1998 - Socransky - Microbial Complexes in Subgingival Plaque

,/ Clin Periodontol IWS: 25: 134-144 Copyright © Munksguard 1998
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juuTof
Clinical perindontcioyy
Microbial complexes in S.S.Socransky\ A.D.Haffaiee\

M.A.Cugini\ C.Smith^ and
R. L.Kent Jr.2
subgingival plaque Departments of, 'Periodontology, ^Biostalistics,

Forsyth Dental Cenier, Boston, MA, USA
Socran.sky SS. Haffajee AD. Cugini MA, Smith C, Kent Jr. RL: Microbial
complexes in subgingival plaque. J Clin Periodontol 1998; 25: 134-144.
© Munksgaard, 1998.
Abstract. It has been recognized for some time thai bacterial species exist in
complexes in subgingival plaque. The purpose of the present investigation was to
attempt to define such communities using data from large numbers of plaque
samples and different clustering and ordination techniques. Subgingival plaque
samples were taken from the mesial aspect of each tooth in 185 subjects (mean
age 51 ± 16 years) with (o^l60) or without («^25) periodontitis. The presence
and levels of 40 subgingival taxa were determined in 13.261 plaque samples using
whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical
assessments were made at 6 sites per tooth at each visit. Similarities between pairs
of species were computed using phi coefficients and species clustered using an aver-
aged unweighted linkage sort. Community ordination was performed using prin-
cipal components analysis and correspondence analysis. 5 major complexes
were consistently observed using any of the analytical methods. One complex
consisted of the tightly related group: Bacteroides forsythus. Porphvromonas
gingivalis and Treponema denticoht. The 2nd complex consisted of a tightly re-
lated core group including members of the Fusohaderium nucleatum/periodoniicum
subspecies, Prevotella intermedia. Prevotella nigrescens and Peptostreptucoccus
micros. Species associated with this group included: Eubacterium nodatum.
Campylobacter rectus, Campylobacter .showae. Streptococcus consteltatus and
Campylobacter graellis. The 3rd complex consisted of Streptoeoeeus sanguis. S.
oralis, S. mitls. S. gordonii and S. intermedius. The 4th complex was comprised
of 3 Capnocytophaga species, Campylobacter concisus, Eikenella eorrodens and
Actinobaciiius actinomycetemcomitans serotype a. The 5th complex consisted of
Veiltonelta parvula and Actitwmyces odontotylicus. A. actinotnycetemcomitans sero-
type b, Selenomonas twxia and Actinomyces naeslundii genospecies 2 {A. viscosusj
were outliers with little relation to each other and the 5 major complexes. The
1st complex related strikingly to clinical measures of periodontal disease particu- Key words: microbiology; bacteria; subgingival
plaque; ecology: periodontal
larly pocket depth and bleeding on probing.
Accepted for publication 14 May 1997
The complexity of the subgingival side in this area. Cursory examination a zone of gram negative and/or motile
microbiota has been recognized since of a series of plaque samples suggests species located adjacent to the epithelial
the 1st microscopic examination of this that there is little order in the micro- lining of the pocket while gram positive
ecosystem by Van Leeuwenhoek in 1683 biota that colonizes gingival sulci or rods and cocci appeared to be forming
(Tal, 1980). Since that time, numerous periodontal pockets. However, examin- a tightly adherent band of organisms on
studies have evaluated the composition ation of sections of plaque by light and the enamel or root surface (Listgarten
of plaque using light and electron electron microscopy indicates a surpris- 1976, Listgarten 1994).
microscopy, cultural techniques and ing degree of order in colonization pat- Cultural, immunologic or DNA
more recently immunologic or DNA terns. For example, early supragingival probe assessments of plaque have dem-
probe techniques. All techniques re- plaque demonstrated columnar ar- onstrated that certain species frequently
inforce Van Leeuwenhoek's initial ob- rangement of morphologically distinct occur together in subgingival plaque
servation that subgingival plaques are bacterial species from the tooth surface samples. For example, Porphyromonas
comprised of a large complex mixture to the outer surface of the plaque gingivalis is almost always observed in
of bacterial species. Indeed it has been (Listgarten et al, 1975), Subgingival samples that arc harboring Bacteroides
estimated that 400 or more species re- plaque was frequently characterized by forsythus. It has been speculated that B.
