Bacterial diversity in aortic aneurysms determined by 16S ribosomal RNA gene analysis.

BACKGROUND: Aortic aneurysms are common vascular conditions that cause considerable morbidity and mortality. Understanding of the mechanisms involved in the pathogenesis of the condition remains limited. Recently, infection has been suggested as possible contributor in the development of the disease. The aim of the present study was to examine aortic aneurysms for the presence of bacterial DNA using polymerase chain reaction (PCR) targeting the 16S ribosomal RNA (rRNA) gene, followed by cloning and sequencing. METHODS: Universal eubacterial primers were used to amplify 16S rRNA bacterial genes in 10 specimens from arterial walls of aortic aneurysms. Subsequently, PCR amplicons were cloned into Escherichia coli and sequencing of the cloned inserts was used to determine species identity or closest relatives by comparison with known sequences in GenBank. RESULTS: Sequences of Stenotrophomonas spp., including S. maltophilia (formerly Pseudomonas homology group V) were detected in six aneurysm samples. Propionibacterium acnes was identified in five samples, and Brevundimonas diminuta (formerly P. diminuta) in four samples. Other species previously assigned to the Pseudomonas genus such as Comamonas testosteroni, Delftia acidovorans, Burkholderia cepacia, Herbaspirillum sp., and Acidovorax sp. were also detected. Some clones fell into other environmental species, including Methylobacterium sp. and Bradyrhizobium elkanii, and others represented bacteria that have not yet been cultivated. DNA sequences from oral bacteria, including Streptococcus sanguinis, Tannerella forsythia, and Leptotrichia buccalis were detected. Sequences from Prevotella melaninogenica and Lactobacillus delbrueckii, which are commonly found in both mouth and gastrointestinal tract, were also detected. Additional species included Dermacoccus spp. and Corynebacterium vitaeruminis. CONCLUSIONS: A wide variety of bacteria, including oral bacteria, was found to colonize aortic aneurysms and may play a role in their development. Several of these microorganisms have not yet been cultivated. CLINICAL RELEVANCE: Although Chlamydophila pneumoniae has been detected in aneurysmal walls, its exact role in the condition remains inconclusive. Overall, there is scarce information about the role of microorganisms in aneurysmal disease. In the present study, we used molecular genetics to detect a diversity of bacteria in arterial walls of aortic aneurysms. The presence of multiple microorganisms in aneurysmal disease may have implications for chemoprophylaxis and antibiotic treatment if directed only at C.pneumoniae.

Detection of Actinobacillus actinomycetemcomitans but not bacteria of the red complex in aortic aneurysms by multiplex polymerase chain reaction.

BACKGROUND: Aortic aneurysms affect an increasing number of elderly patients and cause considerable morbidity and mortality. The understanding of the mechanisms involved in the pathogenesis of aortic aneurysms is unclear and little is known about the role of microorganisms in the development of the condition. The aim of the present study was to examine aortic aneurysm samples for the presence of four putative periodontal pathogens: Actinobacillus actinomycetemcomitans, Treponema denticola, Tannerella forsythensis, and Porphyromonas gingivalis. METHODS: Fifty-six samples from the aneurysm wall were obtained from patients undergoing aneurysm repair. DNA was extracted from tissue by conventional methods. Universal eubacterial primers for general detection of bacteria and species specific primers for detection of the periodontal pathogens were used to amplify part of the 16S rRNA gene by polymerase chain reaction (PCR). RESULTS: Bacterial DNA was detected in 50 of the 56 aneurysm samples (89.2%). A. actinomycetemcomitans was found in four samples (7.1%). None of the samples was positive for T. denticola, T. forsythensis, or P. gingivalis. CONCLUSION: Bacteria are commonly present in aortic aneurysms and may play a role in the development of the condition. Periodontal pathogens are also present.

Characterization of Streptococcus constellatus strains recovered from a brain abscess and periodontal pockets in an immunocompromised patient.

