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Whole Genomic DNA Probe for Detection of Porphyromonas endodontalis
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JOURNAL OF ENDODONTICS Copyright 0 ZOO0 by The American Association of Endodontists
VOL. 26,
No. 4, APRIL 2000
Whole Genomic DNA Probe for Detection of Porphyromonas endodontalis R. Nissan, DMD, S. R. Makkar, DDS, M. N. Sela, DMD, PhD, and R. Stevens, DDS, MS
The purpose of the present study was to develop a DNA probe for Porphyromonas endodontalis. Pure cultures of P. endodontalis were grown in TYP medium, in an anaerobic chamber. DNA was extracted from the P. endodontalis and labeled udng the Genius System by Boehringer Mannheim. The labeled P. endodontalis DNA was used in dot-blot hybridization reactions with homologous (P. endodontalis) and unrelated bacterial samples. To determine specificity, strains of 40 other oral bacterial species (e.g. Porphymmonas gingivalis, Porphymmonas asacchamlytica, and Prevotella intermedia) were spotted and reacted with the P. endodontalis DNA probe. None of the panel of 40 oral bacteria hybridized with the P. endodontalis probe, whereas the blot of the homologous organism showed a strong positive reaction. To determine the sensitivity of the probe, dilutions of a P. endodontalis suspension of known concentration were blotted onto a nylon membrane and reacted with the probe. The results of our investigation indicate that the DNA probe that we have prepared specifically detects only P. endodontalis and can detect at least 3 x lo4 cells.
positive correlation between periapical pathosis and infection by Peptostreptococcus m g n u s and “Bacteroides” species. Cultural methods depend on the viability of the bacteria in question and require elaborate methodology for bacterial culturing and growth. Furthermore some of the bacteria involved in endodontjc infections are nutritionally fastidious, extremely oxygensensitive obligate anaerobes (1). Consequently the potential exists that culturing may fail to detect some of the microorganisms present in endodontic infections. To overcome these technical barriers, several molecular methods are available for the accurate and rapid identification of many bacteria. Two such methods, hybridization of DNA probes and immunoassays, have been suggested as alternatives to culturing for both research and diagnostic purposes. Serological typing of P. endodontalis has demonstrated three serotypes of this species (8). Therefore the availability of speciesspecific antibodies is a prerequisite for immunological detection of P. endodontalis. The existing information regarding the significance of P. endodontalis in root canal infections, as well as endodontic abcesses, has been obtained by cultural methods (2,3). These data suggest that these bacteria may be important in the development of pathosis in the root canal system and periapical area. However, the nutritional fastidiousness and oxygen sensitivity of P. endodontalis make the study of this organism extremely difficult. Therefore it is important to develop a specific, rapid, and reliable noncultural method that will detect these organisms in the in vivo situations. The purpose of the present study was to develop a sensitive, species-specific DNA probe for P. endodontalis.
Bacterial infection is the major cause of pulpal and periapical disease, necessitating endodontic therapy for many thousands of dental patients each year. Of the many different bacterial species isolated from endodontic infections, a few have been closely associated with acute symptoms, such as pain, swelling, and suppuration in endodontic patients. One such organism, Porphyromonas endodontalis, is an assacharolytic, obligatory anaerobic, Gramnegative coccobacillus (1). P. endodontalis has been isolated from infected root canals as part of a mixed microbiota (2) and from periapical abscesses of endodontal origin (3). Using cultural techniques, a correlation has been suggested between the presence of specific anaerobic oral bacteria (such as Peptostreptococcus spp, Bacteroides spp, and Porphyromonas spp) and clinical symptoms in endodontically involved teeth (46). Also, using cultural methods, Yoshida et al. (7) showed a
MATERIALS AND METHODS Bacterial Strains and Culture Conditions P. endodontalis strain 35406 was obtained from ATCC. Pure cultures were grown in a modified TYP medium as described by Herveijer et al. (9): trypticase soy broth (BBL, Cockeysville, MD), 1%; yeast extract (Difco, Detroit, MI), 0.5%; protease peptone (Difco), 1%, adjusted to pH 7.4; cysteine-HC1 (Sigma Chemical Co., St. Louis, MO), 0.5 mg/ml; hemin, 5 pg/ml; and vitamin K,, 1 kg/ml. Cultures were incubated in an anaerobic chamber with an atmosphere of N2-85%, H2-5%, and C02-10%. Forty other bacterial strains (Table 1) were used as negative controls. 217
218
Journal of Endodontics
Nissan et al. TABLE1. Strains used to test probe specificity Bacterial Strain
Porphyromonas endodontalis Peptostreptococcus micros Bacteroides beta Fusobacterium nucleatum Porphyromonas assacchyrolyticus Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivais Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Prevotella intermedia Prevotella intermedia Prevotella intermedia Prevotella intermedia Prevotella intermedia Prevotella intermedia Fusobacterium species Capnocytophaga species Actinobacillus actinomycetemcomitans Eikenella corrodens
Strain No.
