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ORIGINAL ARTICLE
M ultiple Bacteria in CalciŽ c Aortic Valve Stenosis
Kristin M. Kolltveit1, Odd Geiran 2, Leif Tronstad1 and Ingar Olsen1
From the 1Institute of Oral Biology, Faculty of Dentistry, University of Oslo, Norway and the 2Department of
Thoracic and Cardiovascular Surgery, National Hospital, Oslo, Norway
Correspondence to: Leif Tronstad, Institute of Oral Biology, Faculty of Dentistry, University of Oslo, P.O. Box
1052 Blindern, N-0316 Oslo, Norway. Tel.: »47 228 40376; Fax: »47 228 40305; E-mail: [email protected]
M icrobial Ecology in Health and Disease 2002; 14: 110 – 117
CalciŽ c aortic valve stenosis affects an increasin g number of elderly patients and causes considerable morbidity. The etiology of this
disease is unclear . Lately, the issue has been raised that its pathogenesis may be linked to chronic in ammation caused by
microorganisms. The aim of the present study was to investigat e if bacteria are present in calciŽ c stenotic aortic valves. Tissue specimen s
were collected from 19 patients undergoin g aortic valve replacemen t for aortic valve stenosis and from ten patients with regurgitant aortic
valves or clinically healthy valves from explanted hearts (controls). Specimen s were sectioned and subjected to transmission and scanning
electron microscopy, as well as to anaerobic culturing. Bacteria of various morphologies (cocci, rods, spiral and crescent forms) were
detected in 16 of the 19 patients with valve stenosis. Twelve of these patients yielded positive cultures and 12 were positive assessed by
electron microscopy. The bacteria were present in Ž brous tissue in the thickened areas of the valve and in lacuna e of calciŽ ed nodules.
Bacteria seemingly undergoin g cell division were observed . M acrophage digestio n of bacteria was also noticed. No bacteria were detected
in the valves from the control group except in one patient. Bacteria of different morphologies were observed in calciŽ c stenotic aortic
valves. It appear s likely that the bacteria are implicated in the pathogenesis of acquired, calciŽ c aortic valve stenosis. Key words : heart
valve diseases, cardiac surgica l procedures, bacteria, aerobic, bacteria, anaerobic.
INTRODUCTION
CalciŽ c aortic valve stenosis is a common disease in Europe and N orth America affecting an increasing number of
elderly patients and causing considerable morbidity. With
regard to the pathogenesis of this disease, different views
have been held. In reality, its etiology still remains obscure. One hypothesis that has been proposed is that aortic
stenosis is another manifestation of atherosclerotic disease
and that its progression can be explained by classical
cardiovascular risk factors (1). Still, risk factors such as
hypertension, cholesterolemia, and cigarette smoking account for only one-half to two-thirds of the incidence of
cardiovascular disease (2). Therefore, other pre-disposing
factors have been searched for. Evidence is accumulating
linking infection and chronic in ammation to arteriosclerosis (3 –5), and recently it has been suggested that microorganisms such as Chlamydia pneumoniae, cytomegalovirus, herpes simplex 1 and periodontal pathogens
may pre-dispose for cardiovascular disease (6– 8).
R outine culturing of diseased heart valves have yielded
several bacterial species, but this information has mostly
been explained as contamination (9– 11). Nevertheless, C.
pneumoniae has been implicated in the pathogenesis of
non-rheumatoid stenotic aortic valves (12, 13). F urthermore, it is well recognized that infective endocarditis is a
bacterial infection of the heart valves or the endothelium
of the heart, frequently caused by oral pathogens (14). The
© Taylor & F rancis 2002. ISSN 0891-060 X
present study was carried out to re-examine the possibility
that microorganisms reside in tissue of stenotic aortic
valves, supporting the hypothesis that infection is an important factor in the etiology and progression of this
disease.
