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Journal of Clinical Microbiology, January 1998, p. 157-160, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Porphyromonas gingivalis
from Saliva by PCR by Using a Simple Sample-Processing Method
Jaana
Mättö,1,*
Maria
Saarela,1
Satu
Alaluusua,2
Virva
Oja,1
Hannele
Jousimies-Somer,3 and
Sirkka
Asikainen1,4
Research Laboratory, Institute of
Dentistry,1
Department of Pedodontics
and Orthodontics,2 and
Department of
Periodontology,4 University of Helsinki, and
Anaerobe Reference Laboratory, National Public Health
Institute,3 Helsinki, Finland
Received 24 April 1997/Returned for modification 16 July
1997/Accepted 1 October 1997
 |
ABSTRACT |
Simple sample-processing methods for PCR detection of
Porphyromonas gingivalis, a major pathogen causing adult
periodontitis, from saliva were studied. The ability to detect P. gingivalis from 118 salivary samples by PCR after boiling and
Chelex 100 processing was compared with bacterial culture. P. gingivalis was detected three times more often by PCR than by
culture. Chelex 100 processing of saliva proved to be effective in
preventing PCR inhibition and was applied to determine the occurrence
of P. gingivalis in saliva samples from 263 Finnish
subjects between 5 and 80 years of age. The occurrence of P. gingivalis increased with age, and it was detected by PCR in the
saliva of 5.0% of subjects between 5 and 10 years of age, 13.8% of
subjects between 11 and 20 years of age, 13.4% of subjects between 21 and 30 years of age, and 63.3% of subjects between 31 and 80 years of
age. The results indicate that P. gingivalis is a rare
finding in saliva from periodontally healthy children and young adults
but a frequent one in saliva from adult periodontitis patients.
| |
INTRODUCTION |
Porphyromonas gingivalis,
a black-pigmented gram-negative anaerobic rod, is a major pathogen
causing adult periodontitis (18). P. gingivalis
is frequently isolated from subgingival plaque of periodontitis
patients, whereas it can be cultured only occasionally from
periodontally healthy adults and is usually not isolated from children
(4, 8, 9, 13, 15, 25).
Saliva is the most probable vehicle for person-to-person transmission
of oral bacteria. Thus, it is likely that the presence of P. gingivalis in saliva is a prerequisite for its transmission. Saliva represents an easily and noninvasively obtainable sample containing bacteria from all oral sites, e.g., the mucosa and supra-
and subgingival plaque. Furthermore, it is also rather easily
obtainable from the oral cavities of young children. However, the
proportion of shed periodontal bacteria in the salivary microbiota is
relatively low, a fact that makes bacterial culture an insensitive detection method. Selective media, such as kanamycin vancomycin laked
blood agar, which are useful for isolation of other oral black-pigmented gram-negative anaerobes from polymicrobial sources, cannot be used for culturing P. gingivalis, since
Porphyromonas spp. isolates are usually susceptible to
vancomycin (11).
PCR allows the specific amplification of target bacterial DNA in
samples for which the background caused by other species is high. Thus,
PCR could be applicable for the detection of P. gingivalis
from saliva. However, biological samples may contain compounds that are
inhibitory to PCR amplification, leading to false-negative results
(2, 10). Aside from that, PCR-based detection methods
require proper validation and quality control to avoid false-positive
results. PCR-based methods for detection of P. gingivalis
from subgingival samples have been described earlier (3, 14, 21,
22, 26), whereas no reports on PCR detection of periodontal
bacteria from salivary samples are available.
This paper describes a simple sample-processing method which can be
used to prevent PCR inhibition by saliva. The efficacy of the present
PCR method for detection of P. gingivalis from salivary
samples was compared with that of bacterial culture. Additionally, the
present PCR method was applied to determine the occurrence of P. gingivalis in saliva specimens from Finnish subjects 5 to 80 years
of age.
| |
MATERIALS AND METHODS |
Subjects, sampling, and bacterial culture.
The material used
in this study consisted of salivary samples from 263 periodontally
healthy or diseased subjects (age range, 5 to 80 years). The
periodontal status of a subject was defined as healthy when no signs of
periodontal breakdown were found. Periodontitis was diagnosed when the
presence of periodontal breakdown was verified in a clinical and/or
radiological examination. Periodontitis was further classified
according to guidelines of the American Academy of Periodontology
(1).