Source
35406 33270 25846 25586 25260
ATCC' ATCC ATCC ATCC ATCC
866 1217 808 910 897 867 1220 1218 880 900 895 901 896 381 326 335 81 7 1903 21 60 2202 2921 962276 961843 966264 962332 862 1280 1218 594 893 594 895 895 869
OMTSt OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS
1215
OMTS
DNA Probe Reaction
+ -
-
and re-extracted with an equal volume of a 1:1 solution of phenol and chloroform. The aqueous phase was again recovered and re-extracted a final time with an equal volume of chloroform: isoamyl alcohol (24:l). The DNA in the resulting aqueous phase was precipitated by the addition of one-tenth volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol. After storage at -20°C for 30 min, the precipitated DNA was pelleted by centrifugation at 20,000 X g for 30 min. After pouring off the supernatant, the pelleted DNA was rinsed with cold 70% ethanol. After drying under nitrogen, the DNA was resuspended in T.1E buffer (10 m~ Tris-HC1 (pH 7.4):O.l m~ EDTA (pH 8.0)).The extracted DNA was labeled by random-primed incorporation of digoxigeninconjugated deoxyuridine-triphosphate using the Genius Nonradioactive DNA Labeling and Detection Kit (Boehringer Mannheim Biochemicals, Mannheim, Germany). After melting and quenching, 5 p g of the P. endodontalis DNA was added to a labeling reaction mixture (60 p1 total) that consisted of DNA polymerase Klenow fragment (6 units), dATP, dGTP, dCTP (6 nM each), dTTP (3.9 n~),digoxigenin-dUTP (2.1 nM), and a random hexanucleotide primer mixture (Boehringer Mannheim Corp., Mannheim, Germany). After centrifugation in a microcentrifuge for 1 min, the reaction mixture was incubated at 37°C for 18 h. The reaction was terminated by the addition of 6 p1 of a 0.2 M EDTA solution, and the labeled DNA was recovered by precipitation with a one-tenth volume of 4 M lithium chloride and 3 volumes of ethanol, followed by centrifugation (microcentrifuge for 30 min). The pelleted DNA was dried, resuspended in 150 p l of T.lE buffer, and then dissolved in 15 ml of hybridization buffer (750 m~ NaC1, 0.1% sodium N-lauroylsarcosine, 0.02% SDS, 0.5% purified fractionated casein (Boehringer Mannheim Corp.), 75 m~ sodium citrate, pH 7.0) to yield the final P. endodontalis DNA probe solution.
Sample Preparation for Hybridization
-
* ATCC = American Type Culture Collection. OMTS = Oral Microbiology Testing Service
t
Preparation of DNA Probe The P. endodontulis DNA was extracted from cells grown to stationary phase in 400 ml of modified TYP broth. Cells were harvested by centrifugation (15,000 X g for 10 min), washed twice in distilled water, and resuspended in 10 ml of solution I (25 m~ Tris-HC1,lO m~ EDTA, 50 m~ glucose, 5 mg/ml of lysozyme, pH 7.4). After incubation on ice for 10 min, the cells were lysed with the addition of 1 ml of 10% sodium dodecyl sulfate (SDS) and incubation at 37°C for 10 min. The resulting viscous lysate was digested with heat-treated (100°C for 15 min) RNAse A (10 pg/ml) for 45 min at 37"C, and then Proteinase K (50 pg/ml) for 30 min at 45°C to hydrolyze RNA and protein. The lysate was extracted with an equal volume (-1 1 ml) of phenol (equilibrated with 0.3 M sodium acetate). After centrifugation at 10,OOO rpm for 15 min to separate the phases, the aqueous phase was recovered,
A nylon blotting membrane (Zeta Probe, MSI, Westborough, MA) was prewetted with distilled water. The membrane was assembled into a Bio-Dot Microfiltration apparatus (Life Science Group, Hercules, CA) and then was rehydrated with 500 p1 of distilled water per well under 100 mm Hg of vacuum. Five microliters of each bacterial sample (broth culture or suspension in saline) was applied to the membrane through the wells of the Bio-Dot apparatus with a gentle vacuum. After the sample filtered through, 500 p1 of 2X SSC (standard saline citrate; 0.3 M NaCl:30 m~ sodium citrate, pH 7.0) were applied to each well under light vacuum. Cells in each sample were lysed by placing the membrane on a Whatman #2 filter paper saturated with 0.5 M NaOH:1.5 M NaCl (pH 13.2) for 10 min. Membranes were then neutralized by transfer to a Petri dish containing another Whatman #2 filter paper saturated with 1.5 M NaCl:0.5 M Tris-HC1 (pH 7.6) for 5 min. They were then incubated for 30 min at 37°C in 10 ml of proteinase K (100 pg/ml), SDS (0.5%), and Tris-HC1 (10 m ~pH , 7.5). The membranes were washed for 10 min in running tap water and then for 3 X 2 minute washes in 95% ethanol on a rotary mixer. Membranes were air-dried and baked for 30 min at 120°C to fix the DNA.
