients, and this discrepancy is attributed to the
greater sensitivity of the methods described in this work. In patients
with gout it is possible either that the organism arrived in the joint
subsequent to the inflammation or even that it initiated the sodium
urate crystal formation by a biochemical reaction or by acting as a
nucleus for crystal formation. RA patients have significantly more
mycoplasmal rRNA in their synovial fluid than the patients with other
arthritides (9), so they may have a particular difficulty
dealing with these organisms, or special strains may be found in RA
patients alone. M. fermentans may well contribute to the
inflammation and chronicity of RA.
We thank David Pitcher for providing the mycoplasmal DNAs with
the primers were tested and Angus Dalgleish for allowing us to use his
automated sequencer.
This work was supported by the St. George's Hospital Special Trustees.
| 1.
|
Abele-Horn, M.,
U. Busch,
H. Nitschko,
E. Jacobs,
R. Bax,
F. Pfaff,
B. Schaffer, and J. Heesemann.
1998.
Molecular approaches to diagnosis of pulmonary diseases due to Mycoplasma pneumoniae.
J. Clin. Microbiol.
36:548-551[Abstract/Free Full Text].
|
| 2.
|
Arnett, F. C.,
S. M. Edworthy,
D. A. Bloch,
D. J. McShane,
J. F. Fries,
N. S. Cooper,
L. A. Healey,
S. R. Kaplan,
M. H. Liang,
H. S. Luthra,
T. A. Medsger, Jr.,
D. M. Mitchell,
D. H. Neustadt,
R. S. Pinals,
J. G. Schaller,
J. T. Sharp,
R. L. Wilder, and G. G. Hunder.
1988.
The American Rheumatism Association 1987. Revised criteria for the classification of rheumatoid arthritis.
Arthritis Rheum.
31:315-324[Medline].
|
| 3.
|
Cheek, R. F.,
I. Olsak,
S. Madoff, and F. I. Preffer.
1997.
In vitro detection of Mycoplasma fermentans binding to B-lymphocytes in fresh peripheral blood using flow cytometry.
Cytometry
28:90-95[CrossRef][Medline].
|
| 4.
|
Chingbingyong, M. I., and C. V. Hughes.
1996.
Detection of Mycoplasma fermentans in human saliva with a polymerase chain reaction-based assay.
Arch. Oral Biol.
41:311-314[CrossRef][Medline].
|
| 5.
|
Ditty, S. E.,
M. A. Connolly,
B. J. Li, and S.-C. Lo.
1999.
Mycoplasma orale has a sequence similar to the insertion-like sequence of M. fermentans.
Mol. Cell. Probes
13:183-189[CrossRef][Medline].
|
| 6.
|
Hannan, P. C. T.
1998.
Comparative susceptibilities of various AIDS-associated and human urogenital tract mycoplasmas and strains of Mycoplasma pneumoniae to 10 classes of antimicrobial agent in vitro.
J. Med. Microbiol.
47:1115-1122[Abstract].
|
| 7.
|
Hopert, A.,
C. C. Uphoff,
M. Wirth,
H. Hauser, and H. G. Drexler.
1993.
Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines.
J. Immunol. Methods
164:91-100[CrossRef][Medline].
|
| 8.
|
Huang, Y.,
J. A. Robertson, and G. W. Stemke.
1995.
An unusual rRNA gene organization in Mycoplasma fermentans (incognitus strain).
Can. J. Microbiol.
41:424-427[Medline].
|
| 9.
|
Johnson, S. M.,
F. E. Bruckner, and D. A. Collins.
1996.
Mycoplasmal RNA and 5'-nucleotidase in synovial fluid from arthritis patients.
Int. Org. Mycoplasmol. Lett.
4:140.
|
| 10.
|
Katseni, V. L.,
C. B. Gilroy,
B. K. Ryait,
K. Ariyoshi,
P. D. Bieniasz,
J. N. Weber, and D. Taylor-Robinson.
1993.
