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Pathogenicity of stable L-phase variants of staphylococcus aureus reaction of rabbit synovium to intraarticular inoculation.

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Pathogenicityof Stable L-Phase Variants of
Staphylococcus Aureus
Reaction of Rabbit Synovium to lntraarticular Inoculation
Calvin C. Linnemann, Jr., Chatrchai Watanakunakorn and Cheryl Bakie
Stable L-phase variants of Staphylococcus aureus were injected into
the knee joints of rabbits. The recovery of organisms at various intervals
was attempted and the reaction of the synovium studied. Although what
appeared to be L-phase variants could be seen within polymorphonuclear leukocytes in the synovial fluid at 48 hours, they did not grow in
culture. At 1 week, there was a mild synovitis characterized by both
acute and chronic inflammatory changes which was diminished at 2
weeks and resolved by 5 weeks. Joints injected with L-phase variants
and sterile media differed only in the degree of change. Following multiple injections a more diffuse chronic synovitis was present, and in l
experimental animal rheumatoid-like changes were seen. L-phase variants could not be recovered from cultures of the synovial fluid or the
synovial tissue.
T h e role of wall-defective microbial
variants (1) in the initiation and/or persistence of infection has not been defined.
We have previously shown that a strain of
L-phase wriant of Staphylococcus aureus
inoculated into an in situ polyethylene
catheter in the right side of the rabbit
heart failed to colonize endocardia1 vegetations (2). A pathogenic role for wall-defective microbial variants in arthritis has been
suggested, but the evidence is unconvincing. Pease (3) isolated a n L-form of a bacFrom the Infectious Disease Division, Department
of Internal Medicine, University of Cincinnati
Medical Center, Cincinnati, Ohio 45229.
CALVIN c LINNEMANN, MD: Assistant Professor; c
WAI'ANAKUNAKORN, MD: Associate Professor; CHERYL
BAKIE: Research Assistant.
Address reprint requests to Dr Linnemann, University of Cincinnati College of Medicine, Christ
Hospital Institute of Medical Research, 21 39 Auburn Avenue, Cincinnati, Ohio 45219.
Submitted for publication September 13, 1973;
accepted November 26, 1973.
terium resembling Listeria from the serum
and joint fluid of all patients with arthritis
whom she studied, including 24 with rheumatoid arthritis, 4 with osteoarthritis and
12 with a n unspecified arthritis. Identification of the organism was based on microscopic examination and the appearance of
vegetative forms in cultures after prolonged
incubation. Direct examination of the joint
fluid in 29 patients revealed material of
similar appearance to that seen in cultures.
Holmes et al (4)reported a case in which
they isolated Neisseria gonorrhoeae from
the synovial fluid in hypertonic media
when chocolate agar and Thayer-hIartin
media were sterile and suggested that the
isolate was wall-defective. Roberts and Little (5) reported the isolation of bacteria in
hypertonic media from the synovial fluid
of diseased joints of all pigs, sheep, cattle
and chickens after cultures in routine media produced no growth, and suggested
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
603
LINNEMANN E l AL
that these were wall-defective variants.
Only one experimental challenge study to
test the pathogenicity of wall-defective microbial variants in joints has been reported.
Cook, Fincham and Lack (6) produced a
chronic monarticular arthritis in rabbits by
the intraarticular injection of either viable
or heat-killed streptococcal L-forms. However in no instance were the L-forms recovered from cultures of synovial tissue.
T h e present study reports the reaction
of rabbit synovium to the intraarticular inoculation of a strain of stable L-phase variants of S aurezis and attempts to recover
the organism in hypertonic media.
variants, were inoculated into the right knee of
each animal and the left knee was injected with
the diluent, 0.9.1, saline for group I and salt broth
or 5 % saline for group I1 and group 111 animals.
Rabbits in group I were given 1 injection of
vegetative S aweus; group I1 received a single inoculation of stable L-phase variants of S aureus;
and group 111 received multiple inoculations of
the stable L-phase variants of S aureus.