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Mierobial complexes 135
forsythus in some fashion precedes odontitis or acute necrotizing ulcerative acetate. The released DNA was placed
colonization by P. gingivalis since B. gingivitis was included in the study. into the extended slots of a Minislot
forsythus is detected more frequently by (Immunetics, Cambridge MA) and then
itself (Gmur et al. 1989). Other com- concentrated onto a nylon membrane
plexes that have been observed include Ciinicai monitoring (Boehringer Mannheim) by vacuum
P. gingivalis and Treponema denticola Subjects were screened for suitability and fixed to the membrane by exposure
(Simonson et al. 1992b) and Fusobae- and if accepted, were asked to sign in- to ultraviolet light followed by baking
teriuin nucleatwn and Prevotella inter- formed consent forms. All subjects were at 120°C for 20 min. The Minislot de-
media (Ali et al. 1994). clinically monitored at baseline and sub- vice permitted the deposition of 28 dif-
Understanding the relationship jects with periodontitis were monitored ferent plaque samples in individual
among bacterial species is useful in at 3 month intervals post therapy. Plaque "lanes" on a single 15X15 cm nylon
understanding the biology of subgingi- accumulation (0/1). overt gingivitis {0/1). membrane as well as 2 control lanes
val plaque and in planning strategies bleeding on probing (0/1). suppuration containing 10^ or lO** cells of each test
for its control. Knowledge of the eco- (0/1). probing pocket depth and probing species. The membrane with fixed DNA
logical relationships among bacterial attachment level were measured at 6 sites was placed in a Miniblotter 45 {Im-
species can direct and focus investi- per tooth (mesiobuccal. buccal. disto- munetics, Cambridge MA), with the
gations on critical bacterial interac- buccal. distolingual. lingual and mesiol- "lanes" of DNA at 90° to the channels
tions. ingual) at all teeth excluding third mo- of the device. A 30x45 "checkerboard"
lars at each visit. The baseline clinical pattern was produced with 5 of the
Technological developments permit
features of the 185 subjects are presented probe lanes remaining empty to permit
the evaluation of large numbers of bac-
in Table 1. accurate localization. Each channel was
terial species in large numbers of plaque
used as a hybridization chamber for
samples from a wide range of subjects.
separate DNA probes. Signals were de-
Such procedures provide a data base in
iMicrobioiogicai assessment tected by chemiluminescence.
which associations among bacterial
species can be precisely examined. As- Subgingival plaque samples were taken
sociations between a pair of species is from the mesio-buccal aspect of each DNA-DNA hybridization
often performed using contingency tooth in each subject at each monitor- The membranes were prehybridized at
tables, sometimes 2x2 and sometimes ing visit. Counts of 40 subgingival spe- 4 2 T for 1 hr in 50% formamide. 5XSSC
with more levels of each species. Cluster cies were determined in each plaque (lXSSC-150 mM NaCl, 15 mM Na
analysis has been useful in describing sample using the checkerboard DNA- citrate, pH 7.0), 1% casein (Sigma, St.
closely related species when more than DNA hybridization technique (Socran- Louis MO). 5 X Denhardt's reagent, 25
a few pairs of species are examined. sky et al. 1994). After the removal of mM sodium phosphate (pH 6.5) and 0.5
Community ordination is a procedure supragingival plaque. subgingival mg/ml yeast RNA (Boehringer Mann-
that attempts to indicate closely related plaque samples were taken with individ- heim). Digoxigen in-labeled, whole chro-
species within a community and then ual sterile Gracey curettes from the me- mosomal DNA probes were prepared
demonstrate the relatedness among dif- sial aspect of each tooth. The samples using a random primer technique (Eein-
ferent communities of species within the were placed in separate Eppendorf berg& Vogelstein 1983). The probes and
ecosystem of interest. The purpose of tubes containing 0.15 ml TE (10 mM hybridization buffer were placed in indi-
the present investigation was to use Tris-HCI, 1 mM EDTA. pH 7.6). 0.15 vidual lanes of the Miniblotter and the
cluster analysis and community ordi- ml of 0.5 M NaOH was added to each whole apparatus placed in a sealed plas-
nation techniques to examine relation- sample and the sample boiled in a water tic bag. Membranes were hybridized
ships among bacterial species in subgin- bath for 5 min. The samples were neu- overnight at 42°C in a hybridizing solu-
gival plaque samples, and relate the tralized using 0.8 ml 5 M ammonium tion containing 45% formamide.
complexes to clinical parameters of
periodontal disease.
Table L Baseline elinieal eharacteristies of subject group («= 185)

Material and Methods Mean (±SD) Range
Subject population
age (years) 5I±16 20-87
185 subjects ranging in age from 20-87 no. missing teeth 3.1±3.l 0-8
years who were considered to be peri- % males 43
odontatly healthy (n=25) or with evi- mean pocket depth (mm) 3.l±0.7 1.7-6.6
dence of prior attachment loss (n^ 160) mean attaehment level (mm) 2.9±l.2 0.8-6.8
were selected for study. All subjects had % sites with:
