Abstract
In a 16S rRNA gene-directed multiplex PCR, Prevotella intermedia- and Bacteroides forsythus-specific reverse primers were combined with a single conserved forward primer. A 660-bp fragment and an 840-bp fragment that were specific for both species could be amplified simultaneously. A total of 152 clinical samples, subgingival plaque and swabs of three different oral mucosae, from 38 periodontitis patients were used for the evaluation.
The major putative pathogens known to be involved in destructive periodontal diseases include Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, and spirochetes (8). Various methods for the detection of these pathogens have been described (30), but there is no consensus regarding the method of choice. Approaches frequently used for microbiological studies include direct microscopy, cultivation, enzyme tests, enzyme-linked immunosorbent assay, detection of signature sequences by using genomic or oligonucleotide probes, and the PCR. The latter, because of its sensitivity of as few as 3 to 50 CFU, is of particular interest for studying the early colonization of the host with periodontal pathogens or suppression of the pathogens following periodontal treatment. Most PCR studies have concentrated on the detection of a single pathogenic species using various targets for primer annealing: A. actinomycetemcomitans (leukotoxin gene-directed primers [11, 12] and 16/23S rRNA gene [rDNA]-directed primers [19]), B. forsythus (randomly amplified polymorphic DNA [RAPD] marker flanking primers [5, 15] and 16S rDNA [23]), P. gingivalis (RAPD marker flanking primers [3], collagenase gene-directed primers [4, 29], outer membrane protein gene-directed primers [17], and 16/23S rDNA-directed primers [21, 22, 26]), and P. intermedia (RAPD marker flanking primers [16]).
To assess the epidemiology of periodontal pathogens and the diagnosis and treatment of periodontal diseases, most species of etiologic importance need to be detected. In a number of studies, seven or eight putative periodontal pathogens have been detected by individual PCRs (2, 6, 24). To minimize the time and expenditure needed for detection procedures, sets of 16S rDNA-directed primers have been combined to detect more than one species in a single sample. However, this multiplex PCR variant was only evaluated for the species A. actinomycetemcomitans, E. corrodens, and P. gingivalis (13, 25, 28).
The aim of the present study was to develop a multiplex PCR using one 16S rDNA-directed conserved forward primer combined with species-specific reverse primers for simultaneous detection of B. forsythus and P. intermedia. After evaluating the method with 6 B. forsythus and 10 P. intermedia strains, as well as 23 strains of other closely and more distantly related (oral) bacteria, the PCR was applied to a total of 152 clinical samples consisting of 38 samples each of subgingival plaque and swabs of tonsil, cheek, and tongue mucosae.
Bacterial strains and patient specimens.
To evaluate the multiplex PCR, the bacteria used as positive controls were B. forsythus ATCC 43037T, FR001/12-3, FR002/23-2, FR004/13-4, FR007/24-6, and FR009/11-6 and P. intermedia ATCC 25611T, A735, FR023/26, FR028/11, H91-360/1, H91-1880/2, Hg404, Hg1103, Hg1269, and MH6. The bacteria used as negative controls were A. actinomycetemcomitans ATCC 33384T, Actinomyces israelii ATCC 12102T, A. gerencseriae ATCC 23860T, A. odontolyticus DSM43331, Capnocytophaga gingivalis ATCC 33624T, C. granulosa ATCC 51502T, C. haemolytica ATCC 51501T, C. ochracea ATCC 33596, C. sputigena ATCC 33612T, E. corrodens ATCC 23834T, F. nucleatum ATCC 25586T, Haemophilus aphrophilus ATCC 33894T, Peptostreptococcus micros ATCC 33270T, Porphyromonas asaccharolytica ATCC 25260T, P. gingivalis ATCC 33277T, Prevotella corporis A350, P. nigrescens ATCC 33563T, Propionibacterium propionicum NCTC12967, Rothia dentocariosa GH399, Stomatococcus mucilaginosus MCCM00557, Streptococcus intermedius DSM20573, S. mutans NCTC11060, and S. salivarius DSM20068. Strains were obtained from the following sources: ATCC strains, American Type Culture Collection, Manassas, Va.; NCTC strains, National Collection of Type Cultures, London, United Kingdom; DSM strains, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; FR strains, K. Pelz, Institute of Medical Microbiology and Hygiene, Freiburg, Germany; A strains, I. Mitchelmore, St. Bartholomew’s Hospital Medical College, London, United Kingdom; the GH strain, G. Haase, Institute of Medical Microbiology, Aachen, Germany; H strains, C. Hoehne, Institute of Medical Microbiology, Halle, Germany; Hg strains, T. J. M. Van Steenbergen, Academic Center for Dentistry, Amsterdam, The Netherlands; MCCM strains, Medical Culture Collection Marburg, Marburg, Germany; MH strains, M. Haapasaylo, Department of Cariology, University of Helsinki, Helsinki, Finland.
