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J Clin Microbiol. Sep 2007; 45(9): 3039–3049.
Published online Jul 18, 2007. doi:  10.1128/JCM.02618-06
PMCID: PMC2045303

Use of Multiple-Displacement Amplification and Checkerboard DNA-DNA Hybridization To Examine the Microbiota of Endodontic Infections[down-pointing small open triangle]

Abstract

Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine 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 samples were 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 and nonamplified samples were computed. Significance of differences for each species between amplified and nonamplified samples was sought with Wilcoxon signed-rank test and adjusted for multiple comparisons. The amount of DNA in the samples ranged from 6.80 (± 5.2) ng before to 6.26 (± 1.73) μg after MDA. Seventy of the 77 DNA probes hybridized with one or more of the nonamplified samples. All probes hybridized with at least one sample after amplification. Most commonly detected species at levels of >104 in both amplified and nonamplified 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 in nonamplified 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. Species commonly detected in endodontic samples included P. tannerae, Prevotella oris, and A. baumannii.

The microbiology of endodontic infections has been studied for many years (4, 47, 58). However, the association between specific microorganisms found in root canals and the symptoms of endodontic infections is poorly understood. Early studies of the endodontic microbiota indicated a predominance of aerobic and facultative bacterial species (16). This conclusion was questioned by the development of anaerobic culturing techniques which clarified the etiopathogenesis of endodontic infections by demonstrating the common occurrence of obligate anaerobic bacteria (4, 23, 30). Nevertheless, culture-based techniques have limitations, such as the difficulty in detecting fastidious anaerobic microorganisms and moderate sensitivity and specificity (44).

Recently, molecular biology techniques have provided a more cost-effective, specific, and sensitive method to evaluate the microbiological profiles of oral pathologies, including endodontic and periodontal infections (37, 38, 44, 52-54). This technology permits the detection of microbial species that are difficult to grow as well as uncultivated and unrecognized phylotypes (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-throughput method to analyze large numbers of DNA samples by use of a wide range of DNA probes on a single nylon membrane (55). The quantity of bacteria in the samples is an important factor in the checkerboard DNA-DNA hybridization technique, since the level of detection is about 104 bacterial cells of a given species. Samples from endodontic pathologies often contain very few bacterial cells and may be below the level of detection of the checkerboard method without a DNA amplification step. To overcome these limitations, the present study used multiple-displacement amplification (MDA) before hybridizing the samples. MDA allows uniform amplification of the whole genome of DNA targets (3, 11, 33, 63, 64), increasing the amount of DNA obtained from the endodontic bacterial samples. Furthermore, MDA provides enough amplified DNA to perform multiple analyses of the same sample by use of different DNA probe sets. The aim of this study was to combine MDA and checkerboard DNA-DNA hybridization to quantitatively and qualitatively assess the taxa present in root canals during endodontic infections.

MATERIALS AND METHODS

Subject population and sample collection.

Sixty-six subjects ranging in age from 11 to 81 years were recruited in the Department of Endodontics, Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil. The subjects had teeth with endodontic infections, with or without radiographically detected periradicular lesions.

The selection of teeth was based on clinical crown conditions that permitted effective placement of rubber dam isolation in teeth with pulp necrosis. The reason for the primary infection was caries; that was detected in almost all cases, although causes of pulp necrosis are sometimes difficult to be determined clinically. Additionally, there was no history of trauma associated with the selected teeth. 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 were single rooted. In the case of multirooted teeth, the sample was taken from the largest root canal.

After informed consent was obtained, the 66 selected teeth were isolated using a rubber dam. Complete asepsis was employed, using the methodology proposed by Möller (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 #10 K-type hand file (Maillefer, Ballaigues, Switzerland). The file was introduced into the canal to a level approximately 1 mm short of the tooth apex. After removal from the canal, the file was cut off below the handle and dropped into an Eppendorf microcentrifuge tube containing a solution of 20 μl of alkaline lysis buffer (400 mM KOH, 100 mM dithiothreitol, 10 mM EDTA). After 10 min of incubation on ice, 20 μl 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 was performed.

For comparison, a second set of samples was taken from 46 of the 66 root canals. In that set of samples, the files were placed into an Eppendorf microcentrifuge tube containing 100 μl 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, and it was maintained at 4°C until checkerboard DNA-DNA hybridization was performed.

MDA of root canal samples.