al least 20 teeth. Exclusion criteria in- plaque 69 ±30 0-100
red 72 ±30 0-100
cluded pregnancy, periodontal therapy
BOP 49 ±37 0-100
or antibiotics in the previous 3 months, suppuration 1.7±5.4 0^9
any systemic condition which might pocket depth <4 mm 74 ±20 11-100
have affected the progression or treat- pocket depth 4-6 mm 23±i7 0-76
ment of periodontitis and the need for pocket depth >6 mm 3±6 0-57
pre-medication for monitoring or ther- attachment level <4 mm 72 ±24 7-100
apy. No subject with localized juvenile attachment level 4-6 mm 23±i8 0-67
periodontitis, rapidly progressive peri- attachment level >6 mm 5 ±9 0-64
136 Socransky cl al.
F/^, I. Dendrogram of a cluster analysis of 32 subgingival taxa. The

similarity between pairs of species was computed using a phi coef-
ficient, the coefficients were scaled and then sorted using an average
unweighted linkage sort. Clusters were formed with a threshold level
of 60"/i, The color coding used to delineate the groups in this figure
were employed in all figures.
Fig. 2. Community ordination of 32 subgingival taxa using corre-
spondence analysis. The relationships among species were evaluated
using the levels of ihe species at each of lhe sampled sites. Correspon-
dence analysis was performed as described by Ludwig & Reynolds
(1988) and the species were plotted along the first (.v-axis) and second
(_}'-axis) axes. The colors indicaie ta.\a within the same group. A. naes-
lundii genospecies 2 [A. viseosus). A. actintmnceteincomitans serotype 5
b and S. no.xia were not part of any complex.
Fig. j . Community ordination of 32 subgingival taxa using correspondence analysis. The analysis was performed as described in Fig. 2:
however, the r-axis has been added. This presentation demonstrates that species that appeared to be closely associated in Fig, 2 such as F.
nodatum and T deniicola were widely separated in the 3-dimensional presentation.
Fig. 4. Community ordination of 32 subgingival taxa using principal components analysis. The relationships among species were evaluated
using the levels of the species at each of the sampled sites. Principal components analysis was performed as described by Ludwig &. Reynolds
(1988) and the species were plotted along the first (.v-axis) and second (r-axis) principal components.
Fig. 5. Community ordination of 32 subgingival taxa using principal components analysis. The analysis was performed as described in Fig.
4; however, the third principal component (r-axis) has been added. This presentation demonstrated that species that appeared to be closely
associated in Fig, 4 such as F. nodatum and T. denticola were widely separated in the 3 dimensional presentation.
5XSSC. ixDenhardfs reagent. 20 mM SDS, 20 min. twice) in a Disk Wisk ap- 1:25,000 dilution of anti-digoxigenin
Na phosphate (pH 6,5). 0.2 mg/ml yeast paratus (Schleicher and Schuell. Keene antibody conjugated with alkaline
RNA. 20 ng/ml o^i labeled probe. lO'V^. NH). phosphatase using the modification de-
dextran sulfate and VA^ casein. Mem- scribed by Engler-Bium et al. <1993).
branes were washed at low stringency to Dcleclion and cnumeraliini After washing, the rnembranes were in-
remove loosely bound probe and then at To detect hybrids, membranes were cubated in Lumiphos 530 (Lumigen,
high stringency (68°C. O.lxSSC, 0,1% blocked and then incubated with a Southfield, MI) for 45 min at 37X,
Microbial complexes 137
present. Signals were evaluated visually

B forsythus -ve B. forsythus +ve
by comparison with the standards for
Td- Td* Td- Td+
% of sites the test species. They were recorded as:
Pg- 64.2 5.1 7.8 7.3 0. not detected; 1,<IO^ cells; 2. - 1 0 ^ 3,
70
Pg* 1.1 1.3 3.1 10.1 10- to 10^ 4, -10"; 5 >10^ cells.
55
40
20 Statistical analysis
Microbiological data available for each
Observed subject included the level of each of 40
15 —
test species from up to 28 plaque
Expected samples (mean 25,2 samples) at each
visit. The prevalence (the number of
10 sites at which the species was detected)
of each species was also computed for
each subject at each visit. A total of
13,261 plaque samples were evaluated.
Data for 32 of the species examined
were used in the following analyses. The
species were chosen on the basis of fre-
quency of detection; species detected
None Pg Td Bf Bf Bf All in< 5% of sites were omitted. Two by
Pg Td two tables were set up for each of the
Fig. 6. Bar chart of the observed and expected frequencies of mettibers of the red complex 496 pairs of species in which the pres-
(fi, forsvihus (Bf). T. denticola (Td) and P. gingivulis (Pg)) individually and in dilTerent ence or absence of each species was
cotTtbinations, The observed frequencies of the 3 species were summarized in a 2X2X2 contin- summarized. A phi coefficient was com-
gency table and conligural frequency analysis (von Eye 1990) was performed to cotiipute the puted to assess association between
expected frequencies. The inset presents the % of sites in each of the 8 cells. each pair of species and scaled to range
between 0 and lOO'Mi, The resulting
similarities were clustered using an aver-
age unweighted linkage sort (Sneath &
% of sites P. intermedia -ve P. intermedia +ve Sokal, 1973). For comparative pur-
Fnv- Fnv+ Fnv— Fnv* Observed
poses, other sitnilarity coefficients were
50 —
Pm~ 42.3 6.2 7.6 7.9 Expected
employed including the Bray Curtis,
40 — Mahalanobis (/- and the correlation co-
Pm+ 2.0 5.4 2.4 26.0 efhcients.