MATERIAL AND METHODS
Patient material
Two groups of patients, referred to the D epartment
of Thoracic and Cardiovascular Surgery, National Hospital, Oslo, N orway for treatment were included in the
study. The study group consisted of 19 patients with
calciŽ c stenotic aortic valves (Table IA) while the control
group comprised ten patients with regurgitant aortic valves
(n¾ 6) or clinically healthy valves from explanted hearts
(n¾ 4) (Table IB). Patients in the stenosis group were
72.7 9 15 years of age. Six of these were men, 13 were
women. Patients in the control group were 48.99 18 years
of age. Of these, nine were men and one was a woman.
The 19 patients with stenotic aortic valves had a mean
aortic gradient assessed by echocardiography of 62 9 22
mmH g, and the valve area was 0.69 0.2 cm 2. The patients
in this group had only mild aortic valve regurgitation.
Concomitant cardiovascular pathology was seen in 11
patients. The control patients had either a signiŽ cant aortic valve regurgitation necessitating valve replacement or
Microbial Ecology in Health and Disease
Bacteria and aortic valve stenosis
111
Table I
Clinical data
Patient
Clinical diagnosis
Presumed etiology
(A) 19 patients with calciŽ c aortic stenosis
1
AS
2
AS, CAD
3
AS, CAD
4
AS, ASO
5
AS
6
AS
7
AS, M I,TI, CAD
8
AS, CAD
9
AS, CAD
RA
10
AS, M S, TI
RF
11
AS, TAA
CH D
12
AS
CH D
13
AS
14
AS
15
AS
16
AS, CAD
17
AS, CAD
18
AS, CAD
RF
19
AS
CH D
Operation
AVR
AVR ,
AVR ,
AVR ,
AVR
AVR
AVR ,
AVR ,
AVR ,
AVR ,
AVR ,
AVR
AVR
AVR
AVR
AVR ,
AVR ,
AVR ,
AVR
CABG
CABG
ASBP
M VP, TVP, CABG
CABG
CABG
M VR, TVP
AAG
CABG
CABG
CABG
(B) 10 patients with regurgitant or clinically normal aortic valves
A
AI
AVR
B
AI
AVR
C
CM
HTX
D
AI
AVR
E
DONOR
F
AI, TAA
AVR , AAG
G
AI, M I
CH D
AVR , M VR
H
CM
HTX
I
CAD
HTX
J
AI, TAA
AVR , AAG
AAG, ascending aortic graft; AI, aorta insufŽ ciency; AS, aortic valve stenosis; ASBP, aortico-subclavian bypass; ASO, obliterative arteriosclerosis; AVR , aortic valve replacement ; CABG , coronar y artery
bypass grafting; CAD, coronar y artery disease; CH D, congenital aortic valve stenosis (bicuspid); CM ,
cardiomyopa thy; DONOR, unused heart graft; HTX, heart transplantation; M I, mitral valve regurgitation; M S, mitral valve stenosis; M VP, mitral valve plasty; M VR , mitral valve replacement ; R A,
rheumato id arthritis; R F , rheumatic fever; TAA, thoracic aortic aneurysm ; TI, tricuspid valve
regurgitation; TVP, tricuspid valve plasty.
clinically normal valves. Only one of these patients had
arteriosclerotic heart disease. No patients exhibited signs
of endocarditis when the valves were examined before and
during surgery.
All patients received 2 g of cephalotin pre-operatively
and had otherwise standard perioperative care. All patients
survived surgery and the Ž rst post-operative year. One
patient ( 12) had a post-operative bacteremia with
Staphylococcus aureus, which was treated uneventfully.
The study was approved by the Regional Committee on
M edical Ethics.
dissection. Sixteen stenotic valves were tricuspid with minimal fusion of the commissures, three were bicuspid. All
stenotic valves were thickened and irregular with multiple
hard, apparently calciŽ ed nodules in the central area of the
cusps (F ig. 1). The four valves from the explanted hearts
were normally funtioning aortic valves. Six regurgitant
valves of the patients of the control group showed opaque
lea ets with no calciŽ ed areas.
Selection of the material for subsequent examination
was done at random. Thus, one cusp was arbitrarily
selected for culturing and one for electron microscopy.
