JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2007, p. 3039–3049 Vol. 45, No. 90095-1137/07/$08.00�0 doi:10.1128/JCM.02618-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Use of Multiple-Displacement Amplification and CheckerboardDNA-DNA Hybridization To Examine the Microbiota of

Endodontic Infections�

L. C. N. Brito,2* F. R. Teles,1,3 R. P. Teles,1 E. C. Franca,2 A. P. Ribeiro-Sobrinho,2A. D. Haffajee,1 and S. S. Socransky1

Department of Periodontology, The Forsyth Institute, Boston Massachusetts1; Federal University of Minas Gerais School ofDentistry, Belo Horizonte, Minas Gerais, Brazil2; and Department of Oral Medicine, Infection and Immunity,

Harvard School of Dental Medicine, Boston, Massachusetts3

Received 31 December 2006/Returned for modification 23 February 2007/Accepted 6 July 2007

Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes presentin small samples, providing abundant targets for molecular analysis. The purpose of this investigation was tocombine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections.Sixty-six samples were collected from teeth with endodontic infections. Nonamplified and amplified sampleswere analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa.Counts, percentages of DNA probe counts, and percentages of teeth colonized for each species in amplified andnonamplified samples were computed. Significance of differences for each species between amplified andnonamplified samples was sought with Wilcoxon signed-rank test and adjusted for multiple comparisons. Theamount of DNA in the samples ranged from 6.80 (� 5.2) ng before to 6.26 (� 1.73) �g after MDA. Seventy ofthe 77 DNA probes hybridized with one or more of the nonamplified samples. All probes hybridized with atleast one sample after amplification. Most commonly detected species at levels of >104 in both amplified andnonamplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies between 89 and 100%of samples. The mean number of species at counts of >104 in amplified samples was 51.2 � 2.2 and innonamplified samples was 14.5 � 1.7. The endodontic microbiota was far more complex than previously shown,although microbial profiles at teeth with or without periradicular lesions did not differ significantly. Speciescommonly detected in endodontic samples included P. tannerae, Prevotella oris, and A. baumannii.

The microbiology of endodontic infections has been studiedfor many years (4, 47, 58). However, the association betweenspecific microorganisms found in root canals and the symptomsof endodontic infections is poorly understood. Early studies ofthe endodontic microbiota indicated a predominance of aero-bic and facultative bacterial species (16). This conclusion wasquestioned by the development of anaerobic culturing tech-niques which clarified the etiopathogenesis of endodontic in-fections by demonstrating the common occurrence of obligateanaerobic bacteria (4, 23, 30). Nevertheless, culture-basedtechniques have limitations, such as the difficulty in detectingfastidious anaerobic microorganisms and moderate sensitivityand specificity (44).

Recently, molecular biology techniques have provided amore cost-effective, specific, and sensitive method to evaluatethe microbiological profiles of oral pathologies, including end-odontic and periodontal infections (37, 38, 44, 52–54). Thistechnology permits the detection of microbial species that aredifficult to grow as well as uncultivated and unrecognizedphylotypes (34), which would lead to a better understanding

of the oral microbiota, including endodontic infections (19,35, 49, 56).

Checkerboard DNA-DNA hybridization is a high-through-put method to analyze large numbers of DNA samples by useof a wide range of DNA probes on a single nylon membrane(55). The quantity of bacteria in the samples is an importantfactor in the checkerboard DNA-DNA hybridization tech-nique, since the level of detection is about 104 bacterial cells ofa given species. Samples from endodontic pathologies oftencontain very few bacterial cells and may be below the level ofdetection of the checkerboard method without a DNA ampli-fication step. To overcome these limitations, the present studyused multiple-displacement amplification (MDA) before hy-bridizing the samples. MDA allows uniform amplification ofthe whole genome of DNA targets (3, 11, 33, 63, 64), increasingthe amount of DNA obtained from the endodontic bacterialsamples. Furthermore, MDA provides enough amplified DNAto perform multiple analyses of the same sample by use ofdifferent DNA probe sets. The aim of this study was to com-bine MDA and checkerboard DNA-DNA hybridization toquantitatively and qualitatively assess the taxa present in rootcanals during endodontic infections.

