* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Multiple bacteria in aortic aneurysms
Survey
Document related concepts
Quorum sensing wikipedia , lookup
History of virology wikipedia , lookup
Traveler's diarrhea wikipedia , lookup
Infection control wikipedia , lookup
Transmission (medicine) wikipedia , lookup
Phospholipid-derived fatty acids wikipedia , lookup
Microorganism wikipedia , lookup
Carbapenem-resistant enterobacteriaceae wikipedia , lookup
Disinfectant wikipedia , lookup
Triclocarban wikipedia , lookup
Human microbiota wikipedia , lookup
Hospital-acquired infection wikipedia , lookup
Bacterial cell structure wikipedia , lookup
Marine microorganism wikipedia , lookup
Anaerobic infection wikipedia , lookup
Transcript
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% JOURNAL OF VASCULAR SURGERY 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. REFERENCES 1. Lawrence PF, Gazak C, Bhirangi L, Jones B, Bhirangi K, Oderich G, et al. The epidemiology of surgically repaired aneurysms in the United States. J Vasc Surg 1999;30:632-40. 2. MacSweeney STR, Powell JT, Greenhalg RM. Pathogenesis of abdominal aortic aneurysm. Br J Surg 1994;81:935-41. 3. Reed D, Reed C, Stemmermann G. Are aortic aneurysms caused by atherosclerosis? Circulation 1992;85:205-11. 4. Chiu B. Multiple infections in carotid atherosclerotic plaques. Am Heart J 1999;138(suppl):S534-6. 5. Danesh R, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link? Lancet 1997;350:430-6. 6. Haraszthy VI, Zambon JJ, Trevisan M, Zeid M, Genco RJ. Identification of periodontal pathogens in atheromatous plaques. J Periodont 2000;71:1554-60. 7. Roivainen M, Viik-Kajander M, Palosuo T, Toivanen P, Leinonen M, Saikku P, et al. Infections, inflammation, and the risk of coronary heart disease. Circulation 2000;101:252-7. 8. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340:115-26. 9. Blasi F, Denti F, Erba M, Cosentini R, Raccanelli R, Rinaldi A, et al. Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms. J Clin Microbiol 1996;34: 2766-9. 10. Juvonen J, Juvonen T, Laurila A, Alakärppä H, Lounatmaa K, Surcel H-M, et al. Demonstration of Chlamydia pneumoniae in the walls of abdominal aortic aneurysms. J Vasc Surg 1997;25:499-505. 11. Buckels JAC, Fielding JWL, Black J, Ashton F, Slaney G. Significance of positive bacterial cultures from aortic aneurysm contents. Br J Surg 1985;72:440-2. 12. Eriksson I, Forsberg O, Lundqvist B, Schwan A. Significance of positive bacterial cultures from aortic aneurysms. Acta Chir Scand 1983;149: 33-5. 13. Ernst CB, Campbell HC, Daugherty ME, Sachatello CR, Griffen WO. Incidence and significance of intra-operative bacterial cultures during abdominal aortic aneurysmectomy. Ann Surg 1977;185:626-30. 14. Farkas J-C, Fichelle J-M, Laurian C, Jean-Baptiste A, Gigou F, Marzelle J, et al. Long-term follow-up of positive cultures in 500 abdominal aortic aneurysms. Arch Surg 1993;128:284-8. 15. Ilgenfritz FM, Jordan FT. Microbiological monitoring of aortic aneurysm wall and contents during aneurysmectomy. Arch Surg 1988;123: 506-8. 16. McAuley CE, Steed DL, Webster MW. Bacterial presence in aortic thrombus at elective aneurysm resection: is it clinically significant? Am J Surg 1984;147:322-4. Marques da Silva et al 1389 17. Schwartz JA, Powell TW, Burnham SJ, Johnson G Jr. Culture of abdominal aortic aneurysm contents. Arch Surg 1987;122:777-80. 18. van der Vliet JA, Kouwenberg PGM, Muytjens HL, Barendregt WB, Boll APM, Buskens FGM. Relevance of bacterial cultures of abdominal aortic aneurysm contents. Surgery 1996;119:129-32. 