University of Groningen
Comparison of subgingival bacterial sampling with oral lavage for detection and quantification
of periodontal pathogens by real-time polymerase chain reaction
Boutaga, Khalil; Savelkoul, Paul H. M.; Winkel, Edwin G.; van Winkelhoff, Arie J.
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Journal of Periodontology
DOI:
10.1902/jop.2007.060078
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Citation for published version (APA):
Boutaga, K., Savelkoul, P. H. M., Winkel, E. G., & van Winkelhoff, A. J. (2007). Comparison of subgingival
bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time
polymerase chain reaction.
Journal of Periodontology
,
78
(1), 79-86.
https://doi.org/10.1902/jop.2007.060078
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Comparison of Subgingival Bacterial
Sampling With Oral Lavage for Detection
and Quantification of Periodontal
Pathogens by Real-Time Polymerase
Chain Reaction
Khalil Boutaga,* Paul H.M. Savelkoul,
†
Edwin G. Winkel,
‡§
and Arie J. van Winkelhoff*
Background: Saliva has been studied for the presence of subgingival pathogens in periodontitis patients.
With the anaerobic culture technique, the discrepancy between salivary recovery and subgingival presence
has been significant, which makes this approach not suitable for practical use in the microbial diagnosis of
periodontitis patients. The real-time polymerase chain reaction (PCR) technique represents a very sensitive
technique to detect and quantify bacterial pathogens. The aim of the study was to compare the presence and
numbers of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythensis,
Prevotella intermedia, and Micromonas micros in subgingival plaque and mouthwash samples by the anaer-
obic culture and real-time PCR techniques.
Methods: Pooled subgingival plaque samples and 10-ml mouthwash samples were collected from
21 adult patients with periodontitis and analyzed by quantitative anaerobic culture and real-time PCR for
A. actinomycetemcomitans, P. gingivalis, T. forsythensis, P. intermedia, and M. micros.
Results: The detection frequency of A. actinomycetemcomitans, P. gingivalis, and T. forsythensis in sub-
gingival plaque was identical by culture and real-time PCR and was higher for P. intermedia and M. micros by
real-time PCR. The highest detection frequencies for the target bacteria were found in mouthwash samples
by real-time PCR. The additional value of the real-time PCR to detect target bacteria was 38% for P. gingi-
valis, 73% for T. forsythensis, 77% for P. intermedia, and 71% for M. micros. The sensitivity to detect target
species in mouthwash by real-time PCR was 100% for all test species except for P. intermedia (93.8%).
Conclusions: Rapid detection and quantification of periodontal pathogens in mouthwash samples are
possible by real-time PCR. The procedure is significantly less time-consuming than subgingival sampling
with paper points. This approach to detect major periodontal pathogens in mouthwash samples may sim-
plify microbial diagnosis in periodontitis patients and may be used to monitor periodontal treatment. J Peri-
odontol 2007;78:79-86.
KEY WORDS
Dental plaque; mouthwash; periodontitis; polymerase chain reaction.
* Department of Oral Microbiology, Academic Center for Dentistry Amsterdam, University of Amsterdam and Vrije University, Amsterdam, The Netherlands.
† Department of Medical Microbiology and Infection Control, Vrije University Medical Center, Amsterdam, The Netherlands.
‡ Clinic for Periodontology and Implantology, Amsterdam, The Netherlands.
§ Academic Center of Oral Health, Department of Periodontology, University Medical Center Groningen, Groningen, The Netherlands.
doi: 10.1902/jop.2007.060078
J Periodontol • January 2007
79
B
acterial plaque is considered the principle
etiological factor in the onset and progression
of periodontitis.
1,2
Actinobacillus actinomyce-
temcomitans, Porphyromonas gingivalis, Tannerella
forsythensis, Prevotella intermedia, and Micromonas
micros are strong markers of periodontitis in
adults,
2,3
and these species have been linked to the
progression of the disease.
4
In the microbiological
diagnosis of periodontal diseases, subgingival plaque
is commonly used to detect and quantify bacterial
species. Curets, dental floss, and paper points have
been used to sample subgingival plaque. The tech-
nique used may influence the outcome of the mi-
crobiological analysis.
5,6
Also, the number of sample
sites is essential, especially when samples are
pooled.
7
Microbial subgingival sampling is time con-
suming and involves the selection of sampling sites,
isolation and drying of these sites, and sampling of
the subgingival area.