MATERIALS AND METHODS

Subject population and sample collection. Sixty-six subjects ranging in agefrom 11 to 81 years were recruited in the Department of Endodontics, FederalUniversity of Minas Gerais (UFMG), Belo Horizonte, Brazil. The subjects had

* Corresponding author. Mailing address: Federal University of Mi-nas Gerais School of Dentistry, Av. Antonio Carlos, 6627–Sl 3310Bairro: Pampulha Cep:31270-901, Belo Horizonte, MG Brazil. Phone:(55)-31-3441-3915. Fax: (55)-31-3418-1433. E-mail: luitauna@yahoo.com.br.

� Published ahead of print on 18 July 2007.

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teeth with endodontic infections, with or without radiographically detectedperiradicular lesions.

The selection of teeth was based on clinical crown conditions that permittedeffective placement of rubber dam isolation in teeth with pulp necrosis. Thereason for the primary infection was caries; that was detected in almost all cases,although causes of pulp necrosis are sometimes difficult to be determined clin-ically. Additionally, there was no history of trauma associated with the selectedteeth. All sampled teeth had never been treated before and were asymptomatic,without acute abscess.

Fifty-seven teeth were molars and 6 teeth were premolars, while 3 teeth weresingle rooted. In the case of multirooted teeth, the sample was taken from thelargest root canal.

After informed consent was obtained, the 66 selected teeth were isolated usinga rubber dam. Complete asepsis was employed, using the methodology proposedby Moller (32). Hydrogen peroxide (30%) was applied on the isolated crown,followed by 5% iodine that was inactivated by a 5% sodium thiosulfate solution.The samples were taken by scraping or filing the root canal walls with a #10K-type hand file (Maillefer, Ballaigues, Switzerland). The file was introducedinto the canal to a level approximately 1 mm short of the tooth apex. Afterremoval from the canal, the file was cut off below the handle and dropped intoan Eppendorf microcentrifuge tube containing a solution of 20 �l of alkaline lysisbuffer (400 mM KOH, 100 mM dithiothreitol, 10 mM EDTA). After 10 min ofincubation on ice, 20 �l of neutralization solution (400 mM HCl, 600 mM

Tris-HCl, pH 0.6) was added, and the sample was kept at 4°C until MDA wasperformed.

For comparison, a second set of samples was taken from 46 of the 66 rootcanals. In that set of samples, the files were placed into an Eppendorf micro-centrifuge tube containing 100 �l TE buffer (10 mM Tris-HCl, 0.1 mM EDTA,pH 7.6). One hundred microliters of 0.5 M NaOH was added to the sample, andit was maintained at 4°C until checkerboard DNA-DNA hybridization was per-formed.

MDA of root canal samples. The procedure was the same as that described byTeles et al. (60). The DNA content of the amplified samples was measured usingthe Picogreen double-stranded DNA quantification assay (Invitrogen, Carlsbad,CA). The microbiological content of the amplified samples was analyzed usingcheckerboard DNA-DNA hybridization.

Bacterial strains and growth conditions. The 77 reference strains used for thepreparation of DNA probes are listed in Table 1. The majority of strains weregrown on Trypticase soy agar supplemented with 5% defibrinated sheep blood(Baltimore Biological Laboratories [BBL], Cockeysville, MD) with some excep-tions. Tannerella forsythia was grown on Trypticase soy agar supplemented with5% sheep blood and 10 �g/ml N-acetylmuramic acid (Sigma Chemical Co., St.Louis, MO). Porphyromonas gingivalis was grown on Trypticase soy agar supple-mented with 5% sheep blood, 0.3 �g/ml menadione (Sigma), and 5 �g/ml hemin(Sigma). Eubacterium and Neisseria species were grown on fastidious anaerobicagar (BBL) with 5% defibrinated sheep blood. Treponema denticola and Trepo-