Tissue material
Anaerobic culture and identiŽ cation of bacteria
The valves were removed by incising them at their base
(F ig. 1). They were then transferred to a sterile container
and brought to an adjacent room for immediate sterile
The aortic cusp selected for culturing was crushed in a
sterile mortar using a sterile pestle and seeded in a laminar
 ow hood onto non-selective agar plates (Trypticase soy,
112
M. Kolltveit et al.
Fig. 1. Photograph of stenotic aortic valve following surgica l
removal. A thickening of the central areas of the cusps with
nodule-lik e structures is seen.
Brucella, Chocolate) and on selective agar plates (TSBV,
Lactose, Sabouraud D extrose), as well as into tubes with
Thioglycollate or Brain H eart Infusion broth. After seeding, the agar plates and tubes were placed immediately in
evacuation jars (Anoxomat System-Ws 9000, M art, Lichtenvoorde, The N etherlands). In the operating theatre, the
atmosphere of the jars was made anaerobic by using three
packages of G ENbox anaer (bioM érieux, M arcy l’Etoile,
F rance). Within 1 h, the jars with the specimens were
brought to the microbiology laboratory where they were
evacuated and Ž lled three times with a gas mixture consisting of 90% N 2, 5% CO 2 and 5% H 2. Incubation occurred at 37°C for 7– 21 days. If it was still negative after
21 days, the cultures were discarded. Preliminary identiŽ cation of pure cultures was based on aerotolerance,
colony and cellular morphology, colony pigmentation,
cellular motility and G ram staining. M icrobial diagnosis
was achieved by using commercial diagnostic kits (API,
bioM érieux). Reading of the kits occurred automatically
in an ATB reader (API, bioM érieux). The results were
transferred into a numerical code and read in a data
based system for microbial identiŽ cation (API Plus,
bioM érieux).
cessing. After dehydration in ethanol, the tissue was dried
at its critical point with carbon dioxide as the transitional
 uid. The specimens were attached to metal stubs with
silver paste and sputter-coated with gold:palladium
(thickness 30 nM ) in a vacuum evaporator. Coated samples were examined in SEM (Philips XL 30 ESEM , Eindhoven, The N etherlands). In addition samples were
examined by energy dispersive X-ray analysis (EDXA)
using the same microscope furnished with an X-ray analyzer.
Three of the tissue blocks were transferred to vials with
2.5% glutaraldehyde:0.1 M phosphate buffer supplemented with 0.1% ruthenium red for transmission electron
microscopy (TEM ), and then brought to the laboratory
for processing. The biopsies were Ž xed for 24 h at 4°C
and stored in 0.1 M phosphate buffer with ruthenium red
at 4°C until preparation. Post-Ž xation was done in 1%
osmium tetraoxide for 2 h at 4°C. After Ž xation, the
blocks were rapidly dehydrated in a graded series of
acetone solution and embedded in Vestopal W. U ltrathin
sections were cut using a Leica U ltracut microtome. The
sections were treated with uranyl acetate for 15 min,
followed by lead citrate for 3 min. They were examined in
a Philips CM 120 transmission electron microscope.
RESULTS
Bacteriologic examination
Twelve of the specimens from the stenotic valves yielded
positive cultures (Table II). All bacteria detected were
recovered from the thioglycollate or brain heart infusion
broth. N o bacterial growth occurred when the tissue samples were placed directly on the solid media. Culturing of
the valves usually gave only monocultures. Both facultative
anaerobic and strict anaerobic bacteria were found. F or
three of the organisms, identiŽ cation beyond Gram stain
was not possible. Two of them were facultative anaerobic
and one strict anaerobic. The bacterial species identiŽ ed
were Staphylococcus warneri, Propionibacterium acnes, Micrococcus luteus, Pantoea spp., Neisseria gonorrhoea, Listeria spp., Clostridium spp. and Acinetobacter :Pseudomonas
spp. Except in one case that yielded P. acnes, bacteria were
not cultured from the valves in the control group.