30 =
Community ordination was per-

formed using principal components
22 —
analysis and correspondence analysis
(Ludwig & Reynolds 1988). The data
employed were the ranks representing
15- different counts of the test species at the
13,261 sampled sites. The data were
plotted as described by Ludwig & Reyn-
olds (1988).
8—
In order to examine the robustness of
the observed relationships, subsets of
the data were analyzed using the cluster
None
and community ordination procedures.
These subsets included use of first visit
Fig. 7. Bar chart of ihe observed and expected frequencies of representative metnbers of the data only and samples from sites within
orange complex {P. micros (Pm). F. nucleatum ss vincentii (Fnv) and P. intertnedia (Pi/) specified pocket depth ranges including
individually and in different combinations. Observed and expected frequencies were conipnted 0-3 mm, 4-6 mm and >6 mm as well
as described for Fig, 6, as pre- and post-therapy data.
Associations among species within a
complex were examined using config-
placed in a film cassette with Reflection cies by adjusting the concentration of ural frequency analysis. Significance of
NEF filtn (Dupont, Boston MA) for 1 each DNA probe. This procedure was differences between observed and ex-
h at 37°C and then developed. Two carried out in order to provide the same pected values within cells were deter-
lanes in each run contained standards sensitivity of detection for each species. mined as described by von Eye (1990).
at different concentrations. The sensi- Failure to detect a signal was recorded The relationship between the clinical
tivity of this assay was adjusted to per- as zero, although conceivably, counts in parameters and individual species and/
mit detection of 10"^ ceils ofa given spe- the I to 1000 range could have been or complexes was examined after aver-
138 Socransky et al.
aging data for a chosen parameter Proportion of Sites exhibiting

within the subject and then averaging the Red complex
across subjects. For example, the levels
of a species or complex were averaged
for all non bleeding on probing sites in
a subject as well for all bleeding on
probing sites in that subject prior to av-
eraging within BOP categories across 1-4
subjects. Significance of differences for
clinical parameters were sought using
5-8
the Kruskal-Wallis test.
Results 9-12
Associations among bacterial species as
determined by cluster analysis
Fig. I presents a dendrogram of the re- Fig. 8. Area plots depicting the percentage of sites colonized by tiiembers of the orange and
sults of the cluster analysis of all 13,261 red complexes. The data are based on examination of 13,261 subgingival plaque samples. The
left hand panel indicates the percentage of sites colonized by 0, 1-4, 5-8 and 9-12 members
samples using ^ coefficients and an av- of the orange complex. The right hand panel presents ihe same data, but overlaid with the
erage unweighted linkage sort. 5 clus- percentage of sites in each of those categories that exhibited 1, 2 or 3 members of the red
ters were formed with >60% similarity complex.
and included 29 of the 32 taxa evalu-
ated. The red cluster consisted of P. gin-
givalis. B. forsythus and T. denticola.
1 -4 5-8 9-12
The orange cluster consisted of F. nu-
eleatum subspecies, P. intermedia and
P. nigre.scens, Peptostreptoeoccus micros
and Campylobacter rectus, Campvlo-
bacter showae, Campylobacter gracilis,
E. noddttim and S. eonstetlatus. The 3
Capnocytophaga species. Catnpylo-
bacter conclsus. Eikenella corrodens and
Actinobacillus actinomycetetncomitans
serotype a formed the green cluster.
while a group of streptococci made up
the yellow cluster. Streptococcus mitis.
Streptococcus sanguis and Streptococ-
cus oralis were most closely related
within this group. Actinotnvces odonto- Proportion of Sites exhibiting
lyticus and Veillonella parvula formed the Orange complex
the purple cluster. Actinotnyces naeslun- Fig. 9. Area plots depicting the % of sites colonized by members of the red and orange
dii genospecies 2 (Actinomyces complexes. The left hand panel indicates the % of sites colonized by 0, 1, 2 or 3 members of
vLseosus), Selenomonas noxia and A. the red complex. Tbe right hand panel presents the same data, but overlaid with the percentage
aetinomycetemcotnitans serotype b did of sites in each of those categories tbat exhibited 0, 1-4, 5-8 and 9-12 members of the orange
not cluster with other species. complex.
onstrated the relationships among the and A. naeslundii genospecies 2 {_A.