TABLE 1. Strains of bacterial species used to prepare DNA probes and standards

Straina Straina

Acinetobacter baumannii (19606) Haemophilus segnis (33393)Actinomyces georgiae (49285) Lactobacillus oris (49062)Actinomyces gerencseriae (23860) Leptotrichia buccalis (14201)Actinomyces israelii (12102) Mogibacterium timidum (33093)Actinomyces meyeri (35568) Neisseria mucosa (19696)Actinomyces naeslundii genospecies I (12104) Peptostreptococcus anaerobius (27337)Actinomyces naeslundii genospecies II (27044) Parvimonas micra (33270)Actinomyces odontolyticus (17929) Porphyromonas endodontalis (35406)Aggregatibacter actinomycetemcomitansb Porphyromonas gingivalis (33277)Atopobium parvulum (33793) Prevotella heparinolytica (35895)Atopobium rimae (49626) Prevotella intermedia (25611)Campylobacter concisus (33237) Prevotella loescheii (15930)Campylobacter ureolyticus (33387) Prevotella melaninogenica (25845)Campylobacter gracilis (33236) Prevotella nigrescens (33563)Campylobacter rectus (33238) Prevotella oris (33573)Campylobacter showae (51146) Prevotella tannerae (51259)Capnocytophaga gingivalis (33624) Propionibacterium propionicum (14157)Capnocytophaga ochracea (33596) Propionibacterium acnesc

Capnocytophaga sputigena Rothia dentocariosa (17931)Corynebacterium matruchotii (14266) Selenomonas artemidis (43528)Dialister pneumosintes (GBA27) Selenomonas noxia (43541)Eikenella corrodens (23834) Selenomonas sputigena (35185)Enterococcus faecalis (29212) Staphylococcus epidermidis (14990)Escherichia coli (10799) Streptococcus anginosus (33397)Eubacterium brachy (33089) Streptococcus constellatus (27823)Eubacterium limosum (8486) Streptococcus gordonii (10558)Eubacterium nodatum (33099) Streptococcus intermedius (27335)Eubacterium saburreum (33271) Streptococcus mitis (49456)Filifactor alocis (35896) Streptococcus mutans (25175)Fusobacterium naviforme (25832) Streptococcus oralis (35037)Fusobacterium necrophorum (25386) Streptococcus parasanguinis (15912)Fusobacterium nucleatum subsp. nucleatum (25586) Streptococcus salivarius (27945)Fusobacterium nucleatum subsp. polymorphum (10953) Streptococcus sanguinis (10556)Fusobacterium nucleatum subsp. vincentii (49256) Streptococcus vestibularis (49124)Fusobacterium periodonticum (33693) Tannerella forsythia (3037)Gemella haemolysans (10379) Treponema denticola (B1)Gemella morbillorum (27824) Treponema socranskii (S1)Haemophilus aphrophilus (33389) Veillonella dispar (17748)Haemophilus paraphrophilus (29242) Veillonella parvula (10790)

a All strains were obtained from the American Type Culture Collection (ATCC number in parentheses) except for Treponema denticola B1 and Treponema socranskiiS1, which were obtained from The Forsyth Institute.

b ATCC strains 43718 and 29523.c ATCC strains 11827 and 11828.

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nema socranskii were grown in mycoplasma broth (Difco Laboratories, Detroit,MI) supplemented with 1 mg/ml glucose, 400 �g/ml niacinamide, 150 �g/mlspermine tetrahydrochloride, 20 �g/ml Na isobutyrate, 1 mg/ml L-cysteine, 5�g/ml thiamine pyrophosphate, and 0.5% bovine serum. All strains were grownat 35°C under anaerobic conditions (80% N2, 10% CO2, 10% H2).

