J Clin Periodontol 2000; 27: 722–732 Copyright C Munksgaard 2000

Printed in Denmark . All rights reserved

ISSN 0303-6979

Microbial composition of supra- Laurie Ann Ximénez-Fyvie,

Anne D. Haffajee and
Sigmund S. Socransky

and subgingival plaque in Department of Periodontology, The Forsyth

Institute, Boston, MA, USA

subjects with adult periodontitis

Ximénez-Fyvie LA, Haffajee AD, Socransky SS: Microbial composition of supra-
and subgingival plaque in subjects with adult periodontitis. J Clin Periodontol
2000; 27: 722–732. C Munksgaard, 2000.

Abstract
Background, aims: The purpose of the present study was to compare and relate
the microbial composition of supra and subgingival plaque in 23 adult peri-
odontitis subjects (mean age 51∫14 years).
Methods: A total of 1,170 samples of supra and subgingival plaque were collected
from the mesial aspect of every tooth (up to 28 supra and 28 subgingival samples)
from each subject and evaluated for the presence and levels of 40 bacterial taxa
using whole genomic DNA probes and checkerboard DNA-DNA hybridization.
Clinical assessments including dichotomous measures of gingival redness,
bleeding on probing, plaque accumulation and suppuration, as well as duplicate
measures of pocket depth and attachment level, were made at 6 sites per tooth.
The counts (levels), % DNA probe count (proportion) and % of sites colonized
(prevalence) of each species in supra and separately in subgingival plaque were
computed for each subject. Significance of differences between supra and sub-
gingival plaque for each species was sought using the Wilcoxon signed ranks test
and adjusted for multiple comparisons.
Results: All 40 taxa were detected in both supra and subgingival plaque. Acti-
nomyces species were the most prevalent taxa in both habitats. 75 to 100% of
supra and 62 to 100% of subgingival sites were colonized by at least one of the
5 Actinomyces species. Supragingival samples exhibited significantly higher
counts of Actinomyces naeslundii genospecies 1, Actinomyces israelii, Actinomyces
odontolyticus, Neisseria mucosa, Streptococcus gordonii, Capnocytophaga ochracea
and Capnocytophaga sputigena when compared with mean counts in subgingival
samples taken from the same tooth surfaces. Subgingival plaque samples pre-
sented significantly higher counts of Prevotella nigrescens, Prevotella intermedia,
Bacteroides forsythus and Porphyromonas gingivalis. Subgingival samples exhibited
a significantly higher proportion of ‘‘red’’ and ‘‘orange complex’’ species, while
supragingival plaque exhibited higher proportions of ‘‘green’’ and ‘‘purple’’ com-
plex species as well as Actinomyces species. Suspected periodontal pathogens
could be detected in supragingival plaque from sites where subgingival samples
were negative for the same species. Key words: supragingival plaque; subgingival
plaque; microbiology; bacteria periodontal
Conclusions: The data indicate that supragingival plaque can harbor putative diseases; DNA probes.
periodontal pathogens, suggesting a possible rôle of this environment as a reservoir
of such species for the spread or reinfection of subgingival sites. Accepted for publication 3 November 1999

To date, few comprehensive studies of plaque composition on the same tooth in the initiation of periodontal infec-
the microbial composition of supragin- surfaces. In reviewing the literature, the tions (Moore & Moore 1994, Haf-
gival plaque have been carried out. majority of recent studies have focused fajee & Socransky 1994, Zambon 1996).
Further, there appears to be no data primarily on the composition of sub- The role of supragingival plaque in oral
which clearly describe the relationship gingival plaque suggesting a role for a microbial ecology and the initiation of
between supragingival and subgingival number of subgingival microorganisms periodontal diseases is less clear.
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Supra- and subgingival microbiota 723