DNA Hybridization Membranes (containing the bacterial samples) were prehybridized for 1 h at 68 degrees in 10 ml of hybridization solution ( 5 ~
Vol. 26, No. 4, April 2000
p. endodontalis, DNA Probe, and Hybridization
219
SSC, 0.1% sodium lauroyl sarcosine, 0.002% SDS, and 0.5% Blocking reagent (Boehringer Mannheim)). P. endodontalis DNA probe was then added to the membranes, and hybridization was performed overnight at 68 degrees in a sealed plastic bag. After hybridization incubation, unbound probe was removed by 2 X 5 min washes at room temperature in 2X SSC:O.l% SDS, and then 2 x 15 min at 68°C in 0.1 X SSC:O.l% SDS.
Detection of Hybridized DNA Probe Immunological detection of hybridized DNA probe was accomplished by incubation of the membranes in Blocking Buffer (10 g of blocking reagent (Boehringer Mannheim) in 100 ml of buffer #1 (1.16% maleic acid:0.87% NaCl, pH 7.5)) containing alkaline phosphatase-conjugated goat anti-digoxigenin Fab fragments for 30 min at room temperature. Unbound antibody was removed by 2 X 15 min washes in buffer #l. Bound antibody-enzyme conjugate was detected using an enzyme substrate (x-phosphate) and a chromagen (nitroblue tetrazolium salt) as described by the manufacturer. The development of a blue-black spot on the membrane denoted a positive reaction.
FIG 1.Specificityof P. endodontalis DNA probe. Results of a dot-blot hybridization using the P. endodontalis probe against a variety of oral bacteria. Note the strong positive reaction against P. endodontalk cells (dot-blot no. 1)and absence of any reaction against any of the other 23 bacteria species tested (dot-blot nos. 2 to 24).
Probe Specificity Specificity of the P. endodontalis DNA probe was evaluated by testing it against a panel of oral bacteria. The ATCC and clinical isolate strains used in the panel to test probe specificity is shown in Table 1. In all. 40 different oral bacteria were tested.
Probe Sensitivity A series of dilutions (45, 3 5 , 25, 1 5 , l:lO, 1:20, 1:100, and 1:1,OOO) of a broth culture of P. endodontalis was made in saline. The concentration of bacteria in each dilution was determined by using a counting chamber under a microscope. Five microliters of each dilution were spotted onto a membrane in the dot-blot apparatus. Membranes were reacted with the P. endodontalis DNA probe as described. Sensitivity of the DNA probe was determined by the number of P. endodontalis cells in the most dilute sample yielding a positive reaction.
FIG2.Sensitivityof P.endodontalis DNA probe. Dot-blot assay using DNA probe with increasing dilutions of the P. endodontalis suspension. Dot-blot nos. 1 to 9 contain 2.4 x lo’, 1.8 x lo6,1.2 x lo6, 6.0 x lo5,3.0 x lo5,1.5 x lo4,3.0 x lo4, and 3.0 x lo3 cells, respectively. Note the limit of detection was 3.0 x lo4 cells (dot-blot no. 8).
TABLE 2. Sensitivity test results Dilution
RESULTS The positive control (P. endodontalis) showed a strong reaction with the probe (Fig. 1). The intensity of the reaction correlated with the number of the cells in each sample (Fig. 2). None of the 39 non-P. endodontalis bacterial strains gave any evidence of hybridization with the probe (Table 1). Using the dot-blot system, it was determined that the P. endodontalis probe was able to detect as few as 3 X lo4 cells (Table 2).
DISCUSSION Several studies have implicated “Bacteroides” (Porphyromonas) melaninogenicus ss asaccharolyticuslgingivalislendodontalis in the development of clinical signs and symptoms, such as pain, swelling, sinus tract formation, and periapical rarefaction from periapical lesions. However, these studies were conducted at a time
4:5 3:5 2:5 1 :5 1:lO 1 :20 1 :loo 1 :1,000
No. of cells/
5 I.LISample 2.4 X 10’ 1.8 X 10’ 1.2 x 106 6.0 x 105 3.0 x 105 1.5x 104 3.0 x 104 3.0 x 103
Reaction with DNA Probe
+++ +++ +++ ++ ++ + + -
Note: The limit of detection was 3.0 x lo4 cells. A 1:1,OOO dilution (correspondingto 3.0 x 16 P. endodontalis cells) gave no reaction with the probe.
before our understanding that “Bacteroides asaccharolyticus” was a heterogeneous group of organisms that actually consists of several different species (e.g. P. gingivalis, P. endodontalis, P. asaccharolyticus, etc.). Consequently, the results of these studies are somewhat ambiguous, because it is difficult to know whether they pertain to P. gingivalis, or P. endodontalis, although a subsequent study suggests that most Porphyromonas strains isolated from root
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Nissan et al.