DNA isolation and preparation of DNA probes. Bacterial strains were grownanaerobically on the surfaces of blood agar plates (except the two spirochetes,which were grown in broth) for 3 to 7 days. The cells were harvested and placedin 1.5 ml of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.6). Cells werewashed twice by centrifugation in TE buffer at 1,300 � g for 10 min. The cellswere resuspended and lysed with either 10% sodium dodecyl sulfate and pro-teinase K (20 mg/ml) for gram-negative strains or in 150 �l of an enzyme mixturecontaining 15 mg/ml lysosyme (Sigma) and 5 mg/ml achromopeptidase (Sigma)in TE buffer (pH 8.0) for gram-positive strains. The pelleted cells were resus-pended by 15 s of sonication and incubated at 37°C for 1 h. DNA was isolatedand purified using the method of Smith et al. (51). The concentration of thepurified DNA was determined by spectrophotometric measurement of the ab-sorbance at 260 nm. The purity of the preparations was assessed by the ratio ofthe absorbance at 260 nm and 280 nm. Whole-genome DNA probes wereprepared from each of the 77 test strains by labeling 1 to 3 �g of DNA with

digoxigenin (Boehringer Mannheim, Indianapolis, IN) by use of a randomprimer technique (17).

Checkerboard DNA-DNA hybridization. Checkerboard DNA-DNA hybridiza-tion was performed as previously described (26, 54, 55). In brief, following amplifi-cation and quantification, amplified samples and nonamplified samples were boiledfor 10 min. Three microliters (approximately 900 ng of DNA) of the amplifiedsample was placed in an Eppendorf tube containing 1 ml of TE buffer prior toboiling. The nonamplified samples were neutralized by adding 800 �l of 5 M am-monium acetate after boiling. Then, the samples were placed into the extended slotsof a Minislot 30 apparatus (Immunetics, Cambridge, MA), concentrated onto anylon membrane (Boehringer Mannheim) by vacuum, and fixed onto the membraneby cross-linking using UV light (Stratalinker 1800; Stratagene, La Jolla, CA) fol-lowed by baking at 120°C for 20 min. The Minislot device permitted the depositionof 28 different samples in individual lanes on a single membrane, which also had twocontrol lanes containing 105 and 106 cells of each bacterial species tested. Themembrane with fixed DNA was placed in a Miniblotter 45 apparatus (Immunetics)with the lanes of DNA at 90° to the channels of the device. A 30-by-45 “checker-board” pattern was produced. Each channel was used as an individual hybridizationchamber for separate DNA probes. Bound probes were detected by anti-digoxigeninantibody conjugated with alkaline phosphatase and a chemifluorescent sub-

FIG. 1. Checkerboard DNA-DNA hybridization membrane showing the hybridization of 40 of the 77 DNA probes to endodontic samples.Standards containing 105 and 106 cells of each test species are shown in the bottom lanes of the membrane. Signals indicate the detection of eachspecies in pairs of nonamplified (n) or amplified (a) samples. nuc., nucleatum; ss, subspecies.

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strate. Signal intensities of the endodontic samples and the standards (con-taining 1 ng and 10 ng of each bacterial species) on the same membrane weremeasured using a Storm FluorImager (Molecular Dynamics, Sunnyvale, CA).The values were then converted to absolute counts using linear regression.Failure to detect a signal was recorded as zero.

Two membranes were run for each sample, one containing the “standard” 40DNA probes used to examine periodontal samples and a second membrane thatemployed 37 probes to species thought to be important in endodontic samples.Specificity tests were conducted for all probes before performing the checker-board DNA-DNA hybridization with the root canal samples. The protocol tovalidate the specificity of these 37 probes was similar to the one used for the

original set of 40 probes. The probes were tested against purified DNA from allother species, as described by Socransky et al. (54). If cross-reactions wereobserved, those probes were discarded and new probes constructed and vali-dated.

Data analysis. Microbiological data were available for 46 nonamplified and 66MDA-amplified root canal samples, taken from 66 subjects. The microbial data wereexpressed in three ways: counts (levels), proportions (percentages of DNA probecounts), and prevalence (percentage of teeth colonized at levels of �105) of 77bacterial species. Count data were expressed as counts � 105 in each sample andaveraged across subjects. The amplified counts that were presented reflect the“number” of organisms detected after MDA amplification of the sample compared

FIG. 2. Bilateral bar chart of the mean counts (� 105 � SEM) of the 77 test species in nonamplified (n � 46) and amplified (n � 66) root canalsamples. The counts for each species were averaged across subjects and presented in descending order of mean counts detected in the amplifiedsamples. ss, subsp.