Moore et al. (1983), examined 22 su- cytophaga species were higher in supra-
Material and Methods
pra and 38 subgingival plaque samples gingival samples.
Subject population
taken from 22 subjects with moderate Several investigators have sought spe-
periodontitis. It was found that the taxa cific ‘‘subgingival’’ species in supragin- 23 adult periodontitis subjects ranging
which predominated in supragingival gival plaque. Riviere et al. (1992), re- in age from 24–82 years were selected for
plaque included 4 species of Streptococ- ported that spirochetes could be ob- the study. All subjects had at least 20
cus, 9 species of Actinomyces, 3 species served in both supra and subgingival teeth and at least 8 sites with pocket
of Capnocytophaga, Veillonella parvula, plaque from periodontitis subjects with depth greater than 4 mm and/or more
Leptotrichia buccalis, 2 species of Selen- pathogen-related oral spirochetes than 10% of sites with loss of attachment
omonas and Rothia dentocariosa. In (PROS) predominating in both supra greater than 3 mm. Exclusion criteria in-
contrast, 3 Fusobacterium species, 2 and subgingival plaque samples. Other cluded pregnancy, lactation, periodontal
Peptostreptococcus species, 7 Eubacteri- investigators have also observed spiro- or antibiotic therapy in the previous 3
um species, Campylobacter rectus, Por- chetes in both supra and subgingival months, any systemic condition which
phyromonas gingivalis and a number of samples (Armitage et al. 1982, Lindhe could influence the course of peri-
Prevotella species were found to be the et al. 1980, Listgarten & Hellden 1978, odontal disease, or which would require
predominant taxa in subgingival Moore et al. 1982ab, Simonson et al. pre-medication for monitoring pro-
plaque. 1988). Gmür & Guggenheim (1994) cedures, current smokers and subjects
Using predominant cultivable tech- analyzed 168 interdental supragingival with localized juvenile periodontitis,
niques, Zee et al. (1996) examined the plaque samples for the presence of rapidly progressive periodontitis or
composition of 44 supragingival plaque Actinobacillus actinomycetemcomitans, acute necrotizing ulcerative gingivitis.
samples taken from 11 subjects at 4 Bacteroides forsythus, C. rectus, P. gin-
time points. During 14 days of plaque givalis and P. intermedia/Prevotella nig-
Clinical monitoring
accumulation, supragingival plaque rescens using monoclonal antibodies.
shifted from a microbiota predomi- They detected all test species, except P. Subjects were screened for suitability
nated by Gram-positive cocci, primarily gingivalis, in a high percentage of sites and if accepted, signed informed con-
Streptococcus species, to one predomi- (A. actinomycetemcomitans 33%, B. for- sent. Clinical measurements were taken
nated by Gram-positive and negative sythus 38%, C. rectus 38%, P. interme- at 6 sites per tooth (mesiobuccal, buc-
rods including Actinomyces, Fusobac- dia/P. nigrescens 100%), suggesting that cal, distobuccal, distolingual, lingual
terium, Veillonella and Capnocytophaga supragingival plaque may be a reservoir and mesiolingual) at all teeth excluding
species. At day 1 of plaque accumu- for such organisms. third molars (a maximum of 168 sites
lation, the most frequently isolated or- Thus, available data suggest that per subject) as previously described
ganisms included Staphylococcus epid- similar species may be found in supra- (Haffajee et al. 1983). Clinical assess-
ermidis, Veillonella dispar, Actinomyces gingival plaque to those described in ment included plaque accumulation (0/
israelii, Propionibacterium granulosum, subgingival plaque. However, the data 1), overt gingivitis (0/1), bleeding on
Gemella morbillorum, Streptococcus are limited in terms of the number of probing (0/1), suppuration (0/1), prob-
mitis and Streptococcus sanguis. At 14 species examined and the number of ing pocket depth and probing attach-
days, samples were predominated by samples and subjects evaluated. In ad- ment level. Pocket depth and attach-
multiple species including Prevotella in- dition, few studies have attempted to ment level measurements were taken
termedia, Fusobacterium and Capnocy- compare and relate supra and subgingi- twice by the same examiner and the av-
tophaga species. val plaque samples taken from the same erage of the pair of measurements was
Cao et al. (1990) used microscopy tooth surface. For these reasons, the used for analysis. Measurements were
and cultural techniques to compare the purpose of the present study was to recorded to the nearest millimeter using
microbiota of 10 supra and 10 subgingi- compare and relate the microbial com- a North Carolina periodontal probe
val plaque samples from the maxillary position of supra and subgingival (Hu-Friedy, Chicago, IL). The baseline
first molars of 10 Chinese subjects. plaque samples obtained from subjects clinical features of the 23 subjects are
Comparisons were made with data with adult periodontitis. presented in Table 1.
from 10 Caucasian subjects examined
in previous studies. The cultivable
supragingival microbiota of the Cauca-
sian subjects was predominated by Acti-
nomyces and Streptococcus species,
Table 1. Baseline clinical characteristics of the subject group (nΩ23)
while Fusobacterium and ‘‘black-pig-
mented Bacteroides’’ were the most pre- Mean(∫SD) Range
dominant organisms in the Chinese age (years) 51∫11 24–82
subjects. These subjects also exhibited no. missing teeth 2.5∫2.3 0ª7
significantly higher percentages of % males 61
spirochetes and motile rods in supra- mean pocket depth (mm) 2.8∫0.4 2.2∫3.6
gingival plaque than Caucasian sub- mean attachment level (mm) 2.7∫0.8 1.5∫4.3
jects. ‘‘Black-pigmented Bacteroides’’, % sites with:
Actinomyces and Streptococcus species plaque 70∫24 5ª100
were similar in both supra and subgin- gingival erythema 56∫33 0ª100
gival plaque samples of the Chinese bleeding on probing 29∫19 4ª76
suppuration 0.5∫1.2 0ª4.6
subjects, but Fusobacterium and Capno-
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
724 Ximénez-Fyvie et al.

spermine tetrahydrochloride, 20 mg/ml ances at 260 nm and 280 nm. Whole-

Microbial assessment
Na isobutyrate, 1 mg/ml L-cysteine, 5 genomic DNA probes were prepared
Bacterial strains and growth conditions mg/ml thiamine pyrophosphate and from each of the 40 test species by label-
The 40 bacterial strains used in the 0.5% bovine serum. ing 1 mg of DNA with digoxigenin
preparation of DNA probes are pre- (Boehringer Mannheim, Indianapolis,
sented in Table 2. All strains were pur- DNA isolation and preparation of DNA IN) using a random primer technique
chased as lyophilized stocks from the probes (Feinberg & Vogelstein 1983).
American Type Culture Collection Bacterial strains were grown anaer-
(ATCC, Rockville, MD) (exceptions are obically on the surface of blood agar Sample collection and DNA-DNA hy-
noted in the Table). Bacterial stocks plates (except the 2 spirochetes which bridization
were rehydrated in Mycoplasma broth were grown in broth) for 3 to 7 days. Samples of supra and subgingival
(Difco Laboratories, Detroit, MI) and The growth was harvested and placed plaque were individually analyzed by
grown on Trypticase soy agar with 5% in 1.5 ml microcentrifuge tubes contain- checkerboard DNA-DNA hybridiza-
defibrinated sheep blood (BBL, Balti- ing 1 ml of TE buffer (10 mM Tris-HCl, tion (Socransky et al. 1994). After dry-
more Biological Laboratories, Cockeys- 0.1 mM EDTA, pH 7.6). Cells were ing and isolation with cotton rolls,
ville, MD) at 35 æC under anaerobic washed 2¿ by centrifugation in TE buf- supragingival plaque was sampled from
conditions (80% N2, 10% CO2, 10% fer at 3,500 rpm for 10 min. The cells the mesial-buccal aspect of each tooth
H2). Several bacterial strains were were resuspended and lysed either with using sterile Gracey curettes. Each
grown on supplemented or enriched 10% SDS and Proteinase K (20 mg/ml) plaque sample was placed in a separate
media: B. forsythus was grown on Tryp- (Sigma) for Gram-negative strains or in tube containing 150 ml of TE buffer (pH
ticase soy agar supplemented with 5% 150 ml of an enzyme mixture containing 7.6). After removal of the supragingival
defibrinated sheep blood and 10 mg/ml 15 mg/ml lysozyme (Sigma) and 5 mg/ sample and any remaining supragin-
N-acetyl muramic acid (Sigma Chemi- ml achromopeptidase (Sigma) in TE gival plaque, subgingival plaque
cal Co., St. Louis, MO). P. gingivalis buffer (pH 8.0) for Gram-positive samples were taken from the same sites
was grown on a similar medium supple- strains. The pelleted cells were resus- (i.e., the mesial-buccal aspect of each
mented with 5% defibrinated sheep pended by 15 s sonication and incu- tooth) using sterile Gracey curettes and
blood, 0.3 mg/ml menadione (Sigma) bated at 37 æC for 1 h. DNA was iso- placed in a second set of individual
and 5 mg/ml hemin (Sigma). Eubacteri- lated and purified using the method de- tubes. 100 ml of 0.5 M NaOH were
um and Neisseria species were grown on scribed by Smith et al. (1989). The added to each tube and the samples
Fastidious Anaerobic Agar (BBL) with concentration of the purified DNA was were dispersed using a vortex mixer.
5% defibrinated sheep blood. Trepone- determined by spectrophotometric The samples were boiled for 10 min and
ma denticola and Treponema socranskii measurement of the absorbance at 260 neutralized using 800 ml of 5 M am-
were grown in Mycoplasma broth nm. The purity of the preparations was monium acetate. The released DNA
supplemented with 1 mg/ml glucose, assessed by the ratio of DNA to protein was then placed into the extended slots
400 mg/ml niacinamide, 150 mg/ml as measured by the ratio of the absorb- of a Minislot-30 apparatus (Im-