The use of saliva for diagnostic purposes has been
the subject of considerable research.
8
Saliva is easy to
obtain and contains bacteria from different oral sites
including oral mucosal sites and supra- and subgingi-
val plaque. Consequently, studies have evaluated the
presence and levels of bacteria in saliva in relation to
the periodontal status. Asikainen et al.
9
used the cul-
ture technique to investigate the presence of A. actino-
mycetemcomitans in stimulated and unstimulated
saliva samples and found these pathogens in 69.9%
and 35.9% of the samples, respectively, when A. acti-
nomycetemcomitans was detected subgingivally.
Umeda et al.
10
used the polymerase chain reaction
(PCR) technique to detect periodontal pathogens in
pooled subgingival plaque and whole saliva samples.
They found that whole saliva samples were more
frequently positive for P. gingivalis and Treponema
denticola than subgingival plaque samples, whereas
saliva samples underestimated the presence of A. ac-
tinomycetemcomitans and T. forsythensis compared
to subgingival samples.
Real-time PCR is a relatively new molecular tech-
nique to detect and quantify periodontal pathogens
and has been used for oral samples by several au-
thors.
11-17
The sensitivity of this technique allows
the detection of very small numbers of pathogens.
Salivary microbiological diagnosis provides a non-
invasive, inexpensive, and rapid technique for the de-
tection and quantification of periodontal pathogens.
We hypothesized that oral mouthwash samples
(oral lavage) can be used to detect and quantify sub-
gingival pathogens, providing a sensitive detection
technique. Therefore, the purpose of this study was
to compare the frequency of detection and the num-
ber of pathogens in subgingival plaque samples and
mouthwash samples by anaerobic culture and real-
time PCR techniques.
MATERIALS AND METHODS
Study Population
A study group of 21 adult patients with untreated
periodontitis was selected after visiting the Clinic for
Periodontology and Implantology Amsterdam. Pa-
tients had periodontal pockets >5 mm with clinical at-
tachment loss that showed bleeding upon pocket
probing in all four quadrants of the dentition as deter-
mined at the baseline visit. Patients had not used an-
tibiotics in the past 3 months. Subjects were verbally
informed about the purpose of the investigation. Pa-
tients were enrolled in the study from January to March
2005. Patients participated in the study on the basis of
informed consent. The study was approved by the
Medical Ethical Committee of the Vrije University
Amsterdam. Table 1 shows the demographic and
periodontal characteristics of the study subjects.
Sampling
At the second visit to the dental office (at least 2 weeks
after the baseline measurements), patients were
asked to rinse with 10 ml sterile saline for 30 seconds.
The deepest pocket in each quadrant of the dentition
was used as the subgingival sample site. The level of
supragingival plaque at these sites was evaluated at
the gingival margin by means of a periodontal probe
(2 = visible plaque; 1 = plaque on periodontal probe;
and 0 = no visible plaque on periodontal probe). Sam-
ple sites were isolated with cotton rolls, and supragin-
gival plaque was carefully removed with curets, and
the sites were air dried. A subgingival plaque sample
was taken by two sterile paper points that were con-
secutively inserted into the periodontal pocket and
removed after 10 seconds. All paper points were
transferred to a vial containing 1.5 ml reduced trans-
port medium.
18
Mouthwash samples and pooled sub-
gingival plaque samples were processed for anaerobic
microbiological analysis and real-time PCR within
4 hours.
Microbiological Procedures
Samples were vortexed for 2 minutes and split: 100 ml
was used to prepare 10-fold serial dilutions in sterile
phosphate buffered saline (PBS) and used for culture,
and 100 ml was stored at -20C and used for real-time
PCR testing within 3 weeks. For anaerobic culture,
100 ml of appropriate dilutions were plated on blood
agar plates that were supplemented with horse blood
(5% volume/volume [v/v]), hemin (5 mg/l), and men-
adione (1 mg/l) and incubated in 80% N
2
, 10% H
2
, and
10% CO
2
at 37C for up to 14 days. A. actinomycetem-
comitans was grown on trypticase-soy with serum,
bacitracin, and vancomycin trypticase (TSBV) plates
and incubated at 37C in air + 5% CO
2
for 3 days. Iden-
tification was done as described by Boutaga et al.
13i
i No. 2, Oxoid, Basingstoke, U.K.