19. Williams RD, Fisher FW. Aneurysm contents as a source of graft infection. Arch Surg 1977;112:415-6. 20. Johnston KW, Rutherford RB, Tilson MD, Shah DM, Hollier L, Stanley JC. Suggested standards for reporting on arterial aneurysms. J Vasc Surg 1991;13:452-8. 21. Bennett DE, Cherry JK. Bacterial infection of aortic aneurysms: a clinicopathologic study. Am J Surg 1967;113:321-6. 22. Jarrett F, Darling RC, Mundth ED, Austen WG. Experience with infected aneurysms of the abdominal aorta. Arch Surg 1975;110: 1281-6. 23. Möller ÅJR. Microbiological examination of root canals and periapical tissues of human teeth. Odont Tidskr 1966;74:1-380. 24. Wilson MJ, Weightman AJ, Wade WG. Applications of molecular ecology in the characterization of uncultured microorganisms associated with human disease. Rev Med Microbiol 1997;8:91-101. 25. Dewhirst FE, Tamer MA, Ericson RE, Lau CN, Levanos VA, Boches SK, et al. The diversity of periodontal spirochetes by 16S rRNA analysis. Oral Microbiol Immunol 2000;15:196-202. 26. Brook I, Frazier EH. Aerobic and anaerobic microbiology of mycotic aortic aneurysm. Clin Infect Dis 1999;28:928-9. 27. Wade WG, Downes J, Dymock D, Hiom SJ, Weightman AJ, Dewhirst FE, et al. The family Coriobacteriaceae: reclassification of Eubacterium exiguum (Poco et al, 1996) and Peptostreptococcus heliotrinireducens (Lanigan, 1976) as Slackia exigua gen. nov., comb. nov. and Slackia heliotrinireducens gen. nov., comb nov., and Eubacterium lentum (Prévot, 1938) as Eggerthella lenta gen. nov., comb. nov. Int J Syst Bacteriol 1999;49:595-600. 28. Debelian GJ, Olsen I, Tronstad L. Bacteremia in conjunction with endodontic treatment. Endodont Dent Traumatol 1995;11:142-9. 29. Sundqvist G. Associations between microbial species in dental root canal infections. Oral Microbiol Immunol 1992;7:257-62. 30. Cummins CS, Johnson JL, Genus I. Propionibacterium Orla-Jensen 1909, 337AL. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG, eds. Bergey’s manual of systematic bacteriology. Baltimore, Md: Williams & Wilkins, 1981:p 1346-53 Vol 2. 31. Felner JM, Dowell VR. Anaerobic bacterial endocarditis. N Engl J Med 1970;283:1188-92. 32. Galema L, Overbosch EH, Schrander-van der Meer AM, Brom HLF, Roggeveen C, van Dorp WT. Mycotic aneurysm after aortic bifurcation prosthesis. Neth J Med 1996;48:105-8. 33. Eishi Y, Suga M, Ishige I, Kobayashi D, Yamada T, Takemura T, et al. Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis. J Clin Microbiol 2002;40:198-204. 34. Kolltveit KM, Geiran O, Tronstad L, Olsen I. Multiple bacteria in calcific aortic valve stenosis. Microb Ecol Health Dis 2002;14:110-7. 35. Martı́n MC, Andrés MT, Fierro JF, Méndez FJ. Endarteritis and mycotic aortic aneurysm caused by an oral strain of Actinobacillus actinomycetemcomitans. Eur J Clin Microbiol Infect Dis 1998;17:104-7. 36. Moore WEC, Moore LVH. The bacteria of periodontal diseases. Periodontology 2000 1994;5:66-77. 37. Sunde PT, Tronstad L, Eribe ER, Lind PO, Olsen I. Assessment of periradicular microbiota by DNA-DNA hybridization. Endodont Dent Traumatol 2000;16:191-6. 38. Mardis JS, Many WJ Jr. Endocarditis due to Actinomyces viscosus. South Med J 2001;94:240-3. 39. Rodloff AC, Hillier SL, Moncla BJ. Peptostreptococcus, Propionibacterium, Lactobacillus, Actinomyces and other non-spore-forming anaerobic gram-positive bacteria. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, eds. Manual of clinical microbiology, 7th ed. Washington, DC: American Society for Microbiology, 1999:672-89. Submitted Feb 25, 2003; accepted May 30, 2003.