Download Multiple bacteria in aortic aneurysms

Multiple bacteria in aortic aneurysms
Rafael Marques da Silva, DDS,a Per S. Lingaas, MD,b Odd Geiran, MD, PhD,b
Leif Tronstad, DMD, PhD,a and Ingar Olsen, DDS, PhD,a Oslo, Norway
Objective: The purpose of the present study was to reexamine the possibility that bacteria, particularly anaerobes, are
present in aortic aneurysms.
Methods: From December 2000 to November 2001, 53 samples from aneurysm walls were collected from 49 patients
during reconstructive surgery. The tissue specimens were sectioned and cultured under anaerobic conditions. Twentyeight specimens were also subjected to scanning or transmission electron microscopy.
Results: Anaerobic cultivation yielded bacteria in 14 of the 53 samples (26.4%). All bacteria were gram-positive cocci or
rods from nine genera and 12 species. Five cultures (35%) were mixed, containing two bacterial species. Mixed aerobic and
anaerobic species were found in four samples (28.5%). Anaerobic bacteria were recovered from 10 of 14 positive cultures
(71%). Among anaerobes found were Propionibacterium acnes, Propionibacterium granulosum, Actinomyces viscosus,
Actinomyces naeslundii, and Eggerthella lenta. Coaggregating bacteria of different sizes and structure were found on the
aneurysm walls and inside the intravascular plaque at electron microscopy. Bacteria were found in 20 of the 28 samples
(71%) examined with scanning or transmission electron microscopy.
Conclusion: Multiple bacteria, many of which did not belong to the indigenous skin microflora, colonize aortic aneurysms.
It is not clear whether the bacteria contribute to weakening of the aortic wall by eliciting inflammation or whether they
are secondary colonizers of aneurysms. (J Vasc Surg 2003;38:1384-9.)
Aortic aneurysm is potentially life-threatening and can
be associated with significant morbidity and mortality. The
condition is more common in men than in women (4:1).
Age is an important risk factor; incidence rises rapidly after
60 years.1 Although understanding of the mechanisms
involved in the process of aneurysm degeneration remains
limited, atherosclerosis is considered a common underlying
etiologic factor, and risk factors for atherosclerosis are
associated with increased risk for aortic aneurysm.2,3 Recent data suggest that chronic infection may have a role in
pathogenesis of atherosclerosis, and several infectious
agents, including Chlamydia pneumoniae, oral bacteria,
and herpesvirus may be involved.4-8 C pneumoniae has also
been detected in the walls of abdominal aortic aneurysms.9,10 However, the role of microorganisms in pathogenesis and development of aortic aneurysm remains to be
elucidated. Bacteriologic monitoring of samples obtained
from aortic aneurysms has shown positive cultures in 8% to
37% of cases,11-19 and cultivated bacteria are commonly
considered culture contaminants.12-14,18 Moreover, most
of the isolated bacteria are aerobes (Table I). Because
recovery and identification of anaerobic bacteria can be
time-consuming and difficult, the true prevalence of these
organisms in aortic aneurysms may be underestimated.
From the Institute of Oral Biology,a Faculty of Dentistry, University of
Oslo, and Department of Thoracic and Cardiovascular Surgery,b National
Hospital.
Supported in part by research grants from the foundation of Professors Hall
and Stokbak, National Hospital, Oslo, Norway.
Competition of interest: none.
Reprint requests: Rafael M. da Silva, Institute of Oral Biology, Faculty of
Dentistry, University of Oslo, PO Box 1052 Blindern, N-0316 Oslo,
Norway (e-mail: [email protected]).
Published online Oct 22, 2003.
Copyright © 2003 by The Society for Vascular Surgery.
0741-5214/2003/$30.00 ⫹ 0
doi:10.1016/S0741-5214(03)00926-1
1384
Therefore the purpose of the present study was to investigate whether bacteria, particularly anaerobes, reside in the
walls of aortic aneurysms.