Associations among bacterial species as
determined by community ordination
Capnocytophaga species, most of the viscosus) was evident (Figs. 2, 3),
Streptococcus species, E. eorrodens and Figs. 4, 5 present principal compon-
The results of community ordination C coneisus. These species were closely ents analysis of the same data and indi-
using correspondence analysis are pre- related to each other and somewhat re- cate similar groupings of species. This
sented in Fig. 2, This analysis related to the orange complex. A. actino- analysis reinforces the distinction of the
inforced the relationships demonstrated mycetemcomitans serotype b appeared red {P. gingivalis. B. forsythus and T.
in Fig. 1 and showed the relationships to be closely related to C ochracea, but deniicola) and orange clusters as well as
among the different microbial complex- examination of the 3 dimensional plot the separation of these 2 groups from
es. For example, the red complex con- of the correspondence analysis (Fig. 3) the green and yellow complexes.
sisting of P. gingivalis. B. forsythus and indicated that these 2 species were dis-
T. denticola was closely associated with tant from one another. The relationship
the orange complex that included F. nu- between A. odontolyticus and V. parvula Associations among species within
cleatum subspecies. P. intermedia and suggested by cluster analysis was con- complexes
P. nigre.scens, P- micros, E. noiiatum firmed by correspondence analysis. The associations between species in the
and S. constellatus and 3 of the Further, the outlier status of S. noxia, red complex are summarized in Fig, 6.
Campylobacter species. Fig. 2 also dem- A. actinomycetemcomitans serotype b 64"/j of sites harbored none of the spe-
% sites colonized the same data together with the percen-

tage of sites in each category that also
100- P. gingivalis F. nuc. ss vincentii S. sanguis harbored 1, 2 or 3 metnbers of the red
p < 0,0001 p < 0,0001
75- 2411 ,393 complex. It is readily apparent that
6458 174 members of the red complex are rarely
50- found in the absence of members of the
25- orange complex. With increasing colon-
ization by the orange complex, more
n
sites were colonized by increasing num-
100- B. forsyttius p. intermedia 4. viscosus bers of the red complex. Fig. 9 presents
p < 0.0001 g p < 0.0001 the same data but with the left hand
75-
pane! depicting the % of sites colonized
50- by members of the red complex. Only
36% of sites harbored one or more of
25- the red complex species compared with
n_ 72% of sites which harbored members
<3 3 4 5 6-7>7 3 4 5 6-7>7 <3 3 4 5 6-7>7 of the orange complex (Fig 8). The
POCKET DEPTH (MM)
right hand panel indicates that a large
Fig. 10. Bar charts of the mean (±SEM) of the % of sites colonized by 6 subgingival species proportion of sites in which the red
at selected pocket depths. The percentage of sites colonized by each species al each pocket complex was not detected was colon-
depth category was computed for each subject and then averaged across subjects. Significance ized by members of the orange com-
of differences among pocket depth categories was tested using the Kruskal-Wallis test. The plex. Taken together the data suggest
total number of subjects was 185. The number of subjects exhibiting the different pocket that species in the orange complex pre-
depths is shown above the bars in the A, nae.slundii genospecies 2 {A. vi.seosus) panel, while cede colonization by species of the red
the number of sites in each pocket depth category is shown above the bars for S. complex.
% sites colonized
Reiationsiiip between bacterial species
100 -| P. gingivalis F. nuc. ss vincentii -. S. sanguis
and ciinicai parameters
75 -
The relationship of the species in the
50 - different complexes to pocket depth was
examined. Species in the red complex
25 - exhibited a very strong relationship
with pocket depth (Figs, 10. II), For ex-
ample. B. forsythus and P. gingivalis
B. forsytiius P. intermedia A. viscosus (and T. denticola, data not shown) in-
creased in prevalence and numbers with
increasing pocket depth. Similarly, all
species in the orange complex showed
a significant association with increasing
pocket depth as exemplified by P. inter-
media and F. nucleatum ss vincentii. A.