R ESULTS
Group I (Vegetative S aureus)
Four rabbits were inoculated with vegetative S uureus. Two of these received 1.0 ml
containing 1Ou organisms and were sacrificed at 3 days. T h e infected knees were
slightly swollen at the time of sacrifice and
MATERIALS AND METHODS
on opening the joint, the synovium was
discolored and thickened and a gray tenaBacteriologicTechnique
T h e bacteriologic techniques used in this study cious pus filled the joint cavity. Gram-stain
have been reported in detail (2). T h e stable L- of the pus revealed numerous polymorphophase variant used was derived from the August
nuclear leukocytes and gram-positive cocci.
Harmon strain of S aureus phage type 6/7/42D/
Histologic sections of the synovium re54/75, originally isolated from a patient with endovealed an acute synovitis with abscess forcarditis. This was induced by methicillin and has
been propagated on antibiotic-free media for sev- mation, and Gram stain revealed clusters
eral years without reversion to the vegetative bac- of gram-positive cocci. Cultures of both
terial phase. T h e L-phase variant has been shown
the pus and synovium grew S auseus. Two
to be sensitive to lysostaphin, which is specific
rabbits
were inoculated with 0.5 ml confor staphylococci, and to produce staphylococcal
taining 10' colony-forming units of S nzireus
enterotoxin A (7,8). T h e vegetative S aureus used
in these experiments was a phage type 79 isolated
and sacrificed at 7 days. There was an
from the blood of a patient with endocarditis. acute synovitis with polymorphonuclear
Cultures for L-phase variants were made in brainleukocytes and gram-positive cocci, but to
heart infusion (BHI) broth with 5% NaCI, O . Z ~ o
a lesser extent than in the animals receivMgSO, and 5% heat-inactivated horse serum (salt
broth), and in BHI broth with 10% sucrose, 0.2y0 ing the larger inoculum. There was also a
MgSO, and 5% heat-inactivated horse serum. These mononuclear infiltrate in the synovium
were incubated at 37°C and routinely subcultured with evidence of a chronic synovitis. T h e
a t 1 week for two successive subcultures. Cultures
control knees which were injected with
ivhich appeared to be grossly cloudy were stained
by the Gram technique and subcultured on a 0.9yo saline were normal except for minihypertonic agar plate. Acridine orange staining mal focal areas of synovitis in 2 of the 4
was done using the technique of Chattman el a1 (9). animals.
Study Design
T h e knee joints of 8- to 10-pound white New
Zealand rabbits were inoculated with a 28-gauge
needle. Bacteria, either vegetative forms or L-phase
604
Group II (L-Phase Variants)
T e n rabbits were inoculated with L-phase
variants and sacrificed at 1, 2 or 5 weeks.
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
STABLE L-PHASE VARIANTS OF S AUREUS
Table 1. Rabbits Inoculated with a Single lntraarticular Injection
of Stable L-Phase Variants of S aureus
Rabbit
No.
419
415
229
234
228
236
254
253
232
231
Size of
lnoculum
1
2
2
1
2
1
7
7
1
2
x 107
x 107
X 10'
x 107
X lo*
x 107
x 107
X
X
X
10'
10'
10'
Time of
Sacrifice
(Wk)
1
1
1
1
2
2
2
2
5
5
Diluent
of
lnoculum
Salt broth
5% saline
Salt broth
5% saline
Salt broth
5% saline
Salt broth
Salt broth
5% saline
Salt broth
Pathologic Findings*
L-Phase variants
chronic synovitis
Acute iMinimal chronic synovitis
Acute chronic synovitis
Normal
Moderate chronic synovitis
Normal
Minimal chronic synovitis
Minimal chronic synovitis
Normal
Normal
+
Control (diluent alone)
Minimal chronic synovitis
Normal
Minimal chronic synovitis
Normal
Minimal chronic synovitis
Normal
Normal
Normal
Normal
Minimal chronic synovitis
'Normal = no thickening of the synovial cell layer or inflammatory infiltrate; minimal = few areas of
involvement with a 2- or 3-fold increase in the thickness of the synovial cell layer and with scanty
mononuclear infiltrate; and moderate = more marked synovial hyperplasia with many mononuclear
cells. Acute and chronic = polymorphonuclear and mononuclear cell infiltrates, respectively.