Electron microscopy
Six tissue blocks with a size of about 2½ 2 mm were
taken from the central area of the cusps (F ig. 1). The
blocks were cut so that they included areas which clinically appeared calciŽ ed and from areas containing both
non-calciŽ ed and calciŽ ed tissue. In the control group
tissue blocks were cut from the center of the cusps.
Three of the tissue blocks were placed in 2.5% glutaraldehyde:0.1 M Sørensen’s phosphate buffer for scanning electron microscopy (SEM ). The specimens were
then sent to the laboratory and stored at 4°C until pro-
Electron microscopy
Bacteria were not seen in TEM and SEM specimens from
control patients but were detected in ten of the samples
from the study group by using TEM and in eight samples
with SEM (Table II). When both TEM and SEM were
applied, bacteria were found in 12 samples. By using TEM ,
SEM and anaerobic culture bacteria were detected in 16 of
the samples. The thickened parts of the valves consisted of
a Ž brous almost acellular tissue (F ig. 2). The nodules that
clinically felt hard were calciŽ ed (F ig. 3) and were rich in
Bacteria and aortic valve stenosis
113
Table II
Bacteria in stenotic, calciŽ c aortic valves demonstrated by anaerobic cultivation and electron microscopy
Patient
Culture
TEM
SEM
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
–
N. gonorrhoea
Clostridium spp.
Anaerobic G ¼ rod
Listeria spp.
Pantoea spp.
F acultative G » rod
M. luteus
F acultative G ¼ rod
–
S. warneri
P. acnes
–
–
S. epidermidis
–
–
Acinetobacter :Pseudomonas spp.
–
Colonies
–
Colonies
R ods:Spirochetes
Selenomonas sp.
Bacteria in calciŽ ed tissue
Colonies
Bacteria in calciŽ c tissue
Bacteria in calciŽ c tissue
Colony
–
Colony
–
–
–
–
–
–
–
Cocci
–
Spirochetes :rods:cocci
R ods:spirochetes
–
–
R ods:cocci
Spirochetes :cocci
Cocci
–
–
–
–
–
–
–
Cocci:rods
–
Cocci:rods
calcium and phosphorus (EDXA). Bacteria were commonly
seen in the vicinity of the calciŽ ed nodules. M ost often
organisms with different morphologies (spirochetes, cocci
and rods) were seen to coaggregate forming small ecosystems in the tissue (F igs 2 and 3), but apparent monocultures
were found as well (F igs 4 and 5). Bacteria seemingly
undergoing cell division were present. The bacteria were
heavily integrated in the Ž brous tissue (F ig. 4) or inhabited
lacunae within the calciŽ ed tissue (F igs 5 and 6). Sometimes
microcolonies were imbedded in an uncalciŽ ed non-Ž brous
extracellular material (F ig. 2). Bacterial cells with a rim of
calciŽ c deposits were also seen (F ig. 7). One organism was
tentatively identiŽ ed as a Selenomonas sp. because of its
Fig. 2. Scanning electron micrograph from thickened area of
valve. A Ž brous tissue is seen . A microcolony of bacteria embed ded in a non-Ž brous, non-calciŽ ed material apparently has been
dislodged during specimen preparation and is lying on the cut
surface of the Ž brous tissue. Bar ¾ 2 m m.
crescent shape and the  agella tuft on its concave side (F ig.
8). M acrophages were observed in the Ž brous tissue apparently engulŽ ng bacteria (F ig. 9).
DISCUSSION
The present study detected bacteria of different morphologies in calciŽ c stenotic aortic valves. The widespread Ž ndings of bacteria in these valves immediately raise the
question of contamination and laboratory error. H owever,
great care was taken throughout the preparative procedures of the specimens both in the operation theatre and in
the microbiology laboratory to prevent microbial contamination. Also, anaerobic culturing would prevent aerobic
Fig. 3. Scanning electron micrograph from calciŽ ed area of a valve
(nodule). Coaggregatin g microorganism s with differen t morphologies (spirochetes, cocci, rods) are seen on the calciŽ ed surface.
Bar ¾ 2 mm.