canals are P. endodontulis (3). Thus, the significance of P. endodontalis in endodontic infections, is still not resolved. To address this question, we developed a sensitive, P. endodontulis-specific DNA probe that will allow us to quickly and accurately identify P. endodontulis from clinical samples. With our probe, we were able to detect at least 3 X lo4 P. endodontulis cells. This sensitivity is consistent with the findings of Loesche et al. (lo), Strzempko et al. (1 l), and Tay et al. (12), who were able to detect lo3 to lo4 P. gingivalis cells with a whole genomic DNA probe. The efficacy of whole genomic DNA probes for detection and identification of specific bacteria is further supported by the results of Tay et al. (12), who found that a DNA probe-based technique was more sensitive than cultural techniques for detecting P. gingivalis on a nonselective culture medium. To be useful, a detection system must be specific. Our results indicate that the whole genomic P. endodontalis DNA probe did not hybridize to a detectable level with the DNA of any of the other 39 oral bacterial species tested. This is also in agreement with previous reports. DNA probes for B. (P.)gingivalis, B. ( P . ) intermedia, and Huemophilus (Actinobacillus) actinomycetemcomitans failed to demonstrate any cross-reaction with DNA from P. endodontulis (1 1). A study by Suzuki et al. (13) reported a low (2 to 33%) degree of hybridization between P. endodontalis and P. gingivalis DNA. However, this investigation used a quantitative spectrophotometric detection system that might permit the detection of low levels of hybridization that may not be detectable by the dot-blot hybridization techniques used in the present investigation. Compared with many other oral bacteria, P. endodontalis has been studied only to a limited extent. Even so, several lines of evidence point to its potential as a endodontic pathogen. Some strains are capsulated, which may affect the cell’s sensitivity to serum bacteriolysis or phagocytosis (14). It produces IgG, IgM, and IgA proteases (15). It also can degrade complement, gelatin, haptoglobin, type IV collagen, and fibrin (16, 17). The lipopolysaccharide of P. endodontalis can induce the production of interleukin- lp and interleukin-6 in periodontal ligament cells and human dental pulp cells (18, 19). P. endodontulis produces a 50 kDa plasminogen activator that converts prekallikrein to kallikrein (20). Any, or all, of these features may play a role in the pathogenic potential of this organism. The availability of the DNA probe P. endodontalis will enable us to quickly and accurately screen endodontic samples from necrotic teeth with a wide variety of clinical symptomatology. We hope to learn whether P. endodontalis can be unambiguously associated with any one (or more) clinical feature of an endodontic infection. Such information would point to the need to learn more about this organism, and the factors that may be responsible for any role that it may play in periapical disease. This study was supported in part by the Faculty Incentive Award #466 from Temple University, Philadelphia, PA. Dr. Nissan is assistant professor, Department of Endodontology, Temple Unhrersity Dental School, Philadelphia, PA. Dr. Makkar was an endodontic resident in the Department of Endodontology, Temple University Dental
Journal of Endodontics School, and is now in endodontic private practice in North Boston, MA. Dr. Sela is professor and chairman, Department of Oral Biology, School of Dentistry, Hebrew University, Jerusalem, Israel. Dr. Stevens is professor and chairman, Department of Endodontology, Temple University Dental School, Philadelphia, PA. Address requests for reprints to Dr. Roni Nissan, Department of Endodontology, Temple University Dental School, 3223 North Broad Street, Philadelphia, PA 19140-5096.
References 1. Shah H, Collins MD. Proposal for reclassification of Bacteroides asaccharolyficus, Bacteroides gingivalis and Bacteroides endodontalis in a new genus Porphyromonas. Int J Syst Bacteriol 1988;38:128-31. 2. Haapasalo M, Ranta H. Ranta K, Shah H. Black-pigmented Bacteroides spp. in human apical periodontitis. Infect lmmun 1986;53:149-53. 3. van Winkelhoff AJ, Carlee AW, de Graaff J. Bacteroides endodontalis and other black-pigmentedspecies in odontogenic abcesses. Infect lmmun 1985;49:494-497. 4. Hashioka K, Yamasaki M, Nakane A, Horiba N, Nakamura H. The relationship between clinical symptoms and anaerobic bacteriafrom infected root canals. J Endodon 1992;18:558-61. 5. Griffee M, Patterson S, Miller C, Kafrawy A, Newton C. The relationship of Bacteroides melaninogenicus to symptoms associated with pulpal necrosis. Oral Surg Oral Med Oral Pathol 1980;50:457-61. 6. Gomes BP, Drucker DB, Lilley JD. Association of specific bacteria with some endodontic signs and symptoms. Int Endod J 1994;27:291-8. 7. Yoshida M, Fukushima H. Yamamato K, Ogawa K, Toda T, Sagawa H. Correlation between clinical symptoms and microorganisms isolated from canals of teeth with periapical pathosis. J Endodon 1987;13:24-8. 8. van Winkelhoff A. Serological characterization of black-pigmentedBacteroides endodontalis. Infect lmmun 1986;51:972-4. 9. Herveijer JA, Loos BG, Neiders ME. Characterizationof total membrane protein of Porphyromonas endodontalis. J Endodon 1992;18:620-4. 