Table 2. Stains employed for the development of DNA probes

Actinomyces species ‘‘Orange’’ complex Other species and new DNA probes
Actinomyces gerencseriae 23840 Campylobacter gracilis 33236 Eubacterium sabureum 33271
Actinomyces israelii 12102 Campylobacter rectus 33238 Gemella morbillorum 27824
Actinomyces naeslundii genospecies 1 12104 Campylobacter showae 51146 Leptotrichia buccalis 14201
Actinomyces naeslundii genospecies 2 43146 Eubacterium nodatum 33099 Neisseria mucosa 19696
Fusobacterium nucleatum ss nucleatum 25586 Prevotella melaninogenica 25845
‘‘Purple’’ complex Fusobacterium nucleatum ss polymorphum 10953 Propionibacterium acnes **
Actinomyces odontolyticus 17929 Fusobacterium nucleatum ss vincentii 49256 Selenomonas noxia 43541
Veillonella parvula 10790 Fusobacterium periodonticum 33693 Streptococcus anginosus 33397
Peptostreptococcus micros 33270 Treponema socranskii S1
‘‘Yellow’’ complex Prevotella intermedia 25611
Streptococcus gordonii 10558 Prevotella nigrescens 33563
Streptococcus intermedius 27335 Streptococcus constellatus 27823
Streptococcus mitis 49456
Streptococcus oralis 35037 ‘‘Red’’ complex
Streptococcus sanguis 10556 Bacteroides forsythus 43037
Porphyromonas gingivalis 33277
‘‘Green’’ complex Treponema denticola B1
Actinobacillus actinomycetemcomitans *
Capnocytophaga gingivalis 33624
Capnocytophaga ochracea 33596
Capnocytophaga sputigena 33612
Eikenella corrodens 23834
All strains were obtained from the American Type Culture Collection (ATCC) except Treponema denticola B1 and Treponema socranskii S1
which were obtained from Forsyth Dental Center. Microbial ‘‘complexes’’ were described by Socransky et al. (1998). *ATCC strains 43718
and 29523; **ATCC strains 11827 and 11828.
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Supra- and subgingival microbiota 725

munetics, Cambridge MA), concen- for each pocket depth category within a
Results
trated onto a 15¿15 cm positively- subject and averaged across subjects in
Mean counts
charged nylon membrane (Boehringer the 3 categories for supra and subgingi-
Mannheim, Indianapolis, IN) and fixed val plaque samples separately. The sig- The mean total count (¿105∫SEM)
to the membrane by cross-linking under nificance of differences between counts was somewhat higher in supragingival
ultraviolet light followed by baking at or proportions of bacterial species in plaque samples than subgingival plaque
120 æC for 20 min. Two lanes on each supra, and separately, subgingival samples; 133∫18 and 100∫18 respec-
membrane had standards that consisted plaque samples for each pocket depth tively. The difference was not statisti-
of a mixture at 105 and 106 cells of each category was tested using the Quade cally significant. The mean counts
bacterial species tested. The membranes test (Conover 1980). The significance of (¿105∫SEM) of the 40 individual spe-
were prehybridized, then hybridized in differences between counts of the same cies in supra and subgingival plaque
the checkerboard format and signals microbial species in supra and subgingi- samples are presented in Fig. 1. Supra-
detected using chemifluorescence and a val plaque samples in the 3 pocket gingival samples exhibited significantly
fluorimager as described previously depth categories was tested using the higher counts of Actinomyces naeslundii
(Feres et al. 1999). Wilcoxon signed ranks test. Adjust- genospecies 1, A. israelii, Actinomyces
ments were made for multiple compari- odontolyticus, Neisseria mucosa, Strep-
sons as described above. tococcus gordonii, Capnocytophaga och-
Data analysis