METHODS
Between December 2000 and November 2001, 53
samples were collected from 49 consecutive patients undergoing aortic aneurysm repair at the Department of Thoracic and Cardiovascular Surgery, National Hospital, Oslo,
Norway. The patients (32 men, 17 women) had a mean age
of 63 years (range, 20-83 years). The samples were taken
from 35 thoracic aneurysms and 18 abdominal aortic aneurysms. No aneurysms showed clinical signs of infection after
preoperative and intraoperative evaluation, and 29 aneurysms (54.7%) were degenerative, on the basis of clinical
information and macroscopic judgment during surgery.20-22 Two patients had obliterative arteriosclerosis in
peripheral arteries in addition to aneurysm degeneration. In
addition, five patients had coronary artery disease, which
required coronary artery bypass surgery either as an isolated
procedure before or concomitant with repair of the aneurysm. Elective surgery was performed in 48 patients
(90.6%). Four patients (7.5%) were operated on urgently,
because of acute symptoms, and in one patient (1.9%) a
ruptured aneurysm was resected. Patients underwent treatment as judged necessary by the surgical team. All patients
received 2 g of cephalotin during induction of general
anesthesia. The study was approved by the Regional Committee on Medical Ethics. Informed consent was obtained
from patients included in the study.
During excision of the aneurysm, tissue specimens for
investigation were taken from the walls of the aneurysm.
Rests of intravascular plaque removed during surgery were
attached to some specimens. The samples were transferred
to glass tubes containing prereduced anaerobe-sterilized
VMGA III transport medium23 and brought to the micro-
JOURNAL OF VASCULAR SURGERY
Volume 38, Number 6
Marques da Silva et al 1385
Table I. Incidence of aortic aneurysm with positive
bacterial cultures and findings according to literature
Positive results
Study
Year
n
%
van der Vliet et al18
Farkas et al14
Ilgenfritz and
Jordan15
Schwartz et al17
Buckels et al11
McAuley et al16
Eriksson et al12
Williams and
Fisher19
Ernst et al13
Present study
1996 55/216 25.5
1993 185/500 37
1988 11/56 19.6
Isolates (%)
Aerobic Anaerobic
74.5
97
85
24.5
3
15
1985
1985
1984
1983
1977
22/211 10.4
22/275 8
9/64
8
12/85 14
7/68 10
100
100
91
69
100
—
—
9
31
—
1977
2003
12/80
14/53
100
42
—
58
15
26.4
biology laboratory for processing. The tissue specimens
were divided into three groups, which were randomly
selected for cultivation, scanning electron microscopy, or
transmission electron microscopy. Specimens selected for
anaerobic cultivation were ground in a sterile mortar while
kept in a laminar flow hood, and seeded into sterile tubes
with thioglycollate and brain heart infusion broth, respectively. Great effort was taken to prevent contamination of
the samples, both in the operating theater and the microbiology laboratory.
After seeding, the tubes were immediately transferred
to anaerobic jars (Anoxomat System, WS9000; Mart, Lichtenvoorde, The Netherlands), which were evacuated and
filled three times with an anaerobic gas mixture (90%
nitrogen, 5% carbon dioxide, and 5% hydrogen). The samples were incubated at 37°C for up to 21 days. The cultures
were visually examined each day, and were discarded after
21 days if still negative. Positive cultures from the broth
media were seeded onto nonselective and selective agar
plates (trypticase soy, Brucella, and chocolate) and submitted to aerobic (24-48 hours) and anaerobic (4-14 days)
cultivation at 37°C. Preliminary identification of pure cultures was made on the basis of aerotolerance, colony and
cellular anatomy, pigmentation, gram staining, and cellular
motility. Biochemical and enzymatic tests for bacterial
identification were performed with commercial diagnostic
kits (API; bioMérieux, Marcy l’Etoile, France) for the
various categories of bacteria tentatively identified after
robot inoculation (bioMérieux). After automatic reading in
a mini API reader (bioMérieux) the results were transferred
into a numerical code and treated in a database for ultimate
identification (API Plus; bioMérieux).
The two parts of the specimens selected for microscopy
were cut in blocks of about 2 ⫻ 2 mm. The material
analyzed in this study comprised specimens from 28 of the
53 samples subjected to cultivation. Samples for electron
microscopy were selected at random. Tissue blocks were
transferred to 2.5% glutaraldehyde or 0.1 mol/L Sorensen
phosphate buffer for scanning electron microscopy, and
were stored at 4°C until processing. After dehydration in
ethanol, the tissue was critically point-dried with carbon
dioxide as the transitional fluid. The specimens were attached to metal stubs with silver paste and sputter-coated
with gold or palladium (thickness, 30 nmol/L) in a vacuum
evaporator. Coated samples were examined in a scanning
electron microscope (XL 30 ESEM; Philips, Eindhoven,
The Netherlands).