A subset of 118 samples was used for evaluation of the PCR method. The
occurrence of P. gingivalis in Finnish subjects as determined by PCR was studied using all 263 saliva samples. The samples
were collected during several previous studies, between 1985 and 1996, by using paraffin chewing stimulation and preserved at 
70°C until
used in the present study. For the detection of P. gingivalis by bacterial culture, 142 of the 263 saliva samples had
been serially diluted immediately after sampling and cultured on
Brucella agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with lysed horse blood (5%), hemin (5 µg/ml), and vitamin K1 (10 µg/ml). The plates were incubated
anaerobically in jars filled by the evacuation-replacement method with
a mixture of gases (85% N2, 10% H2, 5%
CO2). The isolates were identified as P. gingivalis on the basis of having the typical colony color and
morphology, lacking colony autofluorescence, having positive trypsin-like enzyme activity (19), and having a positive
indole reaction.
Sample processing for PCR.
Two rapid methods of sample
processing for PCR were tested.
(i) Boiling.
A 30-µl aliquot of each of the 118 saliva
specimens was boiled for 10 min and then centrifuged at 10,000 × g for 5 min, and 5 µl (or 0.5 µl) of the supernatant was
used as a template for PCR.
(ii) Chelex 100 treatment.
A 50-µl aliquot of each of the
263 samples of saliva was incubated with 12.5 µl of 25% (wt/vol)
Chelex 100 (Bio-Rad Laboratories, Hercules, Calif.), a cation-chelating
resin, at 56°C for 30 min before being boiled and centrifuged as
described above. A 6-µl aliquot of the supernatant was then used as a
template for PCR.
PCR amplification.
Two P. gingivalis-specific
primers described by Slots et al. (21) were used to amplify
a 404-bp fragment of the 16S rRNA gene: primer 1 (5'-AGG CAG CTT
GCC ATA CTG CG-3') and primer 2 (5'-ACT GTT AGC AAC TAC CGA
TGT-3'). The specificity of the PCR method was investigated by
using purified DNA from 26 clinical P. gingivalis isolates
from unrelated subjects and that from 34 isolates of 30 species/genera
other than P. gingivalis as templates for PCR. The latter
included Porphyromonas asaccharolytica ATCC 25260, four
clinical isolates of P. asaccharolytica (AHN 1728, AHN
10916, AHN 10803, and AHN 10927), Porphyromonas endodontalis ATCC 35406, Porphyromonas macacae ATCC 33141, Porphyromonas salivosa NCTC 11632 (reclassified as P. macacae), Porphyromonas levii ATCC 29147, Porphyromonas canoris NCTC 12835, Porphyromonas
cangingivalis AHN 4138, Porphyromonas cansulci AHN
4364, Prevotella intermedia ATCC 25611, Prevotella
nigrescens ATCC 33563, Prevotella corporis ATCC 33547, Prevotella denticola AHN 9656, Prevotella
loescheii AHN 9806, Prevotella melaninogenica 25AA,
Prevotella oris ATCC 33573, Fusobacterium
nucleatum ATCC 25586, Fusobacterium naviforme AHN 9610, Selenomonas sp. AHN 9988, Capnocytophaga sp. AHN
10373, Bacteroides forsythus R878D, Bacteroides
gracilis AHN 9641, Campylobacter rectus ATCC 33238, Campylobacter concisus ATCC 33237, Eikenella corrodens AHN 9363, Veillonella sp. AHN 5836, Actinobacillus actinomycetemcomitans ATCC 29523, Haemophilus aphrophilus 1659, Peptostreptococcus
micros D3Ja, Streptococcus mutans 75.3, and
Streptococcus sobrinus 475.4.
Amplification reactions were performed in a total volume of 50 µl
consisting of 0.2 mM each deoxynucleoside triphosphate (dATP, dTTP,
dCTP, and dGTP; Pharmacia LKB, Piscataway, N.J.), 1.5 mM MgCl2, 10× Taq buffer (Perkin-Elmer), 1 µM
each primer, 2.5 U of AmpliTaq polymerase (Perkin-Elmer Cetus, Norwalk,
Conn.), and 0.5 to 6 µl of template overlaid with mineral oil. PCR
amplification was performed in a thermocycler (Perkin-Elmer Cetus).