Microbiological data available for each

subject were the counts of each of the
40 test species from up to 28 supragin-
gival and, separately, up to 28 subgingi-
val plaque samples per subject. The
analyses compared microbial data ex-
pressed in 3 ways: counts (levels), %
DNA probe count (proportions) and
prevalence (% of sites colonized). In or-
der to compare the counts of each of
the bacterial species, the data were ex-
pressed as counts ¿105 at each site, av-
eraged within a subject and then aver-
aged across subjects. In a similar
fashion, the % DNA probe count and
prevalence of each species were com-
puted at each site, averaged across sites
within each subject and then across
subjects. Significance of differences be-
tween supra and subgingival plaque for
each species was sought using the
Wilcoxon signed ranks test. Adjust-
ments were made for multiple compari-
sons as described by Socransky et al.
(1991).
To examine the associations between
individual species in supragingival and
subgingival plaque samples on the same
tooth surface, 2¿2 contingency tables
were constructed for each species that
summarized the presence or absence of
that species at all sampled sites for the
23 subjects. Because there were multiple
sites examined in each subject, the sig-
nificance of the associations was exam-
ined using Mantel-Haenszel procedures
in which each subject was a stratum in
the analysis.
To examine the relationship between
microbial species in supra and subgingi-
Fig. 1. Bi-lateral bar chart of the mean counts (¿105∫SEM) of individual species in supra
val plaque samples and pocket depth, and subgingival plaque samples from 23 periodontitis subjects. The mean count was computed
sampled sites were subset at the quar- for each species in supra and subgingival plaque for each subject, and then averaged across
tiles into categories of ⬍3, 3–4, and ⬎4 subjects. The data are ordered on the basis of mean counts in the supragingival plaque
mm pocket depth. Counts and pro- samples. The significance of differences between supra and subgingival counts was tested using
portions of each species were averaged the Wilcoxon signed ranks test. *p⬍0.05, **p⬍0.01 after adjusting for multiple comparisons.
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
726 Ximénez-Fyvie et al.

racea and Capnocytophaga sputigena

when compared with mean counts in
subgingival samples taken from the
same tooth surfaces. Subgingival
plaque samples presented significantly
higher counts of P. nigrescens, P. inter-
media, B. forsythus and P. gingivalis.
None of the species that were detected
in significantly higher counts in supra-
gingival plaque samples were species
thought to be periodontal pathogens.

Mean % DNA probe count

The mean proportions that each species

comprised in the supra and subgingival
plaque samples are presented in Fig. 2.
A. naeslundii genospecies 1, N. mucosa
and A. odontolyticus were in signifi-
cantly greater proportions in supragin-
gival plaque while P. nigrescens and B.
forsythus were at higher proportions in
the subgingival samples. The 40 test
species were grouped into microbial
complexes (Table 2) as described by
Socransky et al. (1998) (Fig. 3). Supra
and subgingival plaque differed primar-
ily in that subgingival samples exhibited
a significantly higher proportion of
‘‘red’’ and ‘‘orange’’ complex species,
while supragingival plaque exhibited
higher proportions of ‘‘green’’ and
‘‘purple’’ complex species as well as Ac-
tinomyces species.

Fig. 2. Bi-lateral bar chart of the mean % DNA probe count (∫SEM) comprised by each Mean % of sites colonized
species in supra and subgingival plaque samples from 23 periodontitis subjects. The % DNA
probe count was computed for each species in each plaque sample, averaged within a subject
Fig. 4 presents the mean prevalence
and then averaged across subjects for the supra and subgingival plaque samples separately. (mean % of sites colonized) of the 40
The data are ordered on the basis of the proportions in the supragingival plaque samples. species in supra and subgingival plaque
The significance of differences between supra and subgingival percentages was tested using samples. All species were detected in
the Wilcoxon signed ranks test. *p⬍0.05, **p⬍0.01 after adjusting for multiple comparisons. both habitats. Actinomyces species such

Fig. 3. Pie charts of the mean % DNA probe

count of microbial groups in supra and sub-
gingival plaque samples from 23 peri-
odontitis subjects. The species were grouped
into the 7 microbial groups presented in
Table 2 based on the description of Socran-
sky et al. (1998). The areas of the pies were
adjusted to reflect the mean total DNA probe
count at the 2 sample locations. The signifi-
cance of differences in mean percentages of
the supra and subgingival complexes was
tested using the Wilcoxon signed ranks test.
The ‘‘red’’, ‘‘orange’’, ‘‘green’’, ‘‘purple’’ and
Actinomyces complexes differed at p⬍0.05
after adjusting for 7 comparisons.
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Supra- and subgingival microbiota 727

as A. naeslundii genospecies 1 and 2,

Actinomyces gerencseriae and A. israelii
were the most prevalent taxa in both su-
pra and subgingival plaque. In individ-
ual subjects, 75–100% of supragingival
samples and 62–100% of subgingival
samples harbored at least one of the 5
Actinomyces species. Ten species includ-
ing 3 species each of Actinomyces,
Streptococcus and Capnocytophaga as
well as N. mucosa were significantly
more prevalent in supragingival plaque.
No species was significantly more
prevalent in the subgingival plaque
samples.