114
M. Kolltveit et al.
Fig. 4. Scanning electron micrograph from thickened, Ž brous area
of a valve. M icroorganisms (cocci) are seen integrated in the
Ž brous tissue (near calciŽ ed nodule). Bar ¾2 mm.
bacteria from inadvertently contaminating the cultures,
and by using electron microscopic techniques, bacteria of
different morphologies and sizes could be seen inside the
tissue. These bacteria, forming small ecosystems in the
tissue, could deŽ nitely not have been brought there during
preparation of the tissue, and the fact that bacteria of
different morphologies formed coaggregates also argued
against contamination.
Combined use of anaerobic culture technique and electron microscopy enabled detection of multiple microorganisms in the stenotic tissue. This was in contrast to previous
studies designed to detect only one single species, e.g., C.
pneumoniae (15). Staining of the samples with ruthenium
red, which has been used extensively to reveal ultrastructural details of bacteria, particularly glycocalyx and other
surface-associated components (16), made the bacteria
stand out in sharp contrast to the surroundin g non-calciŽ ed tissue when examined with TEM . The SEM was
Fig. 5. Scannin g electron micrograph from calciŽ ed area of a
valve (nodule). M icroorganisms (cocci) are seen in lacuna e of the
calciŽ ed tissue. Bar ¾5 mm.
especially useful to demonstrate different cell morphologies (cocci, rods, spiral and crescent shaped forms) and to
show coaggregates of such bacteria in the stenotic valves.
Twelve specimens were positive after culturing, and 12
specimens after microscopy, while 16 were positive for
bacteria when assessed both with culture and electron
microscopy. Thus, the combination of techniques was
useful for the detection of bacteria. Still, in spite of this
combination of techniques it is more than likely that not
all microorganisms in the stenotic lesions were detected
since they were located inside the tissue and could easily
have been missed during specimen preparation and:or
seeding. Except in one case, which showed P. acnes, bacteria were not detected in the valves of the control group.
S. warneri, M. luteus and P. acnes, which were found in
three of the positive cultures from valve stenosis, have
previously been rejected in cultures of heart valves as
contaminants (9 –11). H owever, S. warneri, which represents only about 1% of the staphylococcal  ora normally
found on human skin, is a well-recognized cause of valve
endocarditis (17). M. luteus has been shown to be the
cause of prosthetic valve endocarditis (18). Sixteen additional cases of endocarditis due to Micrococcus spp. are
reported in the literature (18). P. acnes is one of the
anaerobic bacteria that can cause endocarditis (19, 20).
Accordingly, the bacteria listed above should not automatically be rejected as contaminants, at least not when major
precautions are taken to prevent contamination, as in the
present study.
We also cultured Pantoea (Enterobacter ) spp., N. gonor rhoeae, Clostridum spp. and Listeria spp. E. agglomerans
has caused endocarditis in a patient with mitral valve
prolapse (21, 22) and gonococcal endocarditis can lead to
rapid clinical deterioration from valvular destruction and
congestive heart failure (23). Clostridium and Listeria spp.
belong to rare and fastidious bacteria, which can cause
endocarditis (24). A total of 21 cases of clostridial endocarditis have been reported in the literature, the majority
being caused by C. perfringens (25 –27). F ifty-eight cases of
endocarditis have been reported due to L. monocytogenes
(28). A previous valvular disease including rheumatic heart
disease, prosthetic valve endocarditis and mitral valve
prolapse was found in 50% of these patients.
Electron microscopy made it possible to detect ‘hard-tocultivate’ organisms such as selenomonads and spirochetes. The primary body niche of Selenomonas is the oral
cavity, particularly the human gingival crevices and periodontal pockets. In the studies of Zambon et al. (29) and
Chiu (30), oral pathogens were detected inside atheromas.
Similar to our Ž ndings, multiple species (up to four) were
found in the same lesion. The source of these pathogens,
dental plaque, is characterized by coaggregation of a multitude of bacteria with different morphologies, and appears
similar in SEM to the coaggregating bacterial complexes
detected in the present study. Spirochetes have previously
Bacteria and aortic valve stenosis
115
Fig. 6. Transmission electron micrograph from calciŽ ed area of a valve
(nodule). M icroorganisms with different morphologies are seen in the calciŽ ed tissue. Bar ¾500 nm.
