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Previous Article | Next Article 
Journal of Clinical Microbiology, May 1999, p. 1621-1624, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Simultaneous Detection of Bacteroides
forsythus and Prevotella intermedia by 16S rRNA
Gene-Directed Multiplex PCR
Georg
Conrads,1,2,*
Thomas F.
Flemmig,3
Ilse
Seyfarth,1
Friedrich
Lampert,1 and
Rudolf
Lütticken2
Clinic of Conservative and Preventive
Dentistry and Periodontology,1 and
Department of Medical Microbiology,2
University Hospital (RWTH), D-52057 Aachen, and Department of
Periodontology, Julius Maximilian University, D-97070
Würzburg,3 Germany
Received 31 July 1998/Returned for modification 2 November
1998/Accepted 13 February 1999
 |
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.
| |
TEXT |
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.
View larger version (97K):
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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.
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|
View this table:
[in this window]
[in a new window]
|
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
|
|
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.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Medical Microbiology, University Hospital (RWTH), Pauwelsstrasse 30, D-52057 Aachen, Germany. Phone: (49)-241-8089787. Fax:
(49)-241-8888483. E-mail:
conrads{at}alpha.imib.rwth-aachen.de.
| |
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Journal of Clinical Microbiology, May 1999, p. 1621-1624, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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