10. Loesche WJ, Lopatin DE. Stoll J, van Poperin N, Hujoel PP. Comparison of various detection methods for periodontopathic bacteria: can culture be considered the primary referencestandard? J Clin Microbioll992;3041826. 11. Strzempko MN, Simon SL, French CK, Lippke JA, Raia FF, Savitt ED, Vaccaro KKA. Cross-reactivity study of whole genomic DNA probes for Haemophilus actinomycetemcomitans, Bacteroides intemedius, and Bacteroides gingivalis. Dent Res 1987;66:1543-6. 12. Tay F, Liu YB, Flynn MJ, Slots J. Evaluation of a non-radioactive DNA probe for detecting Porphyromonas gingivalis in subgingival specimens. Oral Microbiol lmmunol 1992;7:344-8. 13. Suzuki K, lkeda T, Nakamura H, Yoshimura F. Isolation and characterization of a nonpigmented variant of Porphyromonas endodontalis. Oral Microbiol lmmunol 1997;12:155-61. 14. Mayrand D, Holt SC. Biology of asaccharolytic black-pigmented Bacteroides species. Microbiol Rev 1988;52:134-52. 15. Kilian M. Degredation of immunoglobulin Al, A2,and G by suspected principal periodontal pathogens. Infect lmmun 1981;34:757-65. 16. Shah HN, Gharbia SE. Biochemical and chemical analyses of blackpigmented Gram-negative anaerobes. FEMS lmmunol Med Microbiol 1993; 6:89-96. 17. Shoshani M, Rosen G, Naor R, Sela M. Hydrolysis of the extracellular matrix proteins: fibronectin. fibrinogen and collagen IV by a protease of Porphyromonas endodontalis [IADR Abstract #2826]. J Dent Res 1998;77: 985. 18. Ogura N, Shibata Y, Kamino Y, Matsuda U, Hayakawa M, Oikawa T. Takiguchi H, lzumi H, Abiko Y. Stimulation of interleukin-6 production of periodontal ligament cells by Porphyromonas endodontalis lipopolysaccharide. Biochem Med Metab Biol 1994;53:130-6. 19. Hosoya S, Matsushima K, Ohbayashi E, Yamazaki M, Shibata Y, Abiko Y. Stimulation of interleukin-1beta-independent interleukin-6 production in human dental pulp cells by lipopolysaccharide. Biochem Mol Med 1996;59: 138-43. 20. Oikawa T, Ogura N, Akiba M, Abiko Y, Takiguchi H, lzumi H. Stimulation of plasmin activity in cultured human fibroblast cells by Porphyromonas endodontalis. Int J Biochem 1993;25:1227-31.
JOURNAL OF ENDODONTICS Copyright 0 ZOO0 by The American Association of Endodontists
VOL. 26,
No. 4, APRIL 2000
Whole Genomic DNA Probe for Detection of Porphyromonas endodontalis R. Nissan, DMD, S. R. Makkar, DDS, M. N. Sela, DMD, PhD, and R. Stevens, DDS, MS
The purpose of the present study was to develop a DNA probe for Porphyromonas endodontalis. Pure cultures of P. endodontalis were grown in TYP medium, in an anaerobic chamber. DNA was extracted from the P. endodontalis and labeled udng the Genius System by Boehringer Mannheim. The labeled P. endodontalis DNA was used in dot-blot hybridization reactions with homologous (P. endodontalis) and unrelated bacterial samples. To determine specificity, strains of 40 other oral bacterial species (e.g. Porphymmonas gingivalis, Porphymmonas asacchamlytica, and Prevotella intermedia) were spotted and reacted with the P. endodontalis DNA probe. None of the panel of 40 oral bacteria hybridized with the P. endodontalis probe, whereas the blot of the homologous organism showed a strong positive reaction. To determine the sensitivity of the probe, dilutions of a P. endodontalis suspension of known concentration were blotted onto a nylon membrane and reacted with the probe. The results of our investigation indicate that the DNA probe that we have prepared specifically detects only P. endodontalis and can detect at least 3 x lo4 cells.
positive correlation between periapical pathosis and infection by Peptostreptococcus m g n u s and “Bacteroides” species. Cultural methods depend on the viability of the bacteria in question and require elaborate methodology for bacterial culturing and growth. Furthermore some of the bacteria involved in endodontjc infections are nutritionally fastidious, extremely oxygensensitive obligate anaerobes (1). Consequently the potential exists that culturing may fail to detect some of the microorganisms present in endodontic infections. To overcome these technical barriers, several molecular methods are available for the accurate and rapid identification of many bacteria. Two such methods, hybridization of DNA probes and immunoassays, have been suggested as alternatives to culturing for both research and diagnostic purposes. Serological typing of P. endodontalis has demonstrated three serotypes of this species (8). Therefore the availability of speciesspecific antibodies is a prerequisite for immunological detection of P. endodontalis. The existing information regarding the significance of P. endodontalis in root canal infections, as well as endodontic abcesses, has been obtained by cultural methods (2,3). These data suggest that these bacteria may be important in the development of pathosis in the root canal system and periapical area. However, the nutritional fastidiousness and oxygen sensitivity of P. endodontalis make the study of this organism extremely difficult. Therefore it is important to develop a specific, rapid, and reliable noncultural method that will detect these organisms in the in vivo situations. The purpose of the present study was to develop a sensitive, species-specific DNA probe for P. endodontalis.