At 24 to 48 hours the injected knees appeared slightly swollen, the right greater
than the left. Aspiration of the joints was
attempted in 2 animals and pus was obtained from the right knee of both rabbits.
On Gram stain, gram-negative spheres were
seen in the joint fluid and similar orange
spheres were seen within polymorphonuclear leukocytes on fluorescent microscopy
after staining with acridine orange. However the L-phase variants could not be recovered on culture.
Of the 4 rabbits sacrificed at 1 week, 2
had received % ml of the L-phase variants
and 2 had received 2 ml. T h e inoculum
contained 107 to 108 organisms. I n each
pair of animals the diluent used for the
inoculum and as the control injection was
salt broth for one, and 5% saline for the
other (Table 1). T h e synovium in the control knees inoculated with 5% saline was
normal, whereas the synovium in those inoculated with salt broth showed focal areas
of hyperplasia and some infiltration with
mononuclear cells. T h e synovium of the
knees inoculated with L-phase variants
showed focal areas of hyperplasia and areas
of both acute arid chronic synovitis, which
was more marked in those receiving salt
broth as the diluent.
Four rabbits were sacrificed 2 weeks after
inoculation. By that time all acute changes
in the knees inoculated with L-phase variants had subsided, and only focal areas of
hyperplasia and minimal to moderate
chronic synovitis persisted. T h e synovium
in the control knees was normal in 3, including 2 which had received salt broth.
T h e remaining animal inoculated with salt
broth had minimal chronic synovitis.
T w o animals in this study group were
sacrificed at 5 weeks and the synovium
was normal in all joints except for 1 control knee which showed minimal chronic
synovitis.
L-phase variants were not recovered from
either the synovial fluid or synovial tissue.
A mannitol-negative, coagulase-negative sta-
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
605
LINNEMANN ET AL
phylococcus which could not be phage- articular injection (6). However the same
typed was present in 2 cultures, 1 from a authors indicated that a wide range of macontrol knee and 1 from a study knee. A terials, especially particulate matter, can
mannitol-negative, coagulase-positive phage induce similar arthritic changes. This was
type 79 staphylococcus was present in the seen in the present study in that control
cultures from 2 other study knees. Cultures knees injected with hypertonic saline
of phage type 79 S uiiwzis were being han- showed markedly less synovial reaction
dled in the same laboratory at the time the than those injected with hypertonic BHI
cultures from these experiments were being broth. T h e knees inoculated with the staperformed. Vegetative staphylococcus was ble L-phase variants showed similar but
not recovered from more than 1 culture in more severe changes than the control knees.
each animal and was never present in both This probably represented a nonspecific
the synovial fluid and synovial tissue cul- reaction to the greater mass of antigenic
ture. N o gram-positive cocci were present material contained in the L-phase variant
in any of the synovial fluid or synovial tis- culture or the presence of small amounts
sue specimens. I t is most likely that these of bacterial toxins (8).
staphylococci were contaminants.
T h e rabbit synovium cleared the L-phase
variants rapidly and no organisms could be
recovered at 48 hours. I n contrast the inGroup 111 (L-Phase Variants,
fecting organism could be isolated from the
Multiple Inoculations)
synovial fluid at 3 and 7 days after the
Four rabbits received multiple inoculaanimals were inoculated with the vegetations of L-phase variants at approximately
tive S uuyezis. Even in joints in which
2-week intervals. Two received three injecL-phase variants were repeatedly inocutions and the other 2 a total of five injeclated
and on occasion produced a chronic
tions. Once again the control joints showed
active
synovitis, the organism could not be
only mild hyperplasia of the synovium or
recovered
from the synovial tissue. Thus
chronic synovitis. I n contrast the joints
there
was
no
evidence that colonization of
inoculated with 1,-phase variants showed
the
synovium
by the L-phase variants had
more pronounced changes ranging from
ever
taken
place.