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with nonamplified standards. They are not actual counts of the original sample butthe “DNA equivalents” after amplification. The data shown in the figures in Resultsrepresent 1/280 of the DNA available after amplification. One-fortieth of the orig-inal sample was amplified by MDA. Three-twentieths of the amplified product wasdeposited in lanes of the Minislot device, providing an approximately 280-fold“amplification.”

Significance of differences between nonamplified and amplified samples foreach species was sought using the Wilcoxon signed-rank test. Adjustments weremade for multiple comparisons as described by Socransky et al. (53).

In a similar fashion, mean proportions of each species were determined forroot canal samples taken from teeth with or without radiographically detectedperiradicular lesions. The significance of differences between groups was deter-mined using the Mann-Whitney test and adjusted for multiple comparisons.

RESULTS

Quantification of DNA after MDA of endodontic samples.DNA from the root canal samples was amplified using MDA.The amount of DNA present in the samples before amplifica-tion averaged 6.80 (� 5.2) ng and 6.26 (� 1.73) �g afteramplification, an approximately 1,000-fold amplification. Am-plified samples provided signals far better than those observedusing nonamplified samples (Fig. 1).

Microbial species in root canal samples. The mean numbersof species (� standard errors of the means [SEM]) detected inamplified and nonamplified root canal samples at a thresholdof �104 were 51.2 � 2.2 and 14.5 � 1.7, respectively. If adetection threshold of �105 was employed, then 11.3 � 1.4 and0.8 � 0.2 species were detected in the amplified and nonam-plified samples, respectively. Figure 2 presents the mean counts(�105 � SEM) of the 77 test species in amplified and nonam-plified root canal samples taken from 46 teeth. The specieswere ordered according to mean counts. In nonamplified sam-ples, Prevotella tannerae exhibited the highest mean counts(0.91 � 105 � 0.25), followed by Acinetobacter baumannii andPrevotella oris, while Streptococcus mitis exhibited the lowestmean counts at (0.01 � 105 � 0.001), followed by Streptococcussalivarius and Actinobacillus actinomycetemcomitans. Sevenspecies were not detected in any of the nonamplified samples.In amplified samples, P. tannerae exhibited the highest meancounts � 105, 3.32 � 0.69, followed by P. oris and Streptococcusmutans, while Campylobacter concisus exhibited the lowestmean counts (0.15 � 105 � 0.02), followed by Leptotrichiabuccalis and Streptococcus salivarius.

The mean proportions (percentages of DNA probe counts �SEM) of the 77 test species in nonamplified and amplified rootcanal samples are presented in Fig. 3. In nonamplified samples, P.tannerae and Acinetobacter baumannii were detected in the high-est mean proportions, 11.20 (� 1.48) and 11.14 (� 1.88), whileEscherichia coli (0.05 � 0.03) showed the lowest detected meanproportions, followed by Actinomyces odontolyticus. In amplifiedsamples, P. tannerae was detected in the highest mean propor-tions (5.33 � 0.64) followed by P. oris and S. mutans, and Strep-tococcus salivarius in the lowest mean proportions (0.28 � 0.03)followed by L. buccalis and Lactobacillus oris.

Figure 4 presents the mean percentages of sampled sitesexhibiting counts of the 77 test species at levels of �104 innonamplified and amplified samples. P. tannerae and A. bau-mannii were detected in all amplified samples and in �90% ofnonamplified samples. Other species that were frequently de-tected included Prevotella heparinolytica in both types of sam-ples and Actinomyces meyeri, Streptococcus parasanguinis,

Atopobium rimae, and Porphyromonas endodontalis in MDA-amplified samples. Prevotella oris, Selenomonas sputigena, Hae-mophilus aphrophilus, and Mogibacterium timidum were de-tected in �50% of nonamplified samples.