Bacterial infection is the major cause of pulpal and periapical disease, necessitating endodontic therapy for many thousands of dental patients each year. Of the many different bacterial species isolated from endodontic infections, a few have been closely associated with acute symptoms, such as pain, swelling, and suppuration in endodontic patients. One such organism, Porphyromonas endodontalis, is an assacharolytic, obligatory anaerobic, Gramnegative coccobacillus (1). P. endodontalis has been isolated from infected root canals as part of a mixed microbiota (2) and from periapical abscesses of endodontal origin (3). Using cultural techniques, a correlation has been suggested between the presence of specific anaerobic oral bacteria (such as Peptostreptococcus spp, Bacteroides spp, and Porphyromonas spp) and clinical symptoms in endodontically involved teeth (46). Also, using cultural methods, Yoshida et al. (7) showed a
MATERIALS AND METHODS Bacterial Strains and Culture Conditions P. endodontalis strain 35406 was obtained from ATCC. Pure cultures were grown in a modified TYP medium as described by Herveijer et al. (9): trypticase soy broth (BBL, Cockeysville, MD), 1%; yeast extract (Difco, Detroit, MI), 0.5%; protease peptone (Difco), 1%, adjusted to pH 7.4; cysteine-HC1 (Sigma Chemical Co., St. Louis, MO), 0.5 mg/ml; hemin, 5 pg/ml; and vitamin K,, 1 kg/ml. Cultures were incubated in an anaerobic chamber with an atmosphere of N2-85%, H2-5%, and C02-10%. Forty other bacterial strains (Table 1) were used as negative controls. 217
218
Journal of Endodontics
Nissan et al. TABLE1. Strains used to test probe specificity Bacterial Strain
Porphyromonas endodontalis Peptostreptococcus micros Bacteroides beta Fusobacterium nucleatum Porphyromonas assacchyrolyticus Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivais Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Porphyromonas gingivalis Prevotella intermedia Prevotella intermedia Prevotella intermedia Prevotella intermedia Prevotella intermedia Prevotella intermedia Fusobacterium species Capnocytophaga species Actinobacillus actinomycetemcomitans Eikenella corrodens
Strain No.
Source
35406 33270 25846 25586 25260
ATCC' ATCC ATCC ATCC ATCC
866 1217 808 910 897 867 1220 1218 880 900 895 901 896 381 326 335 81 7 1903 21 60 2202 2921 962276 961843 966264 962332 862 1280 1218 594 893 594 895 895 869
OMTSt OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS OMTS
1215
OMTS
DNA Probe Reaction
+ -
-
and re-extracted with an equal volume of a 1:1 solution of phenol and chloroform. The aqueous phase was again recovered and re-extracted a final time with an equal volume of chloroform: isoamyl alcohol (24:l). The DNA in the resulting aqueous phase was precipitated by the addition of one-tenth volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol. After storage at -20°C for 30 min, the precipitated DNA was pelleted by centrifugation at 20,000 X g for 30 min. After pouring off the supernatant, the pelleted DNA was rinsed with cold 70% ethanol. After drying under nitrogen, the DNA was resuspended in T.1E buffer (10 m~ Tris-HC1 (pH 7.4):O.l m~ EDTA (pH 8.0)).The extracted DNA was labeled by random-primed incorporation of digoxigeninconjugated deoxyuridine-triphosphate using the Genius Nonradioactive DNA Labeling and Detection Kit (Boehringer Mannheim Biochemicals, Mannheim, Germany). After melting and quenching, 5 p g of the P. endodontalis DNA was added to a labeling reaction mixture (60 p1 total) that consisted of DNA polymerase Klenow fragment (6 units), dATP, dGTP, dCTP (6 nM each), dTTP (3.9 n~),digoxigenin-dUTP (2.1 nM), and a random hexanucleotide primer mixture (Boehringer Mannheim Corp., Mannheim, Germany). After centrifugation in a microcentrifuge for 1 min, the reaction mixture was incubated at 37°C for 18 h. The reaction was terminated by the addition of 6 p1 of a 0.2 M EDTA solution, and the labeled DNA was recovered by precipitation with a one-tenth volume of 4 M lithium chloride and 3 volumes of ethanol, followed by centrifugation (microcentrifuge for 30 min). The pelleted DNA was dried, resuspended in 150 p l of T.lE buffer, and then dissolved in 15 ml of hybridization buffer (750 m~ NaC1, 0.1% sodium N-lauroylsarcosine, 0.02% SDS, 0.5% purified fractionated casein (Boehringer Mannheim Corp.), 75 m~ sodium citrate, pH 7.0) to yield the final P. endodontalis DNA probe solution.