Tissue blocks were placed in vials with 2.5% glutaraldehyde or 0.1M Sorensen phosphate buffer supplemented
with 0.1% ruthenium red for transmission electron microscopy. The specimens were fixed for 24 hours at 4°C, and
stored in 0.1 mol/L of phosphate buffer with ruthenium
red at 4°C until preparation. Postfixation was performed in
1% osmium tetraoxide for 2 hours at 4°C. After fixation, the
blocks were rapidly dehydrated in graded series of acetone
solution and embedded in Vestopal W. Ultrathin sections
were cut with a Leica Ultracut microtome. The sections
were treated with uranyl acetate for 15 minutes, followed
by lead citrate for 3 minutes. They were examined in a
transmission electron microscope (CM 120; Philips).
RESULTS
Thirty-day overall mortality for patients operated on
was 3.9%. Two early deaths were the result of multiple
organ failure unrelated to infectious complications. Two
late deaths were recorded; one patient died of a dissecting
aneurysm (no signs of infection) 6 months after repeat
operation, and the other patient died of unknown cause 18
months after the primary operation. All other patients were
followed up postoperatively for 17 to 25 months (mean, 22
months). One patient had postoperative mediastinitis from
Staphylococcus aureus, which was treated successfully. No
late infections, including graft infections, were noted during follow-up in the remaining patients.
Anaerobic cultivation
Microbiologic analysis of the 56 aortic aneurysm specimens yielded bacteria in 9 of 35 thoracic aneurysms
(25.7%) and 5 of 18 abdominal aneurysms (27.8%), for an
overall incidence of 26.4% (14 of 53 aneurysms). Twelve of
48 specimens obtained at elective surgery yielded bacteria,
whereas 2 of 4 specimens obtained at emergent surgery
yielded positive cultures. No growth was observed in material from the ruptured aneurysm. There were no significant differences regarding incidence of positive cultures and
location of the aneurysms or indication for surgery. Only
one aneurysm sample (14%) from a patient with concomitant occlusive disease yielded bacteria.
Nineteen bacterial strains were isolated at anaerobic
culture. All strains were gram-positive cocci or rods, representing 9 genera and 12 species (Table II). Anaerobic
bacteria were recovered from 10 of 14 positive cultures
(71%). Five cultures (35%) yielded two different species.
Mixed cultures of aerobic and anaerobic bacteria were
found in four specimens (28.5%). Propionibacterium species were isolated from 8 patients, with Propionibacterium
acnes detected most often (five aneurysms). Propionibacterium species were recovered from six of seven positive
JOURNAL OF VASCULAR SURGERY
December 2003
1386 Marques da Silva et al
Table II. Bacteria* cultured anaerobically from aortic aneurysm specimens from 14 patients
Age
(y)
Sex
Location of
aneurysm
Type of
surgery
Cause
39
30
43
M
M
M
Ascending
Ascending, arch
Ascending
Elective
Elective
Elective
Congenital
Congenital
Mechanical
Marfan syndrome
48
M
Ascending
Elective
Mechanical
Bicuspid aorta
63
M
Ascending
Elective
Mechanical
Previous infections
endocarditis
21
67
83
F
M
M
Ascending
Descending
Ascending
Elective
Elective
Elective
Inflammatory
Inflammatory
Degenerative
Atherosclerosis
71
77
57
M
F
M
Ascending, arch
Abdominal
Abdominal
Elective
Elective
Elective
Degenerative
Degenerative
Degenerative
Atherosclerosis
Atherosclerosis
Atherosclerosis
75
75
75
M
M
F
Abdominal
Abdominal
Abdominal
Elective
Acute
Acute
Degenerative
Degenerative
Degenerative
Atherosclerosis
Atherosclerosis
Atherosclerosis
Associated condition
Culture results
Bacillus licheniformis
Propionibacterium granulosum
Propionibacterium acnes
Micrococcus luteus
Streptococcus mitis
B licheniformis
Actinomyces naeslundii
Kocuria varians
Eggerthella lenta
Gemella haemolysans
Actinomyces viscosus
Micrococcus spp
P acnes
P granulosum
P granulosum
Staphylococcus epidermidis
P acnes
P acnes
P acnes
*Of the genera isolated, Actinomyces, Propionibacterium, and Eggerthella (Eubacterium) are traditionally listed as anaerobic.39
aneurysms with degenerative or atherosclerotic origin, and
P acnes was recovered in pure culture in two cases in which
the patient was operated on urgently because of acute
symptoms (Table II). Micrococcus species and Bacillus licheniformis were identified in two cases. Other species
isolated were Actinomyces viscosus, Actinomyces naeslundii,
Eggerthella lenta, Streptococcus mitis, Gemella haemolysans,
Staphylococcus epidermidis, and Kocuria varians (Table II).