Relationship of species in supra and

subgingival plaque samples

Fig. 5 presents images of sections of 2

‘‘checkerboard’’ membranes with supra
and subgingival plaque samples taken
from the same sites in one subject. Ex-
amination of supra and subgingival
plaque samples from the same tooth
surface revealed that a given species
could be detected in supra or subgingi-
val plaque only, in both or in neither.
The frequency of detection of each spe-
cies in supra and subgingival plaque
samples was examined using 2¿2 con-
tingency tables. Table 3 is an example
of data for P. gingivalis, A. naeslundii
genospecies 2, P. intermedia and V. par-
vula. Data for all 40 species are sum-
Fig. 4. Bi-lateral bar chart of the mean prevalence (% of sites colonized,∫SEM) of individual marized in Fig. 6. While the presence or
species in supra and subgingival plaque samples from 23 periodontitis subjects. The prevalence absence of many species in supragin-
was computed for each species in supra and subgingival plaque individually for each subject gival samples was significantly associ-
and then averaged across subjects. The data are ordered on the basis of the prevalence in the ated with their presence or absence in
supragingival plaque samples. The significance of differences between supra and subgingival subgingival samples (Table 3, Fig. 6),
mean values was tested using the Wilcoxon signed ranks test. *p⬍0.05, **p∞0.01 after ad- the association was not significant for
justing for multiple comparisons. many species including A. actinomyce-
temcomitans, B. forsythus and T. dentic-
ola. Species were frequently detected in
supragingival plaque or subgingival
plaque samples only.
Fig. 7 presents the ratio of the mean
counts for each species in supragingival
plaque compared to subgingival plaque
in the 23 periodontitis subjects. The
circles to the left of the mid-line indi-
cate species whose mean counts were
higher in supragingival than subgingi-
val plaque while species to the right of
the mid-line had higher mean counts in
sub than supragingival plaque. For ex-
ample, mean counts (¿105) for Capno-
cytophaga gingivalis were 3.59 and 0.75
Fig. 5. Checkerboard DNA-DNA hybridization membranes showing reactions of 40 DNA
in supra and subgingival plaque
probes with14 supra and 14 subgingival plaque samples from a single periodontitis subject. samples respectively providing a ratio
Mixed standards at 105 and 106 cells are shown in the last 2 lanes of each membrane. Signals of 4.79. Similarly, mean counts (¿105)
for 6 putative periodontal pathogens are indicated according to the detection of each species for B. forsythus were 1.69 and 0.43 in
in either supra or subgingival plaque samples only, or in both. sub and supragingival samples respec-
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
728 Ximénez-Fyvie et al.

Fig. 6. Stacked bar chart of the % of sites at which each species was
detected in either supra or subgingival plaque samples, both or nei-
ther. Plaque samples were taken from the same tooth surface at 585
sites in 23 subjects with adult periodontitis. 2¿2 contingency tables Fig. 7. Plot of the ratios of mean counts of each species in supra and
were prepared for each species that indicated for each surface whether subgingival plaque samples. The counts of each species were averaged
the species was detected at supragingival, subgingival, both or neither in supra and subgingival plaque samples separately and then averaged
(Table 3). The values were converted to percents prior to plotting. across subjects. The circles to the left of the mid-line represent the
The species are ordered on the basis of the % of sites at which a ratios for species which were found at higher levels in the supragin-
species was detected in both supra and subgingival samples. Mantel- gival samples while circles to the right of the mid-line represent ratios
Haenszel adjusted odds ratios are presented to the left of the bars. for species found at higher levels in subgingival samples.
Mantel-Haenszel p values were adjusted for multiple comparisons.
*p⬍0.05, **p⬍0.01, ***p⬍0.001.

tively providing a ratio of 3.93. These

data suggest that species such as B. for-
sythus, Eubacterium nodatum and P.
gingivalis achieved higher levels in the
subgingival environment in adult peri-
odontitis subjects while C. gingivalis, C.
sputigena, S. gordonii and A. odontolyt-
icus achieved higher levels in the supra-
gingival plaque.

Relationship of microbial composition of

supra and subgingival plaque to pocket
depth

Fig. 8 presents mean counts of each

bacterial species tested in supra and
subgingival plaque samples in pockets
⬍3, 3–4 and ⬎4 mm. Mean counts were
generally high at sites with greater
pocket depth for both the supragingival Fig. 8. Bi-lateral bar charts of the mean counts (x 105,∫SEM) of individual species in supra
and subgingival samples. After ad- and subgingival plaque samples from 23 periodontitis subjects at sites with pocket depths ⬍3,
justing for multiple comparisons, only 3–4 and ⬎4 mm. The mean count was computed for each species in supra and subgingival
T. socranskii and Eubacterium saburre- plaque in each pocket depth category for each subject, and then averaged across subjects in
um were significantly related to in- the 3 pocket depth groups. The data are ordered on the basis of mean counts in the supragin-
gival plaque samples from sites with pocket depths ⬍3 mm. The significance of differences
creased pocket depth in supragingival
between supra and subgingival counts in each pocket depth category was tested using the
plaque. In contrast, mean counts of 17 Wilcoxon signed ranks test. .p⬍0.05, ..p⬍0.01 after adjusting for multiple comparisons. The
species were significantly higher at significance of differences of mean counts of each species in supra and separately subgingival
deeper periodontal pockets for the sub- plaque samples among pocket depth categories was tested using the Quade test. Supragingival
gingival plaque samples. In particular, plaque: ππp⬍0.01; Subgingival plaque: *p⬍0.05, **p⬍0.01, ***p⬍0.001 after adjusting for
members of the ‘‘red’’ and ‘‘orange’’ multiple comparisons.
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Supra- and subgingival microbiota 729