Sample Preparation for Hybridization
-
* ATCC = American Type Culture Collection. OMTS = Oral Microbiology Testing Service
t
Preparation of DNA Probe The P. endodontulis DNA was extracted from cells grown to stationary phase in 400 ml of modified TYP broth. Cells were harvested by centrifugation (15,000 X g for 10 min), washed twice in distilled water, and resuspended in 10 ml of solution I (25 m~ Tris-HC1,lO m~ EDTA, 50 m~ glucose, 5 mg/ml of lysozyme, pH 7.4). After incubation on ice for 10 min, the cells were lysed with the addition of 1 ml of 10% sodium dodecyl sulfate (SDS) and incubation at 37°C for 10 min. The resulting viscous lysate was digested with heat-treated (100°C for 15 min) RNAse A (10 pg/ml) for 45 min at 37"C, and then Proteinase K (50 pg/ml) for 30 min at 45°C to hydrolyze RNA and protein. The lysate was extracted with an equal volume (-1 1 ml) of phenol (equilibrated with 0.3 M sodium acetate). After centrifugation at 10,OOO rpm for 15 min to separate the phases, the aqueous phase was recovered,
A nylon blotting membrane (Zeta Probe, MSI, Westborough, MA) was prewetted with distilled water. The membrane was assembled into a Bio-Dot Microfiltration apparatus (Life Science Group, Hercules, CA) and then was rehydrated with 500 p1 of distilled water per well under 100 mm Hg of vacuum. Five microliters of each bacterial sample (broth culture or suspension in saline) was applied to the membrane through the wells of the Bio-Dot apparatus with a gentle vacuum. After the sample filtered through, 500 p1 of 2X SSC (standard saline citrate; 0.3 M NaCl:30 m~ sodium citrate, pH 7.0) were applied to each well under light vacuum. Cells in each sample were lysed by placing the membrane on a Whatman #2 filter paper saturated with 0.5 M NaOH:1.5 M NaCl (pH 13.2) for 10 min. Membranes were then neutralized by transfer to a Petri dish containing another Whatman #2 filter paper saturated with 1.5 M NaCl:0.5 M Tris-HC1 (pH 7.6) for 5 min. They were then incubated for 30 min at 37°C in 10 ml of proteinase K (100 pg/ml), SDS (0.5%), and Tris-HC1 (10 m ~pH , 7.5). The membranes were washed for 10 min in running tap water and then for 3 X 2 minute washes in 95% ethanol on a rotary mixer. Membranes were air-dried and baked for 30 min at 120°C to fix the DNA.
DNA Hybridization Membranes (containing the bacterial samples) were prehybridized for 1 h at 68 degrees in 10 ml of hybridization solution ( 5 ~
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SSC, 0.1% sodium lauroyl sarcosine, 0.002% SDS, and 0.5% Blocking reagent (Boehringer Mannheim)). P. endodontalis DNA probe was then added to the membranes, and hybridization was performed overnight at 68 degrees in a sealed plastic bag. After hybridization incubation, unbound probe was removed by 2 X 5 min washes at room temperature in 2X SSC:O.l% SDS, and then 2 x 15 min at 68°C in 0.1 X SSC:O.l% SDS.
Detection of Hybridized DNA Probe Immunological detection of hybridized DNA probe was accomplished by incubation of the membranes in Blocking Buffer (10 g of blocking reagent (Boehringer Mannheim) in 100 ml of buffer #1 (1.16% maleic acid:0.87% NaCl, pH 7.5)) containing alkaline phosphatase-conjugated goat anti-digoxigenin Fab fragments for 30 min at room temperature. Unbound antibody was removed by 2 X 15 min washes in buffer #l. Bound antibody-enzyme conjugate was detected using an enzyme substrate (x-phosphate) and a chromagen (nitroblue tetrazolium salt) as described by the manufacturer. The development of a blue-black spot on the membrane denoted a positive reaction.
FIG 1.Specificityof P. endodontalis DNA probe. Results of a dot-blot hybridization using the P. endodontalis probe against a variety of oral bacteria. Note the strong positive reaction against P. endodontalk cells (dot-blot no. 1)and absence of any reaction against any of the other 23 bacteria species tested (dot-blot nos. 2 to 24).
Probe Specificity Specificity of the P. endodontalis DNA probe was evaluated by testing it against a panel of oral bacteria. The ATCC and clinical isolate strains used in the panel to test probe specificity is shown in Table 1. In all. 40 different oral bacteria were tested.
Probe Sensitivity A series of dilutions (45, 3 5 , 25, 1 5 , l:lO, 1:20, 1:100, and 1:1,OOO) of a broth culture of P. endodontalis was made in saline. The concentration of bacteria in each dilution was determined by using a counting chamber under a microscope. Five microliters of each dilution were spotted onto a membrane in the dot-blot apparatus. Membranes were reacted with the P. endodontalis DNA probe as described. Sensitivity of the DNA probe was determined by the number of P. endodontalis cells in the most dilute sample yielding a positive reaction.
FIG2.Sensitivityof P.endodontalis DNA probe. Dot-blot assay using DNA probe with increasing dilutions of the P. endodontalis suspension. Dot-blot nos. 1 to 9 contain 2.4 x lo’, 1.8 x lo6,1.2 x lo6, 6.0 x lo5,3.0 x lo5,1.5 x lo4,3.0 x lo4, and 3.0 x lo3 cells, respectively. Note the limit of detection was 3.0 x lo4 cells (dot-blot no. 8).