The procedure was the same as that described by Teles et al. (60). The DNA content of the amplified samples was measured using the Picogreen double-stranded DNA quantification assay (Invitrogen, Carlsbad, CA). The microbiological content of the amplified samples was analyzed using checkerboard DNA-DNA hybridization.

Bacterial strains and growth conditions.

The 77 reference strains used for the preparation of DNA probes are listed in Table Table1.1. The majority of strains were grown on Trypticase soy agar supplemented with 5% defibrinated sheep blood (Baltimore Biological Laboratories [BBL], Cockeysville, MD) with some exceptions. Tannerella forsythia was grown on Trypticase soy agar supplemented with 5% sheep blood and 10 μg/ml N-acetylmuramic acid (Sigma Chemical Co., St. Louis, MO). Porphyromonas gingivalis was grown on Trypticase soy agar supplemented with 5% sheep blood, 0.3 μg/ml menadione (Sigma), and 5 μg/ml hemin (Sigma). Eubacterium and Neisseria species were grown on fastidious anaerobic agar (BBL) with 5% defibrinated sheep blood. Treponema denticola and Treponema socranskii were grown in mycoplasma broth (Difco Laboratories, Detroit, MI) supplemented with 1 mg/ml glucose, 400 μg/ml niacinamide, 150 μg/ml spermine tetrahydrochloride, 20 μg/ml Na isobutyrate, 1 mg/ml l-cysteine, 5 μg/ml thiamine pyrophosphate, and 0.5% bovine serum. All strains were grown at 35°C under anaerobic conditions (80% N2, 10% CO2, 10% H2).

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

DNA isolation and preparation of DNA probes.

Bacterial strains were grown anaerobically 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 placed in 1.5 ml of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.6). Cells were washed twice by centrifugation in TE buffer at 1,300 × g for 10 min. The cells were resuspended and lysed with either 10% sodium dodecyl sulfate and proteinase K (20 mg/ml) for gram-negative strains or in 150 μl of an enzyme mixture containing 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 resuspended by 15 s of sonication and incubated at 37°C for 1 h. DNA was isolated and purified using the method of Smith et al. (51). The concentration of the purified DNA was determined by spectrophotometric measurement of the absorbance at 260 nm. The purity of the preparations was assessed by the ratio of the absorbance at 260 nm and 280 nm. Whole-genome DNA probes were prepared from each of the 77 test strains by labeling 1 to 3 μg of DNA with digoxigenin (Boehringer Mannheim, Indianapolis, IN) by use of a random primer technique (17).

Checkerboard DNA-DNA hybridization.

Checkerboard DNA-DNA hybridization was performed as previously described (26, 54, 55). In brief, following amplification and quantification, amplified samples and nonamplified samples were boiled for 10 min. Three microliters (approximately 900 ng of DNA) of the amplified sample was placed in an Eppendorf tube containing 1 ml of TE buffer prior to boiling. The nonamplified samples were neutralized by adding 800 μl of 5 M ammonium acetate after boiling. Then, the samples were placed into the extended slots of a Minislot 30 apparatus (Immunetics, Cambridge, MA), concentrated onto a nylon membrane (Boehringer Mannheim) by vacuum, and fixed onto the membrane by cross-linking using UV light (Stratalinker 1800; Stratagene, La Jolla, CA) followed by baking at 120°C for 20 min. The Minislot device permitted the deposition of 28 different samples in individual lanes on a single membrane, which also had two control lanes containing 105 and 106 cells of each bacterial species tested. The membrane 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 “checkerboard” pattern was produced. Each channel was used as an individual hybridization chamber for separate DNA probes. Bound probes were detected by anti-digoxigenin antibody conjugated with alkaline phosphatase and a chemifluorescent substrate. Signal intensities of the endodontic samples and the standards (containing 1 ng and 10 ng of each bacterial species) on the same membrane were measured 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” 40 DNA probes used to examine periodontal samples and a second membrane that employed 37 probes to species thought to be important in endodontic samples. Specificity tests were conducted for all probes before performing the checkerboard DNA-DNA hybridization with the root canal samples. The protocol to validate 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 all other species, as described by Socransky et al. (54). If cross-reactions were observed, those probes were discarded and new probes constructed and validated.

Data analysis.