Cook and co-workers (6)
mild to severe chronic synovitis. I n 1 of
also
failed
to
recover
the organism from
the animals receiving three inoculations
synovial
tissue
of
rabbits
after intraarticuthere were rheumatoid-like changes with
vascular proliferations, hyperplasia of the lar inoculation of viable streptococcal Llining cells of the synovium, cellular infil- forms.
Stable L-phase variants were chosen for
tration, and aggregates of mononuclear
the
present study in order to distinguish
cells. No L-phase variants were recovered
the
pathogenic role of the wall-defective
from cultures of synovial tissue from these
form
from the vegetative form. Reversion
animals.
to the vegetative form and subsequent tissue damage could be excluded. There was
DISCUSSION
no convincing evidence of in vivo reversion
It has been reported that viable or heat- in the present study. However there was no
killed streptococcal L-forms induced a absolute proof that this had not occurred.
chronic monarticular arthritis after intra- T h e lack of stable biologic markers of the
606
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October1974)
STABLE L-PHASE VARIANTS OF S AUREUS
reverted staphylococci made it almost impossible to identify these organisms correctly (10).
This study indicates that a large inoculum of stable L-phase variants of S aweus
could induce a nonspecific arthritis, but
the normal clearance mechanisms of the
rabbit synovium prevented the colonization
and persistence of the organism. It is possible that wall-defective variants other than
stable L-phase variants may be pathogenic
when present in the synovium or contribute to the persistence of infection despite
antibiotic therapy. Such an hypothesis is extremely difficult to prove, even though
there have been isolated case reports of the
recovery of bacteria from synovial fluid
cultures in hypertonic media when routine
cutures failed to yield the organisms.
ACKNOWLEDGMENTS
This investigation was supported by Public
Health Service training grant T1-AI-87 and research grant AI-09755 from the National Institute
of Allergy and Infectious Diseases, and by the
Morton Hamburger Memorial Fund.
Judith Carleton made the stable L-phase variant
of S azcreus available for this investigation. T h e
assistance of On Ja Kim in the interpretation of
histopathology is gratefully acknowledged.
REFERENCES
1. McGee ZA, Wittler RG, Gooder H, et al:
Wall-defective microbial variants: terminol-
ogy and experimental design. J Infect Dis
123:433438, 1971
2. Linnemann CC Jr, Watanakunakorn C,
Bakie C: Pathogenicity of stable L-phase
variants of Staphylococcus aureus: Failure to
colonize experimental endocarditis in rabbits. Infect Immunity 7:725-730, 1973
3. Pease P: Bacterial L-forms in the blood and
joint fluids of arthritis subjects. Ann Rheum
Dis 28:270-274, 1969
4. Holmes KK, Gutman L T , Belding ME, et al:
Recovery of Neisseria gonorrhoeae from
“sterile” synovial fluid in gonococcal arthritis. N Engl J Med 284:318-320, 1971
5. Roberts DH, Little TWA: A note on spheroplasts and mycoplasma in synovial fluid. Br
Vet J 127:143-147, 1971
6. Cook J, Fincham WJ, Lack CH: Chronic
arthritis produced by streptococcal L-forms.
J Pathol 99:283-297, 1969
7. Watanakunakorn C, Browder HP: Effects of
lysostaphin and its two active components
on stable wall-defective forms of Staphylococccus aureus. J Infect Dis 121:124-128, 1970
8. Czop JK, Bergdoll MS: Synthesis of enterotoxin by L-forms of Staphylococcus aureus.
Infect Immunity 1:169-173, 1970
9. Chattman MS, Mattman LH, Mattman PE:
L-forms in blood cultures demonstrated by
nudeic acid fluorescence. Am J Clin Pathol
51:41-50, 1969
10. Watanakunakorn C, Bakie C: Coagulase
production, mannitol fermentation, penicillinase elaboration and phage typability of
Staphylococcus aureus reverted from L-phase
variants. J Infect Dis 127: 571-575, 1973
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
607
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