BACKGROUND: There have been a number of reports of brain abscesses suggesting an odontogenic etiology. However, no efforts have been made to compare brain abscess isolates with isolates from the oral cavity using highly discriminative methods. We report a brain abscess caused by Streptococcus constellatus in an immunocompromised patient where oral infection (periodontitis) was suspected to be implicated. METHODS: The brain abscess and oral isolates were compared by means of one phenotypic and three genetic (restriction fragment length polymorphism [RFLP], ribotyping, and random amplified polymorphic DNA [RAPD]) fingerprinting techniques. RESULTS: The phenotypic method and RFLP showed identical profiles between brain and periodontal isolates, while ribotyping and RAPD showed very close similarity, with only one band difference in one of the three ribotypes and in one of the three polymorphic RAPD. CONCLUSIONS: Gene transfer by genetic recombinational events in the periodontal pocket might have been responsible for the emergence of a strain variant of S. constellatus that had the potential to cause an abscess at a distant site (brain). The importance of odontogenic sources as potential foci of infection for brain abscesses is discussed.

Multiple bacteria in aortic aneurysms.

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. Twenty-eight 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.

Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-filled teeth.

Whether micro-organisms can live in periapical endodontic lesions of asymptomatic teeth is under debate. The aim of the present study was to visualize and identify micro-organisms within periapical lesions directly, using fluorescence in situ hybridization (FISH) in combination with epifluorescence and confocal laser scanning microscopy (CLSM). Thirty-nine periapical lesions were surgically removed, fixed, embedded in cold polymerizing resin and sectioned. The probe EUB 338, specific for the domain Bacteria, was used together with a number of species-specific 16S rRNA-directed oligonucleotide probes to identify bacteria. To control non-specific binding of EUB 338, probe NON 338 was used. Alternatively, DAPI (4',6'-diamidino-2-phenylindole) staining was applied to record prokaryotic and eukaryotic DNA in the specimens. Hybridization with NON 338 gave no signals despite background fluorescence of the tissue. The eubacterial probe showed bacteria of different morphotypes in 50 % of the lesions. Rods, spirochaetes and cocci were spread out in areas of the tissue while other parts seemed bacteria-free. Bacteria were also seen to co-aggregate inside the tissue, forming microcolonies. Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis and treponemes of phylogenetic Group I were detected with specific probes. In addition, colonies with Streptococcus spp. were seen in some lesions. A number of morphotypes occurred that could not be identified with the specific probes used, indicating the presence of additional bacterial species. CLSM confirmed that bacteria were located in different layers of the tissue. Accordingly, the FISH technique demonstrated mixed consortia of bacteria consisting of rods, spirochaetes and cocci in asymptomatic periapical lesions of root-filled teeth.

Microbiota of periapical lesions refractory to endodontic therapy.

The periapical microbiota of 36 teeth with refractory apical periodontitis was investigated. None of the teeth had responded to conventional endodontic or long-term (> 6 months), calcium-hydroxide treatment. Eight patients had received antibiotics systemically. After anaerobic culture, a total of 148 microbial strains were detected among 67 microbial species. One of the 36 lesions was culture-negative. Approximately half (51.0%) of the bacterial strains were anaerobic. Gram-positive species constituted 79.5% of the flora. Facultative organisms, such as Staphylococcus, Enterococcus, Enterobacter, Pseudomonas, Stenotrophomonas, Sphingomonas, Bacillus, or Candida species were recovered from 27 of the lesions (75%). Sulfur granules were found in 9 lesions (25%). In these granules Actinomyces israelii, A. viscosus, A. naeslundii, and A. meyeri were identified. Other bacterial species, both gram-positive and gram-negative, were detected in the granules as well. Two sulfur granules did not contain Actinomyces. Scanning electron microscopy demonstrated rod- and spirochete-like cells in the granules, and transmission electron microscopy revealed organisms with copious amounts of extracellular material. Outer membrane vesicles were also seen. Some of the granules were calcified. This study demonstrated a wide variety of microorganisms, particularly gram-positive ones, in the periapical lesions of teeth with refractory apical periodontitis.