Microbiological data were available for 46 nonamplified and 66 MDA-amplified root canal samples, taken from 66 subjects. The microbial data were expressed in three ways: counts (levels), proportions (percentages of DNA probe counts), and prevalence (percentage of teeth colonized at levels of >105) of 77 bacterial species. Count data were expressed as counts × 105 in each sample and averaged across subjects. The amplified counts that were presented reflect the “number” of organisms detected after MDA amplification of the sample compared with nonamplified standards. They are not actual counts of the original sample but the “DNA equivalents” after amplification. The data shown in the figures in Results represent 1/280 of the DNA available after amplification. One-fortieth of the original sample was amplified by MDA. Three-twentieths of the amplified product was deposited in lanes of the Minislot device, providing an approximately 280-fold “amplification.”

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

In a similar fashion, mean proportions of each species were determined for root canal samples taken from teeth with or without radiographically detected periradicular lesions. The significance of differences between groups was determined 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 amplification averaged 6.80 (± 5.2) ng and 6.26 (± 1.73) μg after amplification, an approximately 1,000-fold amplification. Amplified samples provided signals far better than those observed using nonamplified samples (Fig. (Fig.11).

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 ...

Microbial species in root canal samples.

The mean numbers of species (± standard errors of the means [SEM]) detected in amplified and nonamplified root canal samples at a threshold of >104 were 51.2 ± 2.2 and 14.5 ± 1.7, respectively. If a detection threshold of >105 was employed, then 11.3 ± 1.4 and 0.8 ± 0.2 species were detected in the amplified and nonamplified samples, respectively. Figure Figure22 presents the mean counts (×105 ± SEM) of the 77 test species in amplified and nonamplified root canal samples taken from 46 teeth. The species were ordered according to mean counts. In nonamplified samples, Prevotella tannerae exhibited the highest mean counts (0.91 × 105 ± 0.25), followed by Acinetobacter baumannii and Prevotella oris, while Streptococcus mitis exhibited the lowest mean counts at (0.01 × 105 ± 0.001), followed by Streptococcus salivarius and Actinobacillus actinomycetemcomitans. Seven species were not detected in any of the nonamplified samples. In amplified samples, P. tannerae exhibited the highest mean counts × 105, 3.32 ± 0.69, followed by P. oris and Streptococcus mutans, while Campylobacter concisus exhibited the lowest mean counts (0.15 × 105 ± 0.02), followed by Leptotrichia buccalis and Streptococcus salivarius.

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 canal samples. The counts for each species were averaged across subjects and presented in ...

The mean proportions (percentages of DNA probe counts ± SEM) of the 77 test species in nonamplified and amplified root canal samples are presented in Fig. Fig.3.3. In nonamplified samples, P. tannerae and Acinetobacter baumannii were detected in the highest mean proportions, 11.20 (± 1.48) and 11.14 (± 1.88), while Escherichia coli (0.05 ± 0.03) showed the lowest detected mean proportions, followed by Actinomyces odontolyticus. In amplified samples, P. tannerae was detected in the highest mean proportions (5.33 ± 0.64) followed by P. oris and S. mutans, and Streptococcus salivarius in the lowest mean proportions (0.28 ± 0.03) followed by L. buccalis and Lactobacillus oris.

FIG. 3.
Bilateral bar chart of the mean percentages of the DNA probe counts (±SEM) for 77 bacterial species in nonamplified (n = 46) and amplified (n = 66) root canal samples. The percentage of the DNA probe count was computed for each ...

Figure Figure44 presents the mean percentages of sampled sites exhibiting counts of the 77 test species at levels of >104 in nonamplified and amplified samples. P. tannerae and A. baumannii were detected in all amplified samples and in >90% of nonamplified samples. Other species that were frequently detected included Prevotella heparinolytica in both types of samples and Actinomyces meyeri, Streptococcus parasanguinis, Atopobium rimae, and Porphyromonas endodontalis in MDA-amplified samples. Prevotella oris, Selenomonas sputigena, Haemophilus aphrophilus, and Mogibacterium timidum were detected in >50% of nonamplified samples.

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 ...

Figures Figures55 and and66 demonstrate the mean percentages of DNA probe counts for the 77 test species in nonamplified and amplified root canal samples, respectively, taken from teeth with or without a radiographically apparent periapical lesion. There were no significant differences between clinical groups after adjusting for multiple comparisons for either the nonamplified or amplified samples.

