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To discriminate between viable and non-viable Enterococcus faecalis, the predominant pathogen in apical periodontitis, a real-time PCR method combined with propidium monoazide (PMA) was developed and evaluated. PMA had no antimicrobial effect on E. faecalis cells and permitted enumeration of both viable and non-viable cells. Therefore, E. faecalis cells from the root canals of nine patients with apical periodontitis were analyzed to evaluate the diagnostic usefulness of this approach. Viable and non-viable E. faecalis cells were successfully discriminated in these clinical specimens. A real-time PCR assay combined with PMA will contribute to the precise diagnosis of apical periodontitis.
Enterococci are present in small numbers in the oral cavities of healthy individuals; however, they dominate the oral cavity in patients with apical periodontitis, which is primarily caused by anaerobic oral bacteria surviving on the teeth in apical biofilms post-treatment. The enterococci recovered from biofilms in the root canals of patients with apical periodontitis are often antimicrobial-resistant (1, 2). E. faecalis is a major pathogen in apical periodontitis (3); thus, monitoring this organism in periapical biofilms during the treatment of apical periodontitis is crucial.
Quantitative PCR-based methods have been developed for enumerating bacteria (4, 5); however, DNA-based detection methods cannot differentiate between signals originating from live and dead bacteria. Such differentiation is diagnostically important, especially for antimicrobial-resistant organisms. Therefore, a PCR-based method that can discriminate between DNA derived from viable and dead bacterial cells is needed. Recently, the DNA-binding dyes EMA and PMA were used for PCR-based differentiation of viable and dead bacterial cells (6–8). These dyes exclusively penetrate dead cells following membrane damage and cross-link the DNA via photo-activation, thereby inhibiting amplification (9). However, recent data has shown that EMA cross-linking during genomic DNA extraction renders the DNA insoluble and causes its loss in concert with cellular debris (7). EMA can also penetrate live cells of some bacterial species (6); however, it is toxic to viable cells (8, 10).
In this study, we evaluated a PMA-based quantitative detection method that distinguished viable from non-viable E. faecalis cells in root canals.
The bacteria used in this study are listed in Table 1. Enterococcus faecalis was grown anaerobically in trypticase soy broth (Becton-Dickinson, Sparks, MD, USA). Bacterial DNA was isolated using a PureGene DNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA). E. faecalis-specific primers targeting azoA (encoding azoreductase; sense: Ef azoAF 5′-CCAATCAAATGGCGGCTTCTACG-3′, antisense: Ef azoAR 5′-GCGATCAGGGAAATGATCGATTCC-3′) were designed (11). Primer specificity was confirmed by PCR using chromosomal DNA from 28 oral bacteria (Table 1). SYBR green-based quantitative real-time PCR was performed in a total volume of 20 μL containing 5 μL of various concentrations of extracted genomic DNA with or without PMA treatment, 5 × SYBR Green Master (Roche Diagnostics, Mannheim, Germany), and 0.5 μM of each primer. Amplification was done using the LightCycler Carousel-Based System (Roche Diagnostics) at 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 53°C for 10 s, and 72°C for 12 s. To confirm the formation of a single product, melting curve analysis was performed at 95°C for 1 min and 55°C for 1 min, with a subsequent temperature increase from 55.0–95.0°C at 0.5°C per 10 s (data not shown). The sizes of the products were confirmed using 2% agarose gels. Using this method, bacterial CFU were detected linearly from 15 to 3.0 × 107 per mixture. The relationship between live cells and Ct values for real-time PCR is as follows: Y = 10−0.293X±11.056 (where Y = log10CFU, X = Ct value, R2= 0.997). Bacterial cell numbers were calculated using this formula.