Fig. 7. Transmission electron micrograp h from border zone between
calciŽ ed and non-calciŽ ed areas of a
valve. Bacterial cell with rim of calciŽ ed deposit s is seen. Bar ¾ 500
nm.
been found in the myocardium in Lyme disease (31).
Interestingly, spirochetes may constitute up to 56% of the
subgingival plaque  ora in adult marginal periodontitis
(32), from which bacteria can reach the heart through
bacteremia (8).
There was a marked difference in age, gender and valve
pathology between the study group and the control group.
The control patients had either clinically normal valves or
suffered from signiŽ cant aortic valve regurgitation, without clinical suspicion of acute endocarditis. Although presence of bacteria was not to be expected in the normal
valves, it should be stressed that all patients studied had
antibiotic prophylaxis during surgery. Bacterial endocarditis was not suspected in any case and regurgitant aortic
valves may also be due to non-infectious factors such as
chronic hypertension, rheumatic valvular disease, congenital disorders or autoimmune diseases. The clinical difference between the study group and the control group is
thus not likely to have in uenced the main conclusion
drawn. It should be acknowledged that optimal controls
for this type of clinical study are not easy to obtain.
In the present study, bacteria might have contributed to
generation of the mineral deposits detected (33). A parallel
to the calciŽ cations in the valves is infected kidney stones
116
M. Kolltveit et al.
Fig. 8. Transmission electron micrograph from calciŽ ed area of a valve
(nodule). The microorganism seen is
tentatively identiŽ ed as a Selenomonas
sp. because of its crescent shape and
the  agella tuft on its concave side.
Bar ¾ 500 nm.
where bacteria are incorporated throughout the stone matrix (34). Previous studies have found T-lymphocytes in the
immediate surroundings of calciŽ ed deposits in stenotic
valves (35, 36). This indicated that bacteria had provoked
an in ammatory response. The macrophage activity observed in the present study supported this notion.
Arteriosclerosis is now widely accepted as an in ammatory disease (3– 5). The idea that microorganisms can
cause in ammatory or immune-mediated, ‘non-infectious’
diseases is not new. Examples include Helicobacter pylori
in peptic ulcer, Epstein –Barr virus in nasopharyngeal carcinoma, Tropheryma whippleii in Whipple’s disease and
mycobacteria in Crohn’s disease and sarcoidosis (37). Similarly, infection may be one pathogenic factor for in ammation and arteriosclerosis. F rom our Ž ndings, we may
suggest that a chronic bacterial infection in the body,
including periodontitis, could disseminate bacteria into the
bloodstream from where they reach the heart valves and
produce an in ammatory basis for stenosis.
Infectious agents and hosts are in a constant  ux, partaking in a continuing evolutionary drama (38). A possible
example of this was the elderly patient with N. gonorrhoeae
who probably had attracted this organism much earlier in
life. Possibly, its virulence had declined over time or the
host had acquired resistance to the damaging effects of the
pathogen. G onococcal infection is not always symptomatic, and a reservoir of infected, asymptomatic individuals maintains this organism in the general population (39).
It must be admitted that the presence of bacteria in
diseased valves does not necessarily imply a causal relationship, and our Ž ndings may suggest a non-speciŽ c trapping
of bacteria in areas of tissue already damaged. H owever,
purely commensal existence of replicating bacteria in in amed tissue is not very plausible. Since the bacteria were
viable, they could have maintained the in ammatory response for a prolonged period of time. If so, the bacteria
were probably more a causal than a coincidental link to the
stenotic lesions.
ACKNOWLEDGEMENTS
We are indebted to R enate Hars and Steinar Stølen for skilful
technical assistance with TEM and SEM , respectively. This study
was supported in part by research grants from the foundations of
Professor Hall and Stokbak, National H ospital, Oslo, N orway.
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Fig. 9. Scannin g electron micrograph from thickened area of a
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