Periodontal Pathogens in Oral Mouthwash Volume 78 • Number 1
80
The strains used in this study were as follows: P. gin-
givalis (W83), T. forsythensis (clinical isolate), A. ac-
tinomycetemcomitans (National Collection of Type
Cultures [NCTC] 9710), P. intermedia (American
Type Culture Collection [ATCC] 25611), and M. mi-
cros (clinical isolate). Reference strains were grown
as recommended by the ATCC. Determination of
the number of the total colony forming units (CFU)
per milliliter of the bacterial suspensions was per-
formed by growing the bacteria 2 to 3 days in brain-
heart infusion (BHI) supplemented with 5 mg/l hemin
and 1 mg/l menadione, and plating serial dilutions as
described by Boutaga et al.
13
Quantitative Real-Time PCR
DNA isolation from plaque samples, mouthwash,
and bacterial reference cultures. From plaque sam-
ples, mouthwash, and bacterial culture dilutions,
100 ml was used for automated DNA extraction and
purification with a DNA isolation kit.
¶
The protocol
included a 1-hour pretreatment with proteinase
K (20 mg/ml) at 56C and 10 minutes deactivation
at 100C. Afterwards, isolation DNA was eluted in
100 ml elution buffer.
Real-time PCR. The primer/probe sets and real-
time PCR conditions were performed as described
previously.
13
Briefly, real-time PCR amplification was
performed in a total reaction mixture volume of
25 ml. The reaction mixtures contained 12.5 ml2· uni-
versal PCR master mix (PCR buffer, deoxynucleoside
triphosphates, DNA polymerase,
#
reference signal
[6-carboxy-X-rhodamine], uracil N-glycosylase, and
MgCl
2
),** 300 to 900 nM pathogen-specific primer,
50 to 100 nM pathogen-specific probe, and 5 mlpurified
DNA from plaque samples. The samples were sub-
jected to an initial amplification cycle of 50Cfor2min-
utes and 95C for 10 minutes, followed by 45 cycles at
95C for 15 seconds and 60C for 1 minute. The data
were analyzed with sequence detection software.
††
For quantification, the results from unknown plaque
samples were projected on the counted pure-culture
standard curves of the target bacteria.
The possible inhibition of the real-time PCR was de-
termined by comparing the results of spiked amplifi-
cations to the original samples. Each sample was
spiked with 1,000 CFU Escherichia coli DH5a (i.e.,
50 CFU equivalents/PCR) before DNA isolation. In
addition, negative amplifications were spiked with
the target bacteria in numbers close to the threshold
line. The primers and probe used for amplification
of E. coli have been described by Huijsdens et al.
19
RESULTS
The prevalence of the target bacteria in subgingival
plaque and mouthwash samples based on the anaer-
obic culture and RT- PCR techniques is shown in
Figure 1.
Culture
Subgingival plaque samples were clearly more often
culture positive for P. gingivalis, T. forsythensis, P. in-
termedia, and M. micros than culture mouthwash
samples. The differences in prevalence ranged
between 23.8% for P. gingivalis to 57.1% for T. forsy-
thensis. A. actinomycetemcomitans was the least
frequently (<10%) recovered species with both
techniques from subgingival plaque. For A. actinomy-
cetemcomitans, mouthwash samples were more often
culture positive.
Real-Time PCR
Mouthwash samples were more often real-time PCR
positive than subgingival plaque samples for all target
species except for M. micros for which the prevalence
was 100% in both mouthwash and plaque samples. It
was striking that the prevalence of A. actinomycetem-
comitans (9%), P. gingivalis (33.3%), and T. forsy-
thensis (85.7%) in real-time PCR plaque-tested
samples was identical to culture plaque-tested sam-
ples . The frequency of detection of P. gingivalis in
mouthwash samples increased from 9.5% by culture
to 47.6% by real-time PCR. The frequency of detection
of T. forsythensis in mouthwash increased from 28.6%
by culture to 100% by real-time PCR. The prevalence
of P. intermedia in mouthwash showed a marked
difference: 4% by culture to 82% by real-time PCR.
M. micros was also more frequently detected in mouth-
wash by real-time PCR than by culture (76.2% versus
28.6%).
Ta b l e 1 .