3 4 5 6-7>7 <3 3 4 5 6-7>7 naeslundii genospecies 2 {A. viscosus)
POCKET DEPTH (MM) and S. .sanguis provide examples of the
remaining species which showed no
statistically significant relationship with
Fig. II. Stacked bar charts of the % of sites colonized by different levels of 6 subgingival pocket depth.
species at selected pocket depths. The number of subjects and sites providing the data were
presented in Fig, 10, Since species in the red complex were
so strongly related to pocket depth, the
relationship between individual and
cies in this complex, while \0"A> of sites cies were detected less frequently than combinations of species in this complex
harbored all 3 species. These percen- expected. Similar results were obtained and pocket depth was examined further
tages were strikingly elevated as deter- for the yellow and green complexes (Fig, 12), Sites with none of the species
mined by configural frequency analysis. (data not shown). exhibited the shallowest mean pocket
In contrast, the individual species were depth, while sites harboring all 3
detected less frequently than expected. showed the deepest. It is interesting to
The same analysis was repeated using Relationship among complexes note that sites harboring P. gingivalis
representative members of the orange The relationship between the red and alone or in combination with the other
complex (Fig. 7). In a fashion similar to orange complexes is illustrated in Figs, 2 species exhibited the deepest mean
the red complex, the % of sites harbor- 8. 9. The left hand pane! of Fig, 8 de- pocket depths. The red complex and the
ing none or all of the species were sig- picts the "/i of sites colonized by 0. 1-4, individual species in that group were
nificantly higher than expected, while 5-8 and 9-12 members of the orange also strongly associated with bleeding
the individual species and pairs of spe- complex. The right hand panel presents on probing (Fig. 13).
140 Socran.skr el al.
Mean PD (mm) Fig. 12. Bur chart of mean (±SEM) pocket depth at
sites harboring none, all or different eomhinations of
4.5 the speeies in the red eomplex {B. forsythus (Bfj. T.
dcnticota (Tdj and P. gingivalis (Pg/). The average
pocket depth for each of the microbial combinations
was compuied within it subject and then averaged
3.4 across subjects.
Td Bf Pg Bf+Td Pg+Td
Counts
No-BOP
p<0.01
Fi^. ti. Bar chart of the mean (±SEM) % of species

in each complex at sites that bled or did noi bleed on
probing. The "/i< of species in a complex present at each
site was computed. The values were averaged for each
BOP category witbin each subject and then averaged
across subjects. The only significant difference between
bleeding and non bleeding sites was observed for tbe
red complex. The individual members of that complex
Av purple yellow green orange red Pg Td Bf differed significantly as dcpicied in the right panel.
A. naeslundii 2
IA viscosus)
V.panulu
C, gtaciUi
S. constellitus
Fig. 14. Diagrammatic representation of the relation-

ships of species within microbial complexes and be-
tween the microbial complexes. This diagram was
based on the results of multiple cluster and community
ordination analyses using the entire data base as well
as subsets of data.
Microbial compte.xes 141
species evaluated, only C .showae and monson et al. 1992b, Kojima et al.
Discussion C. rectus cross-reacted but at a homo- 1993, Wolff et al. 1993. Ah et al. 1994,
The goal of the present investigation logous:heterologous ratio > 100:1. Kamma et al, 1995, Kigure et al. 1995).
was to attempt to understand the na- There were no cross-reactions among In particular, Kigure et al. (1995) using
ture of the microbial complexes that the 3 Capnocytophaga species. Cross-re- immunohistochemical techniques, pro-
exist in subgingival plaque. Any rep- actions within the Sireplococci were vided graphic demonstration of the re-
resentation of these complexes whether minimal always exceeding 100:1 for lationship between T. denticola and P.
by cluster analysis or community ordi- homologousiheterologous species, al- gingivalis in biopsies of subgingival
nation techniques suffers the limitation though S. oralis. S. mitis and S. sanguis plaque, epithelial and connective tissues
that one is attetiipting to represent mul- showed no cross-reactions with 5, in- from different pocket depths in human
ti-dimensional relationships in two or tertnedius and S. constellatus. Thus, the periodontitis subjects. They demon-
three dimensions. Thus, different pres- majority of the observed relationships strated that both species were predomi-
entations of relationships are bound to were unlikely to be due to cross-reac- nant in pockets > 4 mm. In 4 6 mm
suggest somewhat different associations tions of the DNA probes although pockets, T. denticola was detected at the
among species. In spite of these reser- some influence cannot be entirely ruled surface layer of the plaque, while P. gin-
vations, the associations observed were out. A second concern might have been givalis cells were detected in the layer
quite robust, in that different similarity the fact that pre- and post-therapy beneath. In deeper pockets, the species
coefficients, different methods of com- samples were included for periodontitis co-existed in large numbers. Other
munity ordination or subsets of the subjects, but only first visit data were studies demonstrated a reduction in the
data, such as flrst visit data only or data used for periodontally healthy subjects. species of this complex after scaling and
from sites with different baseline pocket It must be emphasized that the relation- root planing (Simonson et al. 1992b,
depths, provided essentially identical ships depicted in this manuscript were Haffajee et al. 1997). Possible mechan-
groupings. The only exceptions were observed when only 1st visit data were isms of pathogenicity of species in the
the occasional movement of E. nodittwn employed; i.e., each subject contributed red complex have been reviewed (Soc-
into the red complex. A. actinomyce- approximately equal numbers of ransky & Haffajee 1991, Haffajee &
temcomitans serotype a joining A. acti- samples. The authors chose to present Socransky 1994); however, it is of inter-
nomycetetncomitans serotype b and A. the entire data base since it included est that this trio has been shown to pro-
naeslundii genospecies 2 (A. viscosus) pre- and post-therapy samples adding a duce proteolytic enzymes such as those
Joining the purple complex. Fig, 14 is degree of robustness to the observed re- sought in the "BANA" test (Loesche et
an attempt to summarize the complexes lationships. al, 1992) and in the proposed SK013
and the relationships among complexes peptidase test (Seida et al. 1992). The
observed in multiple analyses of the full The relationship of P. gingivalis. T. biological basis of the association
data base and subsets of the base. The denticola and B. forsythus (red complex) among P. gingivalis, T. denticola and B.