Electron microscopy
Bacteria were detected with both transmission electron
microscopy and scanning electron microscopy, and were
observed in 20 of 28 specimens examined (71%). Eighteen
positive specimens (90%) yielded negative results at anaerobic cultivation.
Scanning electron microscopy. Bacteria were observed at the surface of the intravascular plaque lining the
lumen of the aneurysm (Fig 1), at the aneurysm walls (Fig
2), and in and among remnants of intravascular plaque left
on the aneurysm walls after surgery (Figs 3, 4). In one
specimen, bacteria were seen among delicate fibers that
appeared to be a wound surface (Fig 5). Most often,
bacteria with different morphologic features (cocci, rods,
filaments) and dividing cells coaggregated, forming small
ecosystems at a distance from each other (Fig 3), but
apparent monocultures of bacteria were seen as well (Fig
4). Spirochetes of various sizes were commonly present,
(Fig 2) and macrophages were observed, engulfing bacteria
(Fig 6).
Transmission electron microscopy. Bacteria with
various morphologic features were observed at the aneurysm walls and in remnants of intravascular plaque adhering
to the walls (Figs 7, 8). Bacterial cells with a distinct capsule
were commonly seen (Fig 7).
DISCUSSION
In the present study the combination of anaerobic
cultivation and electron microscopy enabled detection of
multiple bacteria in aortic aneurysms. Electron microscopy
was useful for demonstrating bacteria of various sizes and
morphologic features, and coaggregating organisms. These
findings provide evidence that bacteria were located at the
aneurysm walls and inside intravascular plaque at the walls.
These bacteria cannot be considered culture contaminants.
Cell division indicated they were multiplying actively, and
the presence of capsule suggested they might be more or
less resistant to phagocytosis.
Little agreement was found between microscopic and
culture findings. When both transmission and scanning
electron microscopy were applied, bacteria were found in
18 samples that were negative at anaerobic cultivation. The
reasons for this are not clear, but that different parts of the
surgical specimens were subjected to different techniques
may be important. In addition, many microorganisms can
resist culture.24 In this regard, detection of spirochetes in
this study was particularly intriguing. It is estimated that
more than 80% of spirochetes found in the oral cavity have
not yet been cultured.25
The incidence of positive bacterial cultures was 26.4%,
which is in accordance with culture frequency reported
previously.11-19 Gram-positive organisms have outnumbered gram-negative organisms in most previous series. In
the present study, exclusively gram-positive bacteria were
isolated. This agrees with the results of van der Vliet et al.18
Our analysis of the relative distribution of aerobic and
anaerobic bacteria revealed a predominance of anaerobic
bacteria. Anaerobes represented 58% of isolates, whereas
previous studies show frequency varying from 0% to 31%
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Volume 38, Number 6
Marques da Silva et al 1387
Fig 1. Scanning electron micrograph of bacteria in intravascular
plaque lining lumen of aneurysm. Bar represents 5 ␮m.
Fig 3. Scanning electron micrograph of co-aggregation of microorganisms with different morphologic features in remnants of
intravascular plaque on aneurysm wall. Bar represents 2 ␮m.
Fig 2. Scanning electron micrograph of spirochete-like cell on
aneurysm wall. Bar represents 2 ␮m.
Fig 4. Scanning electron micrograph of microorganisms (cocci)
with remnants of intravascular plaque on aneurysmal wall. Bar
represents 2 ␮m.
(Table I). Inadequate methods of transportation and culturing of anaerobic bacteria may account for the infrequent
recovery of these organisms from aortic aneurysms.
The presence of anaerobic bacteria colonizing aortic
aneurysms was highlighted in a study of infectious (mycotic) aneurysms.26 The authors found it reasonable that
many of the organisms were anaerobic because of the close
proximity of the aorta to the gastrointestinal tract, where
anaerobes predominate. The enteric source of the organisms was supported by isolation of Enterobacteriaceae, Bacteroides fragilis, and Clostridum perfringens, which normally colonize the gut. Most often these bacteria reach
aortic aneurysms from the digestive tract via bacteremia. E
lenta, found in this study, is one of the predominant
microorganisms in the intestinal microflora, and is isolated
from human feces, wounds, and abscesses27 and frequently
from endodontic infections.28,29
P acnes was the most frequently cultured organism,
found in five positive cultures, followed by Propionibacte-
rium granulosum in three cultures. In a study of aortic
aneurysms by Eriksson et al,12 Propionibacterium species
were isolated from 3 patients, and were considered contaminants from the skin of the patients or the surgical team.