All bacteria were grown on Columbia agar with 5% sheep blood for 3 to 5 days at 37°C. After sufficient growth, 1 to 10 colonies (depending on their size) of the pure bacterial cultures were suspended in 250 μl of reduced transport medium (20) and the suspensions were kept frozen (−70°C) until investigation.
A total of 152 clinical samples were taken from 38 adult patients with untreated periodontitis, representing consecutive samples recruited from the Department of Periodontology, Julius Maximilian University, Würzburg, Germany. The mean age of the patients was 51.8 ± 11.0 years, and 21 were female and 17 were male. The mean percentage of sites with a periodontal probing depth of 4 to 6 mm was 31.4 ± 11.9, the mean percentage of sites with a periodontal probing depth of ≥7 mm was 4.5 ± 6.3, and the mean percentage of sites with bleeding on probing was 53.8 ± 32.3. Patients who had used systemic antibiotics in the previous 6 months were excluded from the study. All patients enrolled in the study signed the informed consent form approved by the Ethics Committee of the Medical Faculty, Julius Maximilian University. Subgingival plaque samples were obtained with a sterile curette from the four deepest periodontal pockets, pooled, and placed in 1 ml of reduced transport medium. Samples from oral mucous membranes, i.e., the dorsum of the tongue, both tonsils, and both buccal mucosae, were collected with sterile cotton swabs, and each was suspended in 1 ml of reduced transport fluid and kept at −70°C until investigation. An aliquot of approximately 250 μl of each suspension was used for the B. forsythus-P. intermedia multiplex PCR.
Sample processing for PCR.
Three different methods of sample preparation for PCR were tested with pure cultures, 12 clinical specimens (for each method, one subgingival plaque sample and one swab sample each from the tonsils, the buccal mucosa, and the dorsum of the tongue), and spiked subgingival plaque from healthy volunteers.
(i) Boiling.
Deep-frozen 250-μl suspensions (bacterial cultures or patient specimens) were incubated for 10 min at 37°C. Four glass beads (2 mm in diameter) were then added, the samples were vortexed for 20 s and centrifuged, the supernatant was removed, and the pellet was resuspended in 100 μl of distilled water. After an additional vortexing-and-centrifugation step, the pellet was resuspended in 500 μl of distilled water and the suspension was heated for 10 min at 94°C with a thermocycler. The vials were then stored for 5 min on ice and centrifuged, and 5-μl aliquots of the supernatant were further used in the PCR assay.
It has recently been reported that Chelex 100 resin (Bio-Rad Laboratories, Hercules, Calif.) (9) processing of oral specimens prior to boiling most effectively decreases PCR inhibition and thus increases sensitivity (21). Therefore, we performed an additional experiment by using subgingival plaque and mucosal swabs taken from three additional patients, spiking the samples with B. forsythus and P. intermedia, and processing them with Chelex 100.
(ii) Lysozyme-phenol protocol.
A second way of isolation was performed by using a previously described protocol (6). After lysozyme-phenol processing of 250-μl suspensions in accordance with this protocol, the aqueous phase was adjusted to 2.5 M ammonium acetate and the nucleic acid was precipitated at −20°C by adding 2.5 volumes of ice-cold 70% ethanol. After centrifugation, the nucleic acid pellet was washed with 250 μl of 70% ice-cold ethanol and dissolved in 500 μl of distilled water.
(iii) QIAamp Tissue Kit protocol.
Suspensions of 250 μl were pelleted by centrifugation for 10 min at 5,000 × g, resuspended in 180 μl of enzyme incubation buffer (20-mg/ml lysozyme, 20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 1.2% Triton X-100), and incubated for 30 min at 37°C. A 20-μl volume of proteinase K stock solution (20 mg/ml) was added, and the sample was mixed by vortexing and incubated at 55°C in a shaking water bath until it was completely lysed. Afterwards, isolation was performed as recommended by the manufacturer (QIAGEN GmbH, Hilden, Germany).
PCR amplification.