Cycling parameters were as follows: an initial denaturation at 95°C
for 1 min; 36 cycles consisting of 95°C for 30 s, 65°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 2 min.
To avoid contamination during PCR amplification, the reagents were
premixed and sterile tips with aerosol barriers were used.
P. gingivalis ATCC 33277 cells (50 cells per PCR) were used as
a
positive control for the PCR, and 5 µl of water constituted
the
negative control. Positive and negative controls were included
in each
PCR set and in all sample processings.
The amplification products were subjected to electrophoresis in a 1%
agarose gel containing ethidium bromide (0.5 µg/ml) and
photographed
under UV illumination by using the Polaroid MP4 system.
A 1-kb DNA
ladder (Gibco BRL Life Technologies, Inc., Gaithersburg,
Md.) was used
as a molecular size standard.
PCR detection limits and inhibitory effect of saliva on PCR.
The detection limits of PCR after boiling and after Chelex 100 processing were determined by using known numbers of P. gingivalis ATCC 33277 cells (0, 1, 2, 5, 50, and 500 cells per PCR
as determined by viable-cell counts) suspended in sterile distilled
water or in saliva with no cultivable P. gingivalis.
To investigate the possible inhibition of PCR amplification by saliva,
P. gingivalis ATCC 33277 cells (50 cells per PCR) were
added
to 118 saliva samples processed by the boiling method. Inhibition
was
recorded when no or a very weak amplification signal was detected.
The
samples that showed inhibition when processed by boiling were
analyzed
additionally by spiking a 10-fold dilution of saliva
processed by
boiling, as well as by Chelex 100 processing of spiked
saliva.
| |
RESULTS |
Specificity of PCR.
Both the specificity and sensitivity of
the PCR primers were 100%. No amplification was detected for any of
the 34 isolates that were not P. gingivalis, whereas all 26 clinical P. gingivalis isolates gave an amplification
product of the expected size.
Comparison of bacterial culture and PCR techniques.
PCR, after
both boiling and Chelex 100 processing, was used on 118 salivary
samples that were also cultured for the presence of P. gingivalis (Table 1). P. gingivalis-positive samples were detected three times more often
by PCR than by bacterial culture: 11 of 118 (9.3%) samples were
P. gingivalis-positive by culture, whereas 40 of 118 (33.9%) samples were P. gingivalis-positive by PCR after
being boiled and 37 of 118 (31.4%) were positive by PCR after being
subjected to Chelex 100 processing. The PCR results obtained after
Chelex 100 processing of the sample correlated better with bacterial
culture than did those obtained after boiling, since all 11 culture-positive samples were also positive by PCR after Chelex 100 processing whereas 4 culture-positive samples failed to give
amplification products after being processed by boiling (Table 1).
There was some discrepancy between the results obtained after sample
boiling and those obtained after Chelex 100 processing
(Table
2). Of the 118 samples, 28 (23.7%) were
P. gingivalis-positive
and 69 (58.5%) were
P. gingivalis-negative by both methods, whereas
21 (17.8%) samples
repeatedly gave discrepant results.
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TABLE 2.
Comparison of two PCR sample processing methods used for
the detection of P. gingivalis in saliva samples from
118 subjects
|
|
Inhibitory effect of saliva on PCR.
Of the 118 saliva samples
processed by the boiling method, 23 (19.5%) showed a distinct
inhibition of PCR amplification when 50 P. gingivalis cells
were added to each 50-µl PCR mixture. Dilution of the saliva
decreased the inhibitory effect, since all but 3 of the 23 inhibitory
samples showed good amplification when 0.5 µl instead of 5 µl of
saliva (in both cases with 50 P. gingivalis cells) was used
as a template for PCR. Although dilution resulted in amplification of
most of the 23 salivary samples, the simultaneously occurring 10-fold
rise in the detection level is not desirable. When Chelex 100 was
applied to process the 23 samples that showed inhibition after boiling,
18 samples gave distinct amplicons and 5 samples gave weak amplicons.