One unique aspect of this study was the

sampling of supra and subgingival
plaque from the same tooth surface per-
mitting the direct comparison of species
at the same tooth site.
Another important aspect of the cur-
rent investigation was the use of 3
methods of data evaluation in order to
describe more comprehensively the
composition of supra and subgingival
plaque. The prevalence or % of sites
colonized provided an estimate of the
extent of colonization of the individual
species. Counts provided the level of
colonization of each species at individ-
ual sites while the % DNA probe count
indicated the proportion that each spe-
cies comprised of the total DNA probe
count in each sample. Each method
provided different information. While
Fig. 9. Pie charts of the mean % DNA probe count of microbial groups in supra and subgingi- there was some relatedness among these
val plaque samples from 23 periodontitis subjects at sampled sites subset at the quartiles into measures, detection of a species at a
pocket depth catgories of ⬍3, 3–4 and ⬎4 mm. The species were grouped into the 7 microbial large proportion of sites did not necess-
groups presented in Table 2 based on the description of Socransky et al. (1998). The areas of arily imply that this species was present
the pies were adjusted to reflect the mean total DNA probe count at each sample location.
in high numbers or represented a large
The significance of differences in mean percentages of the supra and subgingival complexes
was tested using the Wilcoxon signed ranks test for each pocket depth category. Differences
proportion of the total count. For ex-
between supra and subgingival plaque samples for individual complexes in each pocket depth ample, A. israelii was found at a large
category are indicated as follows: *p⬍0.05, **p⬍0.01 after adjusting for 7 comparisons. Only proportion of both supra and subgingi-
the ‘‘orange’’ complex differed significantly among pocket depth categories both for supra val sites; however, levels of this species
and subgingival samples, while the Actinomyces differed among pocket depth categories for were significantly higher in the supra
the subgingival plaque samples. than the subgingival plaque samples. In
contrast, B. forsythus was found less
commonly both supra and subgingi-
complexes, including Fusobacterium deep pockets. Actinomyces species were vally but counts were significantly
species, P. intermedia, P. nigrescens, C. significantly lower in the subgingival higher in subgingival plaque samples.
rectus, B. forsythus, P. gingivalis and T. samples of the deep sites. At shallow One of the most striking findings of
denticola, were detected at higher num- sites, ‘‘green’’ complex species were in the current investigation was the pre-
bers at pockets ⭓3 mm. At sampled higher mean proportions in supragin- dominance of the Actinomyces species
sites ⬍ 3 mm, 6 species were signifi- gival samples, whereas, ‘‘orange’’ and in both supra and subgingival plaque
cantly higher in supragingival samples ‘‘red’’ complex species were in higher samples. Irrespective of the analysis em-
compared with subgingival samples. proportions in subgingival samples at ployed, 4 of the 5 Actinomyces species
These included 2 species each of the the deeper sites. examined, A. naeslundii genospecies 1
genera Actinomyces and Streptococcus and 2, A. gerencseriae and A. israelii,
as well as C. sputigena and N. mucosa. were consistently the most prevalent,
Discussion
Fewer taxa differed between supra and exhibited the highest mean counts and
subgingival plaque samples at pockets The purpose of the current investiga- proportions in both supra and subgin-
⬎ 3 mm. Notable, were the higher mean tion was to examine the microbial com- gival plaque samples. The ubiquitous
counts of B. forsythus and P. intermedia position of supra and subgingival colonization of these species was
at the deep periodontal sites. plaque samples from adult periodontitis further emphasized in that individual
The proportion of microbial com- subjects in order to compare and relate subjects harbored at least one of the Ac-
plexes in supra and subgingival plaque the species found in the 2 environments. tinomyces species in 75 to 100% of
samples at the different pocket depth While many studies have evaluated the supragingival samples and 62 to 100%
categories is presented in Fig. 9. The composition of subgingival plaque, of subgingival samples. However, al-
areas of the pies were adjusted to reflect studies of supragingival plaque compo- though predominant in both supra and
the mean total DNA probe count at sition have been comparatively few. subgingival plaque, the mean total pro-
each of the sample pocket depth cate- Further, these studies were limited in portion of Actinomyces species was sig-
gories. Total counts were highest in terms of the number of samples, sub- nificantly lower (38%) in subgingival
supragingival plaque from pockets ⬎4 jects and species evaluated. The use of plaque compared with 48% in supragin-
mm and lowest in subgingival samples checkerboard DNA-DNA hybridiza- gival plaque samples.
from pockets ⬍3 mm. The proportions tion allowed the examination of 1170 Not surprisingly, species thought to
of ‘‘orange’’ complex species were sig- supra and subgingival plaque samples play a major rôle in the pathogenesis of
nificantly increased in both supra and obtained from 23 adult periodontitis periodontal infections were detected
subgingival samples from sites with subjects for their content of 40 species. less frequently overall compared with
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
730 Ximénez-Fyvie et al.

the Actinomyces and other host com- in the oral cavity (Gibbons 1989, Gib- gators have shown that species such as
patible species. The prevalence of sus- bons et al. 1990). The habitats above B. forsythus and P. gingivalis thrive in
pected periodontal pathogens such as and below the gingival margin both deep periodontal pockets (Gmur et al.
B. forsythus, P. gingivalis and T. dentic- provide a tooth surface for colonization 1989, Haffajee et al. 1998, Socransky et
ola did not differ significantly between but differ in the nature of the boundary al. 1998) and have the potential to
supra and subgingival plaque and one away from the tooth surface. Supragin- colonize the epithelial lining of the peri-
or more of these species were detected givally, the surface of the plaque is not odontal pocket (Childs & Gibbons
on average at greater than 50% of sites. physically constrained but is in direct 1988,1990). In contrast, it appears that
However, counts of B. forsythus and P. contact with saliva. Subgingivally, the Actinomyces attach well to the tooth
gingivalis were significantly higher in plaque is confined within the gingival surface (Gibbons et al. 1990) above and
subgingival plaque and proportions of sulcus or periodontal pocket which pro- below the gum and are a major compo-
these 2 species and T. denticola ap- vides a second, quite different epithelial nent of plaque in both ecosystems. Pre-
peared to be higher subgingivally. Other surface for colonization. The soft tissue liminary data from ongoing studies in-
species thought to be important in peri- wall also provides a source of somewhat dicate that Actinomyces species are
odontal diseases, such as members of different nutrients than those provided found in lower proportions in samples
the genera Fusobacterium and Prevotel- in the supragingival environment. from oral soft tissues such as the
la showed similar patterns of coloniza- Given these differences in habitat, one tongue, cheeks and palate, suggesting
tion. might expect major differences in the that the primary and most important
The data in the present investigation composition of plaque in the 2 do- area for colonization of members of
are in accord with previous studies mains. However, there did not appear this genus may be the tooth surface.
(Gmur et al. 1989, Haffajee et al. 1998, to be an abrupt change in plaque com- The finding of suspected periodontal
Socransky et al. 1998) that indicate that position at the gingival margin. The na- pathogens in supragingival plaque
members of the ‘‘red’’ and ‘‘orange’’ ture of the species colonizing above and samples has been described by a num-
complexes are in higher numbers and below the gum was not strikingly differ- ber of investigators (Cao et al. 1990, Ri-
proportions in subgingival plaque ent when only presence or absence was viere et al. 1992, Gmur & Guggenheim
samples from deeper periodontal evaluated. The major differences were 1994, Zee et al. 1996) and has import-
pockets. However, unique to this inves- in counts and proportions of many of ant implications. In accord with those
tigation was the recognition that certain the species examined. In particular, the studies, the current investigation de-
species, such as T. socranskii and E. sa- proportions of ‘‘red’’ and ‘‘orange’’ tected suspected periodontal pathogens
burreum were at higher mean counts in complex species were significantly in supragingival plaque samples at a
supragingival plaque samples taken higher in subgingival plaque. The pro- significant number of sites, although
from sites with deeper pocket depths portions of the ‘‘red’’ complex species counts and proportions were relatively
than sites with shallower pockets. Mem- (B. forsythus, P. gingivalis, T. denticola) low. Further, these species were found
bers of the ‘‘orange’’ complex were also in subgingival plaque was twice that ob- at supragingival sites where the corre-
at greater mean proportions in supra- served supragingivally (7.0 versus 2.8%) sponding subgingival site did not harb-
gingival samples from deep pocket sites and ‘‘orange’’ complex species com- or the species. For example, P. gingi-
than sites with shallow pockets. Such prised about 18 and 28% of the total valis was found at 18% (69/385) of
data imply an association of supragin- DNA probe count supra and subgingi- supragingival sites where it was not de-
gival plaque composition with pocket vally respectively. This finding was not tected subgingivally (Table 3). Thus, the
depth at the adjacent site although unexpected, since several members of results of the present investigation sug-
cause or effect cannot be discriminated. the ‘‘red’’ and ‘‘orange’’ complexes are gested that supragingival colonization
One of the prerequisites for coloniza- either designated or suspected peri- by such species is more than merely a
tion of a species is its ability to adhere odontal pathogens whose most advan- transient phenomenon and do not sup-
to one or more oral surfaces in order to tageous habitat might be expected to be port the notion of subgingival over-
overcome the potent fluid flow present the subgingival space. Several investi- growth.