potential for human error in interpreta- appears in one guise or another in the forsythus is not known. However, it has
tion of the multi-dimensional data is Figures provided in this manuscript as been shown that members of this com-
high: however. Fig. 14 may serve as a well as when subsets of data were exam- plex coaggregate strongly in vitro (
point of departure for further evalu- ined from pre or post therapy visits or Grcnier 1992a, Onagawa et al, 1994.
ation of subgingival microbial relation- for sites subset in different pocket depth Yao et al. 1996) and one species of the
ships. ranges (data not shown). Aspects of this complex may produce growth factors
complex have been described in the required by another in that complex
One concern in interpreting the data literature (Gmur et al. 1989. Simonson (Grenier 1992b, Nihus et al. 1993).
would be the sensitivity and specificity et al, 1992ab). Gmur et al. (1989) de-
of the DNA probes employed. The sen- scribed a strong relationship between B. A second complex that was observed
sitivity of the assay was set to 10" cells forsythus and P. gingivalis in subgingi- in multiple analyses was the orange
of a species by adjusting the concen- val plaque samples from pockets of dif- complex consisting of the F. nudeatum
tration of each probe in the hybridiza- ferent depths in adult subjects. Both subspecies, F. periodonticwn. P micros.
tion buffer. The specificity of the probes species were detected more frequently P. intermedia. P. nigrescens. Streptococ-
was examined using a recently acquired and in higher numbers in deeper peri- cus constellatus, E. nodatum. C. showae.
Storm Fluorimager (Molecular Dy- odontal pockets. In addition, P. gingi- C gracilis and C rectus. The species in
namics, Sunnyvale, CA, USA) and At- valis was never detected in the absence this group were closely associated with
tophos (Amersham Life Science, Ar- of B. forsythus. Simonson et al. (1992a) one another and this complex appeared
lington Heights. Illinois, USA) instead found a strong association between P. closely related to the red complex.
of Lumiphos 530 (Lumigen, Southfield. gingivalis and T. denticota in plaque Other studies have shown an associ-
Michigan, USA) in the detection step. samples taken from subgingival sites in ation between members of this com-
Over 92% of all probe: heterologous spe- 74 Fijians, 74 Colotnbians and 73 plex. For example, Ali et al. (1994)
cies reactions did not exhibit cross-reac- Americans stationed in the Sinai desert, found that P. intermedia was always de-
tions under the conditions of the assay. Pederson et al, (1994) demonstrated a tected in the presence of F, nudeatum in
Some probes such as those to B. for- similar relationship in a continental US subgingival plaque samples from deep
sythus, P. gingivalis and T. denticola did population. Members of the red com- pockets in a group of adult peri-
not exhibit cross-reactions with any het- plex were found together in high num- odontitis subjects. P. micros and C. rec-
erologous species tested. When cross-re- bers in lesions of adult periodontitis ttis were significantly elevated in
actions were observed they were always (Hosaka et al, 1994. Umeda et al. samples from mobile teeth compared
within genera and were quite limited. 1996); in particular, in sites with deeper with non-mobile teeth (Grant et al.
For example, of the 6 Campylobacter pockets or more advanced lesions (Si- 1995), Von Troil-Lindcn et al. (1995)
142 Socranskv el at.
found that P. intermedia. C rectus and On observing Fig. 14. it is tempting Further, other studies have suggested
P. micros were significantly elevated in to speculate that the relationships ob- that different therapies affect different
saliva samples from subjects with ad- served mimic to some extent the mi- segments of the microbiota and the use
vanced periodontitis compared with crobial succession patterns that may an incorrect therapy may be ineffective
samples from subjects with initial or no exist in developing plaque. It seems in lowering the target species and might
periodontitis. Further, treatment that likely that A. naeslundii genospecies 2 adversely affect other members of the
included systemically administered me- {A. vLscosus) and the strcptococcal spe- microbial community (Haffajee et al.