The detection limit of the PCR after boiling and after Chelex 100 processing of the sample was one
P. gingivalis cell per
PCR
in water. The detection limit in saliva showing no PCR inhibition
was
also one cell per PCR. However, the amplification signal obtained
with
a few cells (1 to 5 cells per PCR) was constantly weaker
in saliva than
in water (Fig.
1).
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FIG. 1.
Detection limit of PCR after Chelex 100 processing.
Lanes 1 to 6, 500, 50, 5, 2, 1, and 0 P. gingivalis ATCC
33277 cells in saliva per PCR, respectively; lanes 7 to 12, 500, 50, 5, 2, 1, and 0 P. gingivalis cells in water per PCR,
respectively; M, 1-kb DNA ladder.
|
|
Occurrence of P. gingivalis.
Since Chelex 100 processing
of the saliva samples decreased PCR inhibition most effectively, it was
used to investigate the occurrence of P. gingivalis in 263 Finnish subjects from 5 to 80 years of age (Table
3). The saliva samples from 142 of these 263 subjects were also cultured for the presence of P. gingivalis (Table 3). The occurrence of P. gingivalis
increased with age, since the organism was detected by PCR in 3 (5.0%)
of the 60 subjects between 5 and 10 years of age, in 12 (13.8%) of the
87 subjects between 11 and 20 years of age, in 9 (13.4%) of the 67 subjects between 21 and 30 years of age, and in 31 (63.3%) of the 49 subjects between 31 and 80 years of age. P. gingivalis was
not found by bacterial culture in any of the 9 subjects between 5 and
10 years of age, whereas it was isolated from 3 (6.1%) of the 49 subjects between 11 and 20 years of age, from 1 (2.3%) of the 44 subjects between 21 and 30 years of age, and from 7 (17.5%) of the 40 subjects between 31 and 80 years of age (Table 3).
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TABLE 3.
Occurrence of P. gingivalis in Finnish
subjects with different periodontal statuses as determined by PCR after
Chelex 100 processing and by bacterial culture
|
|
Data on periodontal status were available for 254 subjects.
P. gingivalis was detected by PCR in 19 (10.3%) of the
185 periodontally
healthy subjects; in 6 (21%) of the 28 subjects with
prepubertal,
localized juvenile periodontitis or some other
type of early-onset
periodontitis; and in 28 (70%) of the 40 subjects
with adult periodontitis.
The corresponding figures obtained by
bacterial culture were 2
(2.6%) of 78, 1 (3.8%) of 26, and 7 (22.6%)
of 31 subjects (Table
3).
| |
DISCUSSION |
In the present study, 20% of the saliva samples showed PCR
inhibition when the boiling method was used for sample processing. PCR
inhibition of other biological samples (e.g., sputum, stool, and
genital ulcer specimens) is well known, and different methods (e.g.,
immunomagnetic separation, phenol-chloroform extraction, and use of
capture resins) have been suggested for the inactivation of PCR
inhibitors (2, 10, 12). However, many of these other methods
are laborious and expensive. In the present study, a simple sample-processing technique involving the use of Chelex 100 resin prior
to PCR amplification proved to be very applicable for the detection of
P. gingivalis in salivary samples. In the Chelex 100 processing method, only one reagent was added to the sample and the
processing was performed in a single tube, which minimizes the number
of steps involving handling and hence the risk of contamination. The
applicability of Chelex 100 for inactivating PCR inhibitors in saliva
probably stems from the ability of Chelex 100 to chelate divalent ions.
Chelex 100 has previously been shown to enhance the efficiency of DNA
extraction, especially from gram-positive and acid-fast bacteria, and
to protect DNA at high temperatures (6).
In the present study, some discrepancies in PCR detection of P. gingivalis from saliva were observed when the two sample
preparation methods were compared. Why some samples were P. gingivalis-positive by PCR after being subjected to Chelex 100 processing but P. gingivalis-negative by PCR after being
boiled can be explained by the better ability of the Chelex 100 method
to abolish PCR inhibition by saliva. The existence of samples that were
P. gingivalis-positive by bacterial culture but P. gingivalis-negative by PCR after being boiled supports this
suggestion. The reason why some samples were P. gingivalis-negative by PCR after being subjected to Chelex 100 processing but P. gingivalis-positive by PCR after being
boiled remained unknown. The results were reproducible, and the finding
can be explained neither by differences in the sensitivities of the two
methods nor by the uneven distribution of very low numbers of P. gingivalis cells in the samples.