FIG. 5.
Bilateral bar chart of the mean percentages of the DNA probe counts (±SEM) for 77 bacterial species in nonamplified root canal samples taken from 20 teeth without radiographically detected periapical lesions (white bars) and 26 teeth with periapical ...
FIG. 6.
Bilateral bar chart of the mean percentages of the DNA probe counts (±SEM) for 77 bacterial species in MDA-amplified root canal samples taken from 36 teeth without radiographically detected periapical lesions (white bars) and 30 teeth with periapical ...

DISCUSSION

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

Another goal of the current investigation was to compare the microbiota in root canals with and without periradicular lesions. Although others have found specific bacterial communities to be associated with asymptomatic or symptomatic endodontic infections (18, 22, 42), the current study found no significant differences between the two clinical states, irrespective of whether the samples were amplified or not. However, this approach might set the stage for further studies in which one species or a set of species might be associated with different symptomatologies that could lead to specific treatment modalities.

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

Among the wide range of taxa detected in this study, species that form black-pigmented colonies (BPB) such as P. tannerae and nonpigmented Prevotella species such as P. oris were in high mean counts. P. tannerae has been reported as an uncultivable organism (15), and its frequency of occurrence in endodontic 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 endodontic samples, P. tannerae was found in only 5% of them, possibly due to limitations in the multiplex technique (38). Other BPB were also present in relatively high mean counts and proportions in amplified samples of the current study, including P. endodontalis, Prevotella loescheii, Prevotella nigrescens, Prevotella intermedia, P. gingivalis, and Prevotella melaninogenica. P. endodontalis has been isolated by cultivation from infected root canals (24, 57), but its prevalence was even higher when molecular techniques were used (19, 22, 40, 48).

Periodontal pathogens of the “red complex” (52), T. forsythia, P. gingivalis, and T. denticola, were detected in amplified and nonamplified samples. T. denticola was present in higher proportions than T. forsythia and P. gingivalis in the amplified samples, a finding similar to that of Haffajee et al. (25) in subgingival microbiota of Brazilian subjects. Using PCR, Roças et al. (36) found that T. denticola was the most prevalent of the three species (44%) in endodontic samples, while Siqueira et al. (49), using checkerboard DNA-DNA hybridization, found that T. forsythia was the most prevalent (39.3%). In other investigations (18, 40), T. denticola was detected in 56% and 79% of samples from infected teeth. The fastidious growth of T. denticola and T. forsythia has led to an underestimation of their prevalence in cultural studies of endodontic infections. Based on their frequent detection by molecular techniques, they might be potential endodontic pathogens (40). Furthermore, T. denticola seems highly pathogenic in monoinfections of the dental pulp in a mouse model system (18).

Members of the “orange complex” were present in endodontic infections, including Fusobacterium nucleatum, which has commonly been isolated from root canal infections (4, 30, 45, 58). In this study, F. nucleatum subspecies were present in relatively high proportions and levels. F. nucleatum is considered a “bridging” species in the formation of dental plaque due to its ability to coaggregate with many species (29). It not only facilitates the survival of obligate anaerobic bacteria in oxygen environments (6) but also enhances colonization by “red complex” 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 of fastidious species, such as T. denticola, T. socranskii, Filifactor alocis, and Dialister pneumosintes ranged from 0.94 to 2.6% of the total DNA probe counts. The frequent detection of A. baumannii in this investigation was in contrast with the lower proportions detected 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). Didilescu et al. (14) found a high prevalence of A. baumannii (85.3%) in dental plaque samples from hospitalized subjects with chronic lung diseases and a lower prevalence (38.7%) in samples from healthy controls. The role of A. baumannii in endodontic infections and the possibility that the oral cavity is a source of the species for medically important infections merit further investigation.

The recognition of greater microbial complexity of root canal infections parallels the greater complexity found in subgingival plaque and other oral samples revealed using molecular techniques. Most previous studies examining the microbiology of root canals reported presence or absence rather than quantitative microbiological data. Furthermore, the data were based on a more limited number of samples and taxa examined. Comprehensive clinical studies of multiple samples quantitatively examined for a plethora of microbial species are necessary to better understand the pathogenesis of endodontic infections and to design targeted therapies.

Acknowledgments

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

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 and Craniofacial Research.

Footnotes

[down-pointing small open triangle]Published ahead of print on 18 July 2007.

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