However, P acnes has been recovered from intestinal contents, wounds, pus, soft tissue abscesses,30 endocarditis,31
infected root canals,28 and mycotic aortic aneurysms.26,32
Debelian et al28 examined spread of microorganisms in the
blood of 26 patients undergoing root canal treatment.
Microorganisms were recovered from both the root canal
and the blood in 11 patients, and P acnes was the most
frequent isolate from both sites. In a study by Eishi et al,33
propionibacterial genomes were abundant in sarcoidal lesions only, excluding the possibility of skin contamination.
Although Propionibacterium species are part of the normal
skin flora, they can clearly act as primary pathogens, and
cannot be automatically dismissed as contaminants.
The results of this study also suggest hematogenous
spread of bacteria from the oral cavity. The source of these
1388 Marques da Silva et al
Fig 5. Scanning electron micrograph of area of aneurysm wall
(seemingly wound surface) with bacteria entangled in meshwork of
delicate fibers. Bar represents 2 ␮m.
Fig 6. Scanning electron micrograph of area of aneurysm wall
with macrophage-engulfing bacteria. Bar represents 2 ␮m.
bacteria, oral biofilm, is characterized by coaggregation of
various organisms. They appear on scanning electron micrographs as similar to bacterial complexes detected in the
present study, as well as inside calcific aortic valves, associated with stenosis, in a previous study by our group.34 Oral
biofilm, with its predominance of anaerobes, may be a
possible source of microbial cells or cell aggregates reaching
not only calcific aortic valves but also aortic aneurysms,
particularly in patients who have or have had periodontitis.
Indeed, Martı́n et al35 reported endarteritis and mycotic
aortic aneurysm caused by an oral strain of Actinobacillus
actinomycetemcomitans. The identity of the blood and oral
isolates was confirmed with phenotypic and genetic analysis, which showed that periodontal pockets were the source
of the organism. A viscosus and A naeslundii, found in the
present study, colonize the oral cavity of human beings and
animals, and can compose major components of dental
plaque. They are frequently isolated in periodontal disease36 and root canal infections,29 and are present in large
JOURNAL OF VASCULAR SURGERY
December 2003
Fig 7. Transmission electron micrograph of microorganism with
distinct capsule in intravascular plaque at aneurysm wall. Bar
represents 50 nm.
Fig 8. Transmission electron micrograph of microorganism
among disintegrating cells at aneurysm wall. Bar represents 100
nm.
numbers in periapical lesions of endodontic origin.37 Both
species can cause cervicofacial and abdominal actinomycosis, and A viscosus has been reported to induce endocarditis.38 Moreover, A naeslundii has been found in only 3 of
14,612 intestinal isolates in patients with periodontal disease,36 indicating that the oral cavity is the main reservoir
for this organism. This is, to the best of our knowledge, the
first time these bacteria were recovered from aortic aneurysms.
The findings of the present investigation emphasize
bacteria as common wall-colonizing organisms of aortic
aneurysms, and negative cultures do not necessarily imply
absence of bacteria. Many of the species detected at anaerobic cultivation did not belong to the indigenous skin
microflora, and cannot be considered culture contaminants. The demonstration of coaggregating bacteria with
different morphologic features on the walls of aneurysms
JOURNAL OF VASCULAR SURGERY
Volume 38, Number 6
and inside intravascular plaque at electron microscopy also
argue against sample contamination.
Thus it may be concluded that aortic aneurysms contain
bacteria. However, the presence of bacteria in aortic aneurysms does not necessarily imply a causal relationship, and
some organisms may actually be secondary colonizers after
aneurysm formation. On the other hand, there is the possibility that infection might contribute in the first place to the
inflammatory response thought to be important in aneurysm
formation. In any case, it is unlikely that viable and multiplying
organisms detected inside the aneurysm wall tissue are harmless. Under favorable conditions, invading organisms generally multiply and produce effects that are injurious, which
might have potential implications for aortic aneurysms. To
prevent detrimental effects of bacteria, prophylactic antibiotic
regimens should be effective in inhibiting their growth.
We thank Renate Hars and Steinar Stølen for skillful
technical assistance with transmission electron microscopy
and scanning electron microscopy, respectively.
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Submitted Feb 25, 2003; accepted May 30, 2003.