PCR amplification was carried out in a volume of 100 μl containing 1× PCR buffer, 1.5 mM MgCl2, 2 U of Taq polymerase (Boehringer, Mannheim, Germany), 0.2 mM each deoxynucleoside triphosphate (Boehringer), 5 pmol of a universal 16S rDNA forward primer (pA; 5′ AGA GTT TGA TCC TGG CTC AG 3′) (10), 5 pmol of either of the two species-specific primers (BFV530 [5′ GTA GAG CTT ACA CTA TAT CGC AAA CTC CTA 3′] for detection of B. forsythus [14] or Pi [5′ GTT GCG TGC ACT CAA GTC CGC C 3′] for detection of P. intermedia [7]) or a universal reverse primer (pH°; 5′ AAG GAG GTG ATC CAG CCG CA 3′ [10]), and 5 μl of either the template (approximately 100 ng) or the reference (100 ng) nucleic acids. All oligonucleotides were synthesized on a DNA synthesizer (OLIGO 1000; Beckman, Munich, Germany). Amplification was performed by using 30 cycles of the following temperature profile: denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and elongation for 2.5 min at 72°C. After the 30 cycles, a final elongation step of 5 min at 72°C was added. Amplification products (aliquots of 10 μl) were separated electrophoretically on a 2% agarose gel (Merck, Darmstadt, Germany) in 1× TPE (80 mM Tris-phosphate, 2 mM EDTA [pH 7.5]). From two independent databases (23a, 24a), the 16S rDNA sequences and the annealing sites of primers were determined for both test species. The search indicated amplicon sizes of 660 bp for P. intermedia and 840 bp for B. forsythus.
Specificity and sensitivity of the PCR.
The specificity of the PCR was evaluated by testing 6 B. forsythus and 10 P. intermedia strains, as well as 23 representatives of closely or more distantly related species (aliquots of 100 ng of nucleic acid). Amplicons appearing to be of the expected sizes (P. intermedia, 660 bp; B. forsythus, 840 bp) were found with all of the strains tested. Neither of these two PCR products or other PCR bands occurred when 100-ng samples of the other 23 representative strains of oral species were used. The sensitivity of the PCR system was evaluated by titrating cultures of B. forsythus ATCC 43037T and P. intermedia ATCC 25611T (106 CFU/ml). We made serial dilutions of the original cultures with reduced transport fluid and plated equal volumes (100 μl) of the dilutions onto Columbia blood agar. The growth of the fastidious bacterium B. forsythus was stimulated by coincubation with F. nucleatum (N-acetylmuramic acid donor). The original cultures of both microorganisms and the dilutions were mixed (1:1) by vortexing before samples were taken for DNA extraction and the subsequent multiplex PCR. The colonies on Columbia blood agar plates were counted after a 3-day incubation for P. intermedia and after a 5-day incubation for B. forsythus (37°C, anaerobically). The detection limit was determined by using known numbers of bacteria diluted either in reduced transport fluid or in subgingival plaque samples from five healthy volunteers previously checked with the multiplex PCR to be free of B. forsythus and P. intermedia.
In the 1:1 mixture of pure cultures of B. forsythus and P. intermedia, the multiplex PCR detected between 50 and 500 CFU of each species. In contrast, the detection limit was slightly increased for the spiked subgingival plaque samples and found to range between 100 and 1,000 CFU. By decreasing the annealing temperature from 55 to 52°C and increasing the concentration of magnesium (1.5 to 4.5 mM), the sensitivity of our procedure could be increased to a single cell but resulted in weak cross-reactivity and nonspecific amplification bands (data not shown). Since subgingival plaque is a mixed community of different species, high specificity is especially important for a PCR. Therefore, 55°C and 1.5 mM MgCl2 were employed in order to maintain the highest possible specificity.
Detection of B. forsythus and P. intermedia in clinical specimens.
The yield of nucleic acids isolated from 12 clinical samples by the three methods described was between 10 μg (boiling) and 50 μg (QIAamp Tissue Kit). It was calculated that DNA extraction from the pure bacterial cells and spiked plaque also yielded similar amounts of nucleic acids. Because isolating pure DNA with the QIAamp Tissue Kit is expensive, boiling was selected as the method of choice to process the remaining 140 specimens.
A representative multiplex PCR result for clinical samples is demonstrated in Fig. 1. The following pattern (Table 1) was found when the 152 clinical samples from different origins were analyzed for the presence of B. forsythus and/or P. intermedia. (i) In 5 of the subgingival plaque samples (13.2%), both species were present, 11 samples (28.9%) demonstrated B. forsythus only, and 1 sample (2.6%) contained P. intermedia only. (ii) Four (10.5%) of 38 tonsil swabs were positive for P. intermedia only, and one specimen harbored both B. forsythus and P. intermedia. (iii) Of the buccal mucosa swab samples, one was positive for both B. forsythus and P. intermedia, six (15.8%) were positive for P. intermedia, and four (10.5%) were positive for B. forsythus. (iv) Finally, 10 (26.3%) of the tongue swabs were positive for P. intermedia but none was positive for B. forsythus. None of the periodontopathogenic species assessed were detected in 21 of 38 subgingival plaque samples, 33 of 38 tonsils swabs, 27 of 38 buccal epithelial swabs, and 26 of 38 tongue swabs. Because these samples contained approximately 30 to 100 different species, this finding supports the specificity of our multiplex PCR. To exclude inhibitory compounds as a principal reason for a negative result, a universal 16S rDNA-directed PCR combining universal primers pA and pH° was used and found to be positive for all culture and clinical specimens. However, it is possible that for some samples with multiplex PCR-negative results, a low concentration of target bacteria (<500 cells) and/or inhibitory compounds, such as cations, caused a false-negative result (1, 18). Since Chelex 100 processing of 12 additional samples resulted in an enhanced sensitivity of 50 CFU/clinical specimen without loss of specificity, we recommend this procedure prior to boiling for future application of the described multiplex PCR.