Mycoplasma fermentans in individuals seropositive and seronegative for HIV-1.
Lancet
341:271-273[CrossRef][Medline].
|
| 11.
|
Kovacic, R.,
V. Launey,
P. Tuppin,
A. Lafeuillade,
V. Feuillie,
L. Montagnier, and O. Grau.
1996.
Search for the presence of six Mycoplasma species in peripheral blood mononuclear cells of subjects seropositive and seronegative for human immunodeficiency virus.
J. Clin. Microbiol.
34:1808-1810[Abstract].
|
| 12.
|
Mårdh, P. A.,
F. J. Nilsson, and A. Bjelle.
1973.
Mycoplasma and bacteria in synovial fluid from patients with arthritis.
Ann. Rheum. Dis.
32:319-325[Free Full Text].
|
| 13.
| O'Dell, J. R., G. Paulsen, C. E. Haire, K. Blakely, W. Palmer, S. Wees, P. J. Eckhoff, L. W. Klassen, M. Churchill, D. Doud, A. Weaver, and G. F. Moore. Treatment of
early seropositive rheumatoid arthritis with minocycline. Arthritis
Rheum. 42:1691-1695.
|
| 14.
|
Pettersson, B.,
T. Leitner,
M. Ronaghi,
G. Bolske,
M. Uhler, and K.-E. Johansson.
1996.
Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons.
J. Bacteriol.
178:4131-4142[Abstract/Free Full Text].
|
| 15.
|
Schaeverbeke, T.,
C. B. Gilroy,
C. Bébéar,
J. Dehais, and D. Taylor-Robinson.
1996.
Mycoplasma fermentans in joints of patients with rheumatoid arthritis and other joint disorders.
Lancet
347:1418[Medline].
|
| 16.
|
Schaeverbeke, T.,
C. B. Gilroy,
C. Bébéar,
J. Dehais, and D. Taylor-Robinson.
1996.
Mycoplasma fermentans, but not M. penetrans, detected by PCR assays in synovium from patients with rheumatoid arthritis and other rheumatic disorders.
J. Clin. Pathol.
49:824-828[Abstract/Free Full Text].
|
| 17.
|
Schaeverbeke, T.,
M. Clerc,
L. Lequen,
A. Charron,
C. Bébéar,
B. de Barbeyrac,
B. Bannwarth,
J. Dehais, and C. Bébéar.
1998.
Genotypic characterization of seven strains of Mycoplasma fermentans isolated from synovial fluids of patients with arthritis.
J. Clin. Microbiol.
36:1226-1231[Abstract/Free Full Text].
|
| 18.
|
Simecka, J. W.,
J. K. Davis,
M. K. Davidson,
S. E. Ross,
C. T. Städtlander, and G. H. Cassell.
1992.
Mycoplasma diseases of animals, p. 391-415.
In
J. Maniloff, R. N. McElhaney, L. R. Finch, and J. B. Baseman (ed.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C.
|
| 19.
|
Wang, R. Y.,
W. S. Hu,
M. S. Dawson,
J. W. Shih, and S. C. Lo.
1992.
Selective detection of Mycoplasma fermentans by polymerase chain reaction and by using a nucleotide insertion sequence within the insertion sequence-like element.
J. Clin. Microbiol.
30:245-248[Abstract/Free Full Text].
|
| 20.
|
Williams, M. H.,
J. Brostoff, and I. M. Roitt.
1970.
Possible role of Mycoplasma fermentans in the pathogenesis of rheumatoid arthritis.
Lancet
ii:277-280[CrossRef].
|
| 21.
|
Williams, M. H., and F. E. Bruckner.
1971.
Immunological reactivity to Mycoplasma fermentans in patients with rheumatoid arthritis. Preliminary communication.
Ann. Rheum. Dis.
30:271-273[Free Full Text].
|
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Abstract
Introduction
Present address: Princess Margaret Hospital, Swindon SN1 4JU,
United Kingdom.