Table 3. Relationship of species in supra and subgingival plaque samples at individual sites.
P. gingivalis A. naeslundii 2
supragingival sugragingival
0 π 0 π
0 316 69 0 42 93
subgingival subgingival
π 106 77 π 19 420
ORΩ2.29, p∞0.05 ORΩ6.43, p∞0.001

P. intermedia V. paravula
supragingival supragingival
0 π 0 π
0 225 81 0 134 153
subgingival subgingival
π 108 149 π 56 214
ORΩ1.75, N.S. ORΩ2.29, p∞0.05
The ‘‘0’’ and ‘‘π’’ row and column headings represent ‘‘not detected’’ and ‘‘detected’’ respectively. The odds ratios and p values were computed
using Mantel-Haenszel procedures with the subject as the stratum. The p-values were adjusted for 40 multiple comparisons.
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Supra- and subgingival microbiota 731

The detection of ‘‘subgingival’’ spe- meticulous supragingival plaque re- forsythus und Porphyromonas gingivalis. Sub-
cies in supragingival sites indicates that moval may be crucial in preventing the gingivale Proben zeigten einen signifikant
such species can colonize supragingival initiation and recurrence of periodontal höheren Anteil der Arten des ‘‘roten’’ und
‘‘orangen’’ Komplexes, während in in supra-
plaque even though the majority of infections, confirming the importance
gingivaler Plaque höhere Anteile der Spezies
them are thought to be oxygen sensitive placed on the use of proper oral hygiene des ‘‘grünen’’ und ‘‘rosa’’ Komplexes sowie
and/or require a low oxidation-reduc- regimes. Actinomyces-Arten beobachtet wurden. Pu-
tion potential for growth. In earlier tative Parodontalpathogene konnten in su-
years, colonization of anaerobic species pragingivvaler Plaque von Stellen gefunden
Acknowledgments
in the supposedly ‘‘aerobic’’ supragin- werden, bei denen diese Arten subgingival
gival plaque was thought to be unlikely. This work was supported in part by re- nicht gefunden wurden.
Recent studies of the micro-organiza- search grants DE-12108, DE-10977 and Schlußfolgerungen: Diese Ergebnisse weisen
tion of the bacterial constituents of DE-04881 from the National Institute darauf hin, daß supragingivale Plaque puta-
tive Parodontalpathogene beherbergen kann,
dental plaque offer reasonable expla- of Dental and Craniofacial Research
was auf eine mögliche Rolle dieser Umge-
nations for detection of anaerobes in and by DGAPA, National Auton- bung als Reservoir dieser Keime für die Wie-
supragingival plaque. Such studies indi- omous University of Mexico (UNAM). derbesiedlung subgingivaler Stellen hin-
cate that in multi-species biofilms, such deutet.
as dental plaque, mixed-species micro-
Zusammenfassung
colonies are formed (Costerton et al. Résumé
1994, 1995). An individual cell within Mikrobielle Zusammensetzung supra- und
a mature multispecies biofilm typically subgingivaler Plaque bei Patienten mit Er- Composition microbienne de la plaque supra
lives in an unique microhabitat where wachsenenparodontitis et sous gingivale chez des sujets atteints de pa-
Einleitung: Diese Studie sollte die mikrobielle rodontite de l’adulte:
nutrients are provided by neighboring
Zusammensetzung supra- und subgingivaler Cette étude se propose de comparer et de
cells and by diffusion, where products Plaque von 23 Patienten mit Erwachsenenpa- mettre en relation la compositon microbien-
are removed by the same processes, and rodontitis (mittleres Alter 51∫14 Jahre) ver- ne de la plaque supra et sous gingivale chez
where antagonists may be kept at a dis- gleichen und in Beziehung setzen. 23 patients atteints de parodontite de
tance by diffusion barriers (Costerton Methoden: Insgesamt wurden 1170 Proben l’adulte (age moyen 51 ans∫14 ans). Un to-
et al. 1987). In situ biofilm measure- supra- und subgingivaler Plaque von den me- tal de 1170 échantillons de plaque supra et
ments of pH, dissolved oxygen, sulfide sialen Stellen eines jeden Zahnes (bis zu 28 sous et 28 échantillons sous gingivaux),
and other components have provided supra- und 28 subgingivale Proben pro Pati- chez chaque sujet et la présence et le taux
critical information about the prop- ent) gewonnen und auf das Vorhandensein de 40 taxons bactériens, à l’aide de sonde
sowie die Konzentration von 40 verschiede- génomique complète et d’hybridiation
erties of these bacterial structures.
nen Bakterienarten mittels Gesamtgenom- DNA-DNA en damier, furent évalués. Les
When a microelectrode was inserted DNS-Sonden und ‘‘Schachbrett’’-DNS- relevés cliniques suivants, appréciation
into a bacterial microcolony within a DNS-Hybridisierung untersucht. Die klini- dichotomique de la rougeur gingivale, sai-
biofilm, the values of dissolved oxygen schen Parameter umfaßten dichotome Beur- gnement au sondage, accumulation de pla-
decreased as the electrode was inserted teilungen der Rötung der Gingiva, des Blu- que et suppuration, ainsi que la mesure du-