Figures 5 and 6 demonstrate the mean percentages of DNAprobe counts for the 77 test species in nonamplified and am-plified root canal samples, respectively, taken from teeth withor without a radiographically apparent periapical lesion. Therewere no significant differences between clinical groups afteradjusting for multiple comparisons for either the nonamplifiedor amplified samples.

DISCUSSION

One of the goals of the current investigation was to increasethe range of bacterial species examined in root canal samplesand to detect species present in low numbers by use of MDA.Previous studies have employed DNA probes to less than 50bacterial species (12, 20, 49, 50, 56), compared with the 77examined in the present study. Thus, a range of taxa far widerthan previously recognized was detected. On average, 51.2species were detected after amplification, more than the 3 to 8species found by others (30, 49), but similar to the figure of 50predicted by Tronstad and Sunde (61) if comprehensive mi-crobiological methods were to be employed. The test specieswere detected more frequently in the MDA-amplified samplesthan in the nonamplified samples, suggesting that this technol-ogy may be useful for endodontic samples containing smallnumbers of bacterial cells.

Another goal of the current investigation was to compare themicrobiota in root canals with and without periradicular lesions.Although others have found specific bacterial communities to beassociated with asymptomatic or symptomatic endodontic infec-tions (18, 22, 42), the current study found no significant differ-ences between the two clinical states, irrespective of whether thesamples were amplified or not. However, this approach might setthe stage for further studies in which one species or a set ofspecies might be associated with different symptomatologies thatcould lead to specific treatment modalities.

Molecular assays have shown that 700 to 1,000 species cancolonize oral biofilms, far more than detected by cultivation (1,34). Root canals are accessible to sources of mixed bacteriafrom carious lesions and periodontal pockets that could colo-nize at low numbers over time. Sample amplification in thisstudy was about 1,000-fold. Since checkerboard DNA-DNAhybridization can detect 104 bacterial cells, it is possible that 10cells of a species were detected. PCR can also detect few cells(18, 19, 45) but requires specific primers and may lead to majoramplification bias (11, 60), which is much lower in MDA (60).

Among the wide range of taxa detected in this study, speciesthat form black-pigmented colonies (BPB) such as P. tanneraeand nonpigmented Prevotella species such as P. oris were inhigh mean counts. P. tannerae has been reported as an uncul-tivable organism (15), and its frequency of occurrence in end-odontic infections was not appreciated until Xia et al. (62)detected it in 60% of samples by use of PCR amplification.When multiplex PCR was used to detect BPB in endodonticsamples, P. tannerae was found in only 5% of them, possiblydue to limitations in the multiplex technique (38). Other BPBwere also present in relatively high mean counts and propor-

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tions in amplified samples of the current study, including P.endodontalis, Prevotella loescheii, Prevotella nigrescens, Pre-votella intermedia, P. gingivalis, and Prevotella melaninogenica.P. endodontalis has been isolated by cultivation from infectedroot canals (24, 57), but its prevalence was even higher whenmolecular techniques were used (19, 22, 40, 48).

Periodontal pathogens of the “red complex” (52), T. for-sythia, P. gingivalis, and T. denticola, were detected in amplifiedand nonamplified samples. T. denticola was present in higher

proportions than T. forsythia and P. gingivalis in the amplifiedsamples, a finding similar to that of Haffajee et al. (25) insubgingival microbiota of Brazilian subjects. Using PCR,Rocas et al. (36) found that T. denticola was the most prevalentof the three species (44%) in endodontic samples, whileSiqueira et al. (49), using checkerboard DNA-DNA hybridiza-tion, found that T. forsythia was the most prevalent (39.3%). Inother investigations (18, 40), T. denticola was detected in 56%and 79% of samples from infected teeth. The fastidious growth

FIG. 3. Bilateral bar chart of the mean percentages of the DNA probe counts (�SEM) for 77 bacterial species in nonamplified (n � 46) andamplified (n � 66) root canal samples. The percentage of the DNA probe count was computed for each species at each sample site and averagedacross subjects. The data are ordered in descending order of mean percentages of DNA probe counts detected in amplified samples. ss, subsp.