Table 1. Strains and amplification results
|Strain||Amplification with the primers for azoA|
|Enterococcus faecalis ATCC 19433||+|
|Streptococcus sobrinus OMZ 176||−|
|Streptococcus salivarius HT9R||−|
|Streptococcus gordonii DL 1||−|
|Streptococcus oralis ATCC 10557||−|
|Streptococcus mutans UA 159||−|
|Streptococcus mitis 903||−|
|Streptococcus sanguinis OMZ 9||−|
|Streptococcus anginosus FW73||−|
|Aggregatibacter actinomycetemcomitans Y 4||−|
|Treponema denticola ATCC 35405||−|
|Treponema vincentii ATCC 35580||−|
|Treponema pectinovorum ATCC 33768||−|
|Porphyromonas gingivalis ATCC 33277||−|
|Prevotella bivia JCM 6331||−|
|Prevotella corporis ATCC 33547||−|
|Prevotella intermedia ATCC 25611||−|
|Prevotella loescheii ATCC 15930||−|
|Prevotella melaninogenica ATCC 25845||−|
|Prevotella nigrescens ATCC 25261||−|
|Prevotella oralis ATCC 33322||−|
|Prevotella oris JCM 8540||−|
|Prevotella pallens ATCC 700821||−|
|Prevotella veroralis ATCC 33779||−|
|Tannerella forsythia ATCC 43037||−|
|Lactobacillus rhamnosus ATCC 7469||−|
|Haemophilus aphrophilus NCTC 5980||−|
|Eschrichia coli DH5α||−|
Propidium monoazide (Biotium, Hayward, CA, USA) was dissolved in 20% DMSO to produce a 24 mM stock solution. Following incubation with the dye for 5 min in the dark, similarly prepared cells were exposed for 5 min to a 600 W halogen light placed 20 cm above 500 μL samples in open microcentrifuge tubes on ice. The toxicity of PMA at 2.4 μM to 2.4 mM to E. faecalis was analyzed at 37°C; however, no toxicity was found (Mann-Whitney U-test, data not shown). In this study, 240 μM of PMA was employed for the analysis.
To investigate the effects of PMA, E. faecalis chromosomal DNA (0.01–100 μg/mL) was analyzed with and without PMA treatment. Real-time PCR was not inhibited by heat-killed cells treated with 240 μM PMA (Fig. 1). To eliminate possible inhibition by the clinical material, E. faecalis samples were spiked with dental plaque and saliva (without E. faecalis) to mimic the oral environment. There was no inhibition of real-time PCR (Fig. 2).
Figure 1. Effect of PMA on heat-killed E. faecalis as assessed by real-time PCR. Serially diluted chromosomal DNA from live cells (#1–5) and live cells spiked with heat-killed cells (#6–9). All cells were treated with PMA as described in the text.
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Figure 2. Inhibition of PMA-treated live E. faecalis cells from (a) E. faecalis-negative dental plaque and (b) saliva.
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Based on these results, nine endodontic samples from eight patients with root-filled teeth and showing radiographic evidence of apical periodontitis were analyzed. The endodontic samples were collected in accordance with the guidelines of the Ethics Committee of Kyushu Dental College Hospital from patients who visited the Department of Preventive Dentistry, Kyushu Dental College Hospital. All patients provided informed consent. Endodontic samples were taken from the infected root canals as described previously (12). The relevant tooth was isolated from the oral cavity with a disinfected rubber dam. The isolated tooth was cleaned and a sterile paper point moistened in sterile saline was placed in the root canal for 10 s. The paper point was then transferred to 200 μL of PBS. The extracted chromosomal DNA served as the PCR template. As shown in Table 2, the prevalence of live E. faecalis cells ranged from 0 to 8.6 × 102 cells (0–73.3%), while that of dead cells ranged from 8.0 × 101 to 1.9 × 104 cells (26.7–100%). In this study, no live cells were observed in the samples from patients 5 and 6. However, previous testing with real-time PCR without PMA had identified these samples as positive for E. faecalis. Thus, real-time PCR and PMA can be used to distinguish live from dead E. faecalis. This method makes it possible to obtain detailed information about apical periodontitis. In this study, we observed no obvious relationship between the clinical symptoms of apical inflammation (pus discharge and percussion pain) and live/dead cell numbers. However, a larger sample number should clarify in more detail the relationship between clinical features and live/dead cell numbers. Our data will help clarify the role of E. faecalis in the etiology of apical periodontitis.
Table 2. Prevalence of live/dead E. faecalis cells in the root canals of patients with apical periodontitis
|Patient No.||Tooth No.||Clinical features||No. of E. feacalis Cells||Percentages of E. feacalis Cells||Live/dead ratio|
|Pus||Percussion Pain||Live (×102)||Dead (×102)||Total (×102)||Live||Dead|
|1||23||−||−||2.1|| 0.8|| 2.9||73.3|| 26.7||2.74|
|2||22||+||+||2.7||187.7||190.4|| 1.4|| 98.6||0.01|
|3||44||−||−||1.4|| 4.9|| 6.3||22.4|| 77.6||0.29|
|4||22||+||+||1.8|| 15.5|| 17.3||10.2|| 89.8||0.11|
|5||24||−||−||0|| 8.4|| 8.4|| 0.0||100.0||0.00|
|6||11||−||−||0|| 2.7|| 2.7|| 0.0||100.0||0.00|
|7||36||−||+||8.6|| 58.3|| 66.9||12.8|| 87.2||0.15|
|8||36||−||+||4.5|| 4.0|| 8.5||52.6|| 47.4||1.11|
|9||17||−||−||1.8|| 2.3|| 4.1||43.3|| 56.7||0.76|