TABLE 2. Sensitivity test results Dilution
RESULTS The positive control (P. endodontalis) showed a strong reaction with the probe (Fig. 1). The intensity of the reaction correlated with the number of the cells in each sample (Fig. 2). None of the 39 non-P. endodontalis bacterial strains gave any evidence of hybridization with the probe (Table 1). Using the dot-blot system, it was determined that the P. endodontalis probe was able to detect as few as 3 X lo4 cells (Table 2).
DISCUSSION Several studies have implicated “Bacteroides” (Porphyromonas) melaninogenicus ss asaccharolyticuslgingivalislendodontalis in the development of clinical signs and symptoms, such as pain, swelling, sinus tract formation, and periapical rarefaction from periapical lesions. However, these studies were conducted at a time
4:5 3:5 2:5 1 :5 1:lO 1 :20 1 :loo 1 :1,000
No. of cells/
5 I.LISample 2.4 X 10’ 1.8 X 10’ 1.2 x 106 6.0 x 105 3.0 x 105 1.5x 104 3.0 x 104 3.0 x 103
Reaction with DNA Probe
+++ +++ +++ ++ ++ + + -
Note: The limit of detection was 3.0 x lo4 cells. A 1:1,OOO dilution (correspondingto 3.0 x 16 P. endodontalis cells) gave no reaction with the probe.
before our understanding that “Bacteroides asaccharolyticus” was a heterogeneous group of organisms that actually consists of several different species (e.g. P. gingivalis, P. endodontalis, P. asaccharolyticus, etc.). Consequently, the results of these studies are somewhat ambiguous, because it is difficult to know whether they pertain to P. gingivalis, or P. endodontalis, although a subsequent study suggests that most Porphyromonas strains isolated from root
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canals are P. endodontulis (3). Thus, the significance of P. endodontalis in endodontic infections, is still not resolved. To address this question, we developed a sensitive, P. endodontulis-specific DNA probe that will allow us to quickly and accurately identify P. endodontulis from clinical samples. With our probe, we were able to detect at least 3 X lo4 P. endodontulis cells. This sensitivity is consistent with the findings of Loesche et al. (lo), Strzempko et al. (1 l), and Tay et al. (12), who were able to detect lo3 to lo4 P. gingivalis cells with a whole genomic DNA probe. The efficacy of whole genomic DNA probes for detection and identification of specific bacteria is further supported by the results of Tay et al. (12), who found that a DNA probe-based technique was more sensitive than cultural techniques for detecting P. gingivalis on a nonselective culture medium. To be useful, a detection system must be specific. Our results indicate that the whole genomic P. endodontalis DNA probe did not hybridize to a detectable level with the DNA of any of the other 39 oral bacterial species tested. This is also in agreement with previous reports. DNA probes for B. (P.)gingivalis, B. ( P . ) intermedia, and Huemophilus (Actinobacillus) actinomycetemcomitans failed to demonstrate any cross-reaction with DNA from P. endodontulis (1 1). A study by Suzuki et al. (13) reported a low (2 to 33%) degree of hybridization between P. endodontalis and P. gingivalis DNA. However, this investigation used a quantitative spectrophotometric detection system that might permit the detection of low levels of hybridization that may not be detectable by the dot-blot hybridization techniques used in the present investigation. Compared with many other oral bacteria, P. endodontalis has been studied only to a limited extent. Even so, several lines of evidence point to its potential as a endodontic pathogen. Some strains are capsulated, which may affect the cell’s sensitivity to serum bacteriolysis or phagocytosis (14). It produces IgG, IgM, and IgA proteases (15). It also can degrade complement, gelatin, haptoglobin, type IV collagen, and fibrin (16, 17). The lipopolysaccharide of P. endodontalis can induce the production of interleukin- lp and interleukin-6 in periodontal ligament cells and human dental pulp cells (18, 19). P. endodontulis produces a 50 kDa plasminogen activator that converts prekallikrein to kallikrein (20). Any, or all, of these features may play a role in the pathogenic potential of this organism. The availability of the DNA probe P. endodontalis will enable us to quickly and accurately screen endodontic samples from necrotic teeth with a wide variety of clinical symptomatology. We hope to learn whether P. endodontalis can be unambiguously associated with any one (or more) clinical feature of an endodontic infection. Such information would point to the need to learn more about this organism, and the factors that may be responsible for any role that it may play in periapical disease. This study was supported in part by the Faculty Incentive Award #466 from Temple University, Philadelphia, PA. Dr. Nissan is assistant professor, Department of Endodontology, Temple Unhrersity Dental School, Philadelphia, PA. Dr. Makkar was an endodontic resident in the Department of Endodontology, Temple University Dental
Journal of Endodontics School, and is now in endodontic private practice in North Boston, MA. Dr. Sela is professor and chairman, Department of Oral Biology, School of Dentistry, Hebrew University, Jerusalem, Israel. Dr. Stevens is professor and chairman, Department of Endodontology, Temple University Dental School, Philadelphia, PA. Address requests for reprints to Dr. Roni Nissan, Department of Endodontology, Temple University Dental School, 3223 North Broad Street, Philadelphia, PA 19140-5096.
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