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of T. denticola and T. forsythia has led to an underestimation oftheir prevalence in cultural studies of endodontic infections.Based on their frequent detection by molecular techniques,they might be potential endodontic pathogens (40). Further-

more, T. denticola seems highly pathogenic in monoinfectionsof the dental pulp in a mouse model system (18).

Members of the “orange complex” were present in endodon-tic infections, including Fusobacterium nucleatum, which has

FIG. 4. Bilateral bar chart of the mean prevalence (% of teeth colonized by counts of �104 � SEM of individual species in nonamplified(n � 46) and amplified (n � 66) root canal samples. The prevalence of each species was computed for each subject and then averaged acrosssubjects. The data are ordered in descending order of prevalence in the amplified samples. ss, subsp.

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commonly been isolated from root canal infections (4, 30, 45,58). In this study, F. nucleatum subspecies were present inrelatively high proportions and levels. F. nucleatum is consid-ered a “bridging” species in the formation of dental plaque dueto its ability to coaggregate with many species (29). It not onlyfacilitates the survival of obligate anaerobic bacteria in oxygenenvironments (6) but also enhances colonization by “red com-plex” species via direct binding (31, 39).

The use of molecular assays has indicated a high prevalence of

species that had infrequently been isolated by cultivation (5, 8, 13,28, 41, 42, 43, 46). In the current study, the average proportions offastidious species, such as T. denticola, T. socranskii, Filifactoralocis, and Dialister pneumosintes ranged from 0.94 to 2.6% of thetotal DNA probe counts. The frequent detection of A. baumanniiin this investigation was in contrast with the lower proportionsdetected by Siqueira et al. (50) in samples from acute abscesses.In the last decade, nosocomial infections caused by multidrug-resistant A. baumannii have been reported (2, 7, 9, 10, 21, 27, 59).

FIG. 5. Bilateral bar chart of the mean percentages of the DNA probe counts (�SEM) for 77 bacterial species in nonamplified root canalsamples taken from 20 teeth without radiographically detected periapical lesions (white bars) and 26 teeth with periapical lesions (black bars). Theproportion of each species was averaged across subjects in the two clinical groups separately. The significance of differences between groups wasdetermined using the Mann-Whitney test and adjusted for multiple comparisons. The data are ordered in descending order of mean percentagesof DNA probe counts detected in teeth with periapical lesions. ss, subsp.

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Didilescu et al. (14) found a high prevalence of A. baumannii(85.3%) in dental plaque samples from hospitalized subjects withchronic lung diseases and a lower prevalence (38.7%) in samplesfrom healthy controls. The role of A. baumannii in endodonticinfections and the possibility that the oral cavity is a source of thespecies for medically important infections merit further investi-gation.

The recognition of greater microbial complexity of root ca-nal infections parallels the greater complexity found in subgin-gival plaque and other oral samples revealed using moleculartechniques. Most previous studies examining the microbiology

of root canals reported presence or absence rather than quan-titative microbiological data. Furthermore, the data werebased on a more limited number of samples and taxa exam-ined. Comprehensive clinical studies of multiple samples quan-titatively examined for a plethora of microbial species arenecessary to better understand the pathogenesis of endodonticinfections and to design targeted therapies.

ACKNOWLEDGMENTS

We thank Maillefer (Ballaigues, Switzerland) for kindly providingthe endodontic files used in the present study.

FIG. 6. Bilateral bar chart of the mean percentages of the DNA probe counts (�SEM) for 77 bacterial species in MDA-amplified root canalsamples taken from 36 teeth without radiographically detected periapical lesions (white bars) and 30 teeth with periapical lesions (black bars).Averaging and statistical testing were as described for Fig. 5. The data are ordered in descending order of mean percentages of the DNA probecounts detected in teeth with periapical lesions. ss, subsp.

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This work was supported in part by grants T32-DE-07327 (F.R.T.),DE-12108, and DE-14242 from the National Institute of Dental andCraniofacial Research.

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