FIG. 1.
PCR analysis for the presence of B. forsythus and P. intermedia at four clinical sites (P, subgingival plaque; T, tonsils; B, buccal membrane; Z, tongue mucosa) from four patients (no. 1 to 4 in Table 1). Lanes: M, AmpliSize standard (50- to 2,000-bp ladder; Bio-Rad Laboratories, Hercules, Calif.); 1, positive control with 100 ng each of B. forsythus and P. intermedia DNAs; 2, negative control lacking template DNA.
TABLE 1.
Detection of B. forsythus and P. intermedia by multiplex PCR in four different specimens taken from each of 38 untreated patients with periodontitisa
| Patient no. | Presence of:
|
||
|---|---|---|---|
| B. forsythus without P. intermedia | P. intermedia without B. forsythus | B. forsythus and P. intermedia | |
| 1 | − | + (T, B, Z) | + (P) |
| 2 | − | − | − |
| 3 | − | − | + (P, B) |
| 4 | + (B) | − | − |
| 5 | − | + (Z, P) | − |
| 6 | − | + (B) | − |
| 7 | + (P) | − | − |
| 8 | − | − | − |
| 9 | + (P) | − | − |
| 10 | + (B) | − | − |
| 11 | + (P) | + (B) | − |
| 12 | − | − | − |
| 13 | − | − | − |
| 14 | − | − | − |
| 15 | − | − | − |
| 16 | − | + (Z) | − |
| 17 | + (P) | − | − |
| 18 | − | + (T, B, Z) | − |
| 19 | − | + (T) | − |
| 20 | − | + (B) | + (P) |
| 21 | + (P) | − | − |
| 22 | − | + (T, Z) | − |
| 23 | + (P, B) | − | − |
| 24 | − | − | − |
| 25 | + (B) | + (Z) | − |
| 26 | − | − | − |
| 27 | − | − | − |
| 28 | − | − | − |
| 29 | + (P) | + (Z) | − |
| 30 | − | + (B, Z) | − |
| 31 | + (P) | + (Z) | − |
| 32 | − | − | − |
| 33 | − | − | − |
| 34 | + (P) | + (Z) | + (T) |
| 35 | − | − | + (P) |
| 36 | + (P) | − | − |
| 37 | − | − | + (P) |
| 38 | + (P) | − | − |
Positive sites in parentheses: P, subgingival plaque; T, tonsil swabs; B, buccal-membrane swabs; Z, tongue mucosal swabs.
Previous studies (14, 23) have focused on subgingival plaque as a likely primary habitat of B. forsythus. In the present study, we investigated the localization of this obligate anaerobic, fastidious species in ecological niches of the oral cavity other than subgingival plaque. Interestingly, the finding that 13.1% of all buccal mucosa swabs were positive for B. forsythus indicates that the buccal membrane is one of its habitats in the oral cavity. In contrast, the prevalence of B. forsythus on the tonsil or tongue mucosa seems to be rather low, even in patients with untreated periodontitis. Surprisingly, the prevalence of P. intermedia among the specimens tested was highest on the dorsum of the tongue (26.3%).
In conclusion, multiplex PCRs may rapidly detect relevant numbers of putative periodontal pathogens cost effectively in clinical specimens. To assist in specific treatment planning, microbiological diagnosis of periodontal infections is increasingly needed. Clinical laboratories which test samples sent in by mail have recently been established, and tests which can be performed in a dentist’s office have also been introduced (27). These tests depend mainly on dot blot hybridization assays using both genomic or oligonucleotide DNA probes.
The method described in this report for the simultaneous detection of B. forsythus and P. intermedia may assist in selecting adjunctive antibiotics for the treatment of aggressive periodontal diseases. Whether the detection of putative periodontal pathogens may also be useful in assessing the risk or progression of periodontal disease onset is unclear and should be established.
Acknowledgments
This work was supported by a grant of the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF no. 01KI9710/5) of Germany.
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