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

Abstract

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-specific16S rRNA-directed oligonucleotide probes to identify bacteria. To control non-specific binding of EUB 338, probe NON 338 was used. Alternatively, DAPI (49,69-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.

Periodontal Pathogens Invade Gingiva and Aortic Adventitia and Elicit Inflammasome Activation in αvβ6 Integrin-Deficient Mice

“…Fluorescence in situ hybridization (FISH) was performed on gingival and aortic sections, as described previously (16), and sections were probed for P. gingivalis and T. denticola together or T. forsythia and F. nucleatum together because of probe hybridization temperature compatibility. The following individual oligonucleotide probes (Invitrogen, Carlsbad, CA) were used to detect 16S rRNA for each species: POGI (5=-CAATACTCGTATCGCCCGTTATTC-3=) for P. gingivalis, TDEN (5=-CATGACTACCGTCATCAAAGAAGC-3=) for T. denticola, B(T)AFO (5=-CGTATCTCATTTTATTCCCCTGTA-3=) for T. forsythia, and FUSO (5=-CTAATGGGACGCAAAGCTCTC-3=) for F. nucleatum (19,20). Probes POGI and B(T)AFO were Alexa Fluor 488 labeled, and probes TDEN and FUSO were Alexa Fluor 568 labeled.…”

Biofilms 2003: Emerging Themes and Challenges in Studies of Surface-Associated Microbial Life

“…The oral microbiological community was well represented at this meeting. Annette Moter described her group's efforts to characterize the unculturable organisms associated with periodontal disease (102). They used fluorescent in situ hybridization (FISH) to characterize subgingival biofilms grown in periodontal pockets.…”

Treponema denticola Activates Mitogen-Activated Protein Kinase Signal Pathways through Toll-Like Receptor 2

“…Direct smears of pericoronitis exudates indicate these acutely infected areas have increased levels of treponemes (37). In addition, spirochetes have been observed in endodontic infections and associated apical regions of the tooth (4,31,33). Clearly, the oral spirochetes are integral components of a polymicrobial anaerobic flora that infect and cause inflammatory diseases within the oral cavity.…”