In the present study, P. gingivalis was detected in saliva
samples three times more often by PCR than by bacterial culture, which
is well in accordance with data from earlier PCR studies using the same
primers for the detection of P. gingivalis in subgingival plaque samples (3, 21). Also, in other studies using primers targeted to different regions of the 16S rRNA gene or to the
collagenase gene, P. gingivalis has been detected in
subgingival samples more frequently by PCR than by bacterial culture
(21, 26). The higher rate of detection by PCR is most likely
due to the higher sensitivity of the PCR technique, which is especially
important in studies on mixed bacterial flora. In addition, in contrast to bacterial culture, PCR amplification also detects nonviable bacterial cells present in the sample. In all earlier studies in which
detection of P. gingivalis by PCR and detection of this bacterium by bacterial culture have been compared, the sample material
has been subgingival plaque. P. gingivalis has been cultured more often and in higher proportions from subgingival plaque than from
saliva (24, 25). However, due to the high detection limit for P. gingivalis in saliva, culture studies may
underestimate its prevalence in saliva samples.
In this study, the occurrence of P. gingivalis as determined
by PCR detection seemed to increase with age of the Finnish subjects. P. gingivalis was infrequently detected in samples from
children under 10 years of age (5%), and it was only rarely detected
in specimens from teenagers and young adults (13.4 to 13.8%), whereas most adults (63.3%) over 30 years of age harbored this bacterium. The
low rate of detection of P. gingivalis in children is in
accordance with a study by Ashimoto et al. (3) in which they
detected P. gingivalis by PCR in 14% of children aged
around 7 years. However, in a study by McClellan et al.
(14), P. gingivalis was detected by PCR in 37%
of subjects under 18 years of age, with similar frequencies
irrespective of age. Differences in the PCR methodologies are a likely
cause for the discrepant results, since the nested-PCR method used by
McClellan et al. (14) is more sensitive than the PCR methods
used in other studies. However, McClellan et al. did not report the
specificity of the nested-PCR method. In culture studies, P. gingivalis has not been isolated (7-9, 13), or has
been isolated only extremely rarely (4, 16, 17), from the
oral cavities of children and young adults, which coincides well with
the low isolation frequency of the present study. Similar to PCR
studies, hybridizations with DNA probes have revealed higher rates of
detection of P. gingivalis in the older age groups as well
as a positive correlation between detection of P. gingivalis and increasing age (20). However, the higher frequency of
detection of P. gingivalis with total chromosomal DNA probes
compared with that of bacterial culture may be explained in part by the
hybridization of these probes to other species, leading to
false-positive results (20, 23).
While P. gingivalis was rarely detected by PCR in saliva
from periodontally healthy subjects or from subjects with localized juvenile periodontitis in the present study, it was common in adult
periodontitis patients. Since the prevalence of periodontitis is very
low in children and adolescents but increases with age, being almost
ubiquitous in middle-aged individuals (5), it is difficult
to find a representative study population in which subjects of various
ages would have similar periodontal statuses. In the present study,
most of the subjects under 30 years of age were periodontally healthy
whereas older subjects commonly exhibited adult periodontitis. The
increased rates of detection of P. gingivalis in the older
age groups may be related to the differences in the periodontal
statuses of the subjects. Also, in previous PCR studies, P. gingivalis has rarely been detected in subjects without
periodontitis (3, 22) but has frequently been found in adult
periodontitis patients (3).
In conclusion, the present study shows the applicability of Chelex 100 processing of salivary samples for PCR amplification. P. gingivalis was rarely detected in saliva from periodontally healthy Finnish children and young adults. The prevalence of P. gingivalis increased with age, suggesting that oral colonization takes place mainly during adulthood.
| |
ACKNOWLEDGMENTS |
This work was supported by the Academy of Finland and by the Emil
Aaltonen Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Dentistry, Research Laboratory, P.O. Box 41, FIN-00014 University of
Helsinki, Finland. Phone: 358-9-19127316. Fax: 358-9-19127519.
| |
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Journal of Clinical Microbiology, January 1998, p. 157-160, Vol. 36, No. 1
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