Demographic Data and Mean (SD) Clinical
Parameters of Sampling Sites
Characteristics N Mean (SD) Range
Males 4 – –
Females 17 – –
Age (years) – 44.80 (9.10) 32-63
PI – 0.91 (0.62) 0-2
BOP – 1.68 (0.40) 0-2
PD (mm) – 6.48 (1.04) 4.75-8.5
Attachment loss (mm) – 4.45 (3.68) 0-8.5
– = not applicable; PI = plaque index; BOP = bleeding on probing; PD =
probing depth.
¶ MagNA Pure DNA Isolation Kit III, Roche Molecular Diagnostics, Almere,
The Netherlands.
# AmpliTaq Gold, Applied Biosystems, Foster City, CA.
** TaqMan, Applied Biosystems.
†† ABI 7000, Applied Biosystems.
J Periodontol • January 2007 Boutaga, Savelkoul, Winkel, van Winkelhoff
81
Numbers of Bacteria
The results of the quantification of tested bacterial
species in mouthwash and subgingival plaque sam-
ples for each method are summarized in Table 2. Sub-
gingival plaque samples yielded more bacterial cells
of each species than mouthwash samples by culture
and real-time PCR. The mean CFU counts of the tested
species by real-time PCR and culture did not differ
more than one log for plaque samples and between
two and four logs for mouthwash samples.
Figures 2 and 3 depict the prevalence per subject
of A. actinomycetemcomitans, P. gingivalis, T. forsy-
thensis, P. intermedia, and M. micros by real-time PCR
and culture, respectively. The results obtained by
real-time PCR in mouthwash samples matched the
results obtained in subgingival plaque samples for
90%, 86%, 86%, 86%, and 95% of the subjects infected
with A. actinomycetemcomitans, P. gingivalis, T. for-
sythensis, P. intermedia, and M. micros, respectively.
The results obtained by anaerobic culture in mouth-
wash samples matched the results obtained in subgin-
gival plaque samples for 95%, 76%, 33%, 57%, and
43% of the subjects infected with A. actinomycetemco-
mitans, P. gingivalis, T. forsythensis, P. intermedia,
and M. micros, respectively.
In Table 3, the calculated sensitivity and specificity
values are presented with the subgingival plaque
samples as reference criteria. With real-time PCR,
the sensitivity was 100% to detect P. gingivalis, T. for-
sythensis, and M. micros in the mouthwash and 93.8%
to detect P. intermedia. For culture, the sensitivity to
detect target bacteria in mouthwash when detected
subgingivally was <35% except for A. actinomycetem-
comitans, which was 100%. The specificity was also
calculated and varied between 0% and 89.5% by
real-time PCR and between 66.7% and 100% by an-
aerobic culture.
Finally, a linear correlation calculated on the basis
of the quantitative results for mouthwash and subgin-
gival plaque samples by real-time PCR (R
2
= 0.202 to
0.601) and anaerobic culture (R
2
= 0.262 to 0.554)
showed that there is a reasonably good correlation
between sample types at the quantitative level.
DISCUSSION
The presence and numbers of periodontal pathogens
in plaque and saliva samples have been studied using
culture, immunoassays, DNA probes, and PCR tech-
niques.
16,20-24
These methods have limited specificity
and sensitivity. Real-time PCR, with species-specific
primers and probe, represents a specific, sensitive,
and quantitative tool to study periodontal pathogens
more accurately and is able to detect as little as a
Table 2.
Number of Bacterial Cells per Milliliter for Each Species Tested by Anaerobic Culture
and Real-Time PCR
Anaerobic Culture and Real-Time
PCR Detected
Mean CFU Count
Culture Real-Time PCR Culture Real-Time PCR
Subgingival Mouthwash
A. actinomycetemcomitans 3.60E+03 2.98E+04 1.21E+03 1.81E+03
P. gingivalis 1.37E+06 4.25E+05 7.14E+04 6.50E+04
T. forsythensis 1.70E+06 2.60E+07 7.95E+04 1.43E+06
P. intermedia 2.14E+06 3.30E+06 1.43E+02 2.59E+06
M. micros 2.99E+06 2.29E+07 6.57E+04 8.67E+06
Figure 1.
Prevalence (%) of A. actinomycetemcomitans (Aa), P. gingivalis
(Pg), T. forsythensis (Tf), P. intermedia (Pi), and M. micros (Mm)in
subgingival plaque samples and mouthwash by anaerobic culture
and real-time PCR.
Periodontal Pathogens in Oral Mouthwash Volume 78 • Number 1
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