further into the microcolony, reaching tens auf Sondieren, Plaqueansammlung und pliquée de la profondeur de poche et du ni-
almost totally anaerobic levels in the Eiterung sowie Doppelmessungen der Son- veau d’attache ont été réalisés sur 6 sites
center. These direct observations of liv- dierungstiefen und klinischen Attachmentle- par dent. Le nombre (niveau), le % du total
ing biofilms may explain the existence, vel an 6 Stellen pro Zahn. Die Anzahl, der des sondes DNA (proportion) et le % de si-
prozentuale Anteil der mit DNS-Sonden tes colonisés (prévalence) de chaque espèce,
and even the physiological activity, of
nachgewiesenen Zahlen (Proportion) und der dans la plaque supragingivale, et séparem-
fastidious anaerobes within mixed bi- prozentuale Anteil kolonisierter Stellen (Prä- ment, dans la plaque sous gingivale, ont été
ofilms in supposedly aerobic environ- valenz) jeder Spezies in supra- und separat in saisis par informatique pour chaque sujet.
ments such as supragingival plaque. subgingivaler Plaque wurden für jeden Pati- La signification des différences entre les
The frequent detection of potential enten berechnet. Die Signifikanz von Unter- plaques supra et sous gingivales pour cha-
periodontal pathogens in supragingival schieden zwischen supra- und subgingivaler que espèce a été recherchée en utilisant le
plaque has important clinical and eco- Plaque für jede Spezies wurden mittels des Wilcoxon signed rank test ajusté pour des
logical ramifications. Until now, the Wilcoxon-Tests bestimmt und für multiple comparaisons multiples. Toutes les espèces
main reason to control supragingival Vergleiche korrigiert. ont été détectées aussi bien dans la plaque
Ergebnisse: Alle 40 Spezies wurden in supra- supra que dans la plaque sous gingivale.
plaque has been to prevent gingivitis.
und subgingivaler Plaque identifizert. Actino- L’espèce Actinomyces était le taxon le plus
The data of the present investigation myces-Spezies waren die Arten, die in beiden prévalent dans les deux habitats, 75 a 100%
suggest a second, perhaps more compel- Lebensräumen am häufigsten vorkamen. 75 des sites supra gingivaux et 62 a 100% des
ling reason for controlling supragin- bis 100% der supra- und 62 bis 100% der sub- sites sous gingivaux étant colonisés par au
gival plaque; i.e., supragingival plaque gingivalen Stellen waren zumindest von einer moins 1 des 5 espèces d’Actinomyces. Les
may be a major reservoir for species von 5 Actinomyces-Arten besiedelt. Supra- échantillons de plaque supragingivale pré-
that ultimately initiate periodontitis. gingivale Proben zeigten signifikant höhere sentaient des comptages plus importants
From this reservoir, species could Zahlen von Actinomyces naeslundii Genospe- pour Actinomyces naeslundi genoespeces 1,
spread to uninfected sites or recolonize zies 1, Actinomyces israelii, Actinomyces Actinomyces israelii, Actinomyces odontoly-
odontolyticus, Neisseria mucosa, Streptococ- ticus, Neisseria mucosa, Streptococcus gor-
subgingival sites which have been peri-
cus gordonii, Capnocytophaga ochracea und donii, Capnocytophaga ochracea et Capnocy-
odontally treated. Further, trans- Capnocytophaga sputigena im Vergleich mit tophaga sputigena par comparaison avec le
mission of species from one subject to subgingivalen Proben der gleichen Stellen. comptage moyen des échantillons sougingi-
another would be more readily achieved Proben subgingivaler Plaqueproben zeigten vaux obtenus sur la même surface dentaire.
if the species were located in the supra- signifikant höhere Zahlen von Prevotella ni- Les échantillons de plaque sous gingivales
gingival plaque. In both instances, grescens, Prevotella intermedia, Bacteioides présentaient des comptage significativement
 1600051x, 2000, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1034/j.1600-051x.2000.027010722.x by UNIVERSIDADE ESTADUAL PAULISTA, Wiley Online Library on [29/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
732 Ximénez-Fyvie et al.

supérieurs de Prevotella nigrescens, Prevo- Feinberg, A. P. & Vogelstein, B. (1983). A Ranney, R. R. (1983). Bacteriology of
tella intermedia, Bacteroides forsythus et technique for radiolabeling DNA restric- moderate (chronic) periodontitis in mature
Porphyromonas gingivalis. Les échantillons tion endonuclease fragments to high speci- adult humans. Infection and Immunity 42,
sous gingivaux présentaient des proportions fic activity. Analytical Biochemistry 132, 6– 510–515.
significativement supérieures d’espèces ap- 13. Moore, W. E. C., Holdeman, L. V., Cato, E.
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Address:
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Costerton, J. W., Cheng, K-J., Geesey, G. G., ert, R. M. (1982a). Bacteriology of experi- Department of Periodontology
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