tronidazole decreased levels of these cies are early colonizers followed by 1996),
species and improved periodontal sta- Capnocytophaga sp,, C. concisus, and E. The data of the present investigation
tus. Members of the orange complex corrodens. A. odontotyticus and V. par- suggest the nature of some of the mi-
have been associated with infections in vula may be bridging species leading to crobial complexes in subgingival
non-periodontal sites. Sundqvist (1992) the orange complex and ultimately, at plaque. Although the number of plaque
sampled 65 infected root canals. The some sites, to the red complex. This samples was quite large and the number
most frequently detected organisms in- pattern of colonization is part specu- of species evaluated was reasonably ex-
cluded E. nudeatum. P. intermedia. P. lation and in part based on data about tensive, the investigation represents an
micros. P. anaerobius. Eubacterium sp. the composition of plaques in various initial attempt at evaluating inter-re-
and C. rectus. Strong associations were clinical conditions (Moore & Moore lationships among subgingival species.
found between F. nucleatum and P. 1994), It is interesting to note that a While some of the associations depicted
micros. E. tnideatum and C rectus and treatment such as SRP has a profound may not be completely accurate, certain
P. intermedia and P. micros. Summanen effect on the species of the red complex associations were seen repeatedly using
et al. (1995) found that the most com- and virtually no effect on the majority different analytical techniques and were
monly detected anaerobes in subcutane- of other species except for an increase in accord with data in the hterature.
ous abscesses of intravenous drug users in A. naeslundii genospecies 2 {A. Further, certain complexes, and mem-
were E. nucleatunt. P. tnicros. P. inter- viscosu.s) and some of the streptococcal bers within the complexes, related
media. P. nigrescens, A. odontotyticus species (Haffajee et al. 1997), strongly to clinical parameters of in-
and V. parvula while E. nucleatum, P.
flammation and periodontal destruc-
intermedia. P. nigrescens. P. micros and The red complex showed the strong-
tion. The data provide a framework for
Euhacterium species were most com- est relationship with the clinical par-
understanding the complex ecology ob-
monly detected in 46 cases of anaerobic ameters considered most meaningful in
served in plaque and could be used to
empyema (Civen et al. 1995). P. inter- periodontai diagnosis. For example, the
guide approaches to diagnosis and ther-
media has been shown to stimulate the individual species in the complex as well
apy of periodontal diseases.
growth of 5. constellatus in vitro and the as ihe complex itself related very
combination of these species often pro- strongly with pocket depth and bleed-
duced fatal pulmonary infections in a ing on probing. As described earlier in
mouse model system (Shinzato & Saito the Discussion, other investigators have
1994). One possible mechanism for co- noted the relationship between pocket Acknowledgments
aggregation between E. nodatum and depth and members of the red complex This work was supported in part by re-
strains of E. nudeatum was described by (Gmur et al. 1989, Kigure et al. 1995. search grants DE-04881 and DE-10977
George and Falkler (1992). The coag- Simonson et al, 1992b). The orange from the National Institute of Dental
gregation was thought to involve a pro- complex also related to pocket depth, Research.
tein receptor on E. nucleatum and a although this relationship and the re-
heat-stable protein or polysaccharide lationship to other clinical parameters
component on E. nodatum. was less striking. Zusammenfassung
Knowledge of the associations be- Mit<rohiette Komplexe in der subgingivalen
While species within complexes were tween subgingival species maybe used Plaque
closely associated, the complexes them- to interpret and guide periodontal ther- Es wurde seit einigcr Zeit erkannt, daB bak-
selves seem to have specific relation- apy. The recognition that an inter-re- terielle Spezies in der subgingivalen Plaque
ships with one another (Fig. 12). Spe- lated complex of species such as the red in Komplexen existieren, Der Zweck der vor-
cies such as A. naeslundii genospecies 2 complex exists suggests that therapies liegenden Unlersuchiing war es, die Defini-
(A. viscosu.s). and members of the yel- that affect 1 of these species may influ- tion solcher Gemeinschaften unter Verwen-
low and green complexes were less com- ence the colonization of the other spe- dung der Daten einer groBen Anzahl von
Plaqueproben sowie verschiedener Cluster-
monly associated with members of the cies in this group. In addition, it might
und Ordnungstecbniken zu versuchen, Bei
red and orange complexes than with be speculated that altering the orange 185 Personen (Altersdurchshniit 5]±f6 Jah-
each other. The 2 species in the purple complex might prevent subsequent re) wurden von der Mesialflache eines jeden
complex were strongly related to each colonization of species in the red com- Zahnes mit (/i=160) oder ohne Parodontitis
other and to a lesser extent to members plex providing the therapist with a sec- ('1 = 25) subgingivale Plagueprobcn genom-
of the orange, green and yellow com- ond route to control this group of or- men, Unter Verwendung von Ganz-Gcnom-
plexes. The reasons for these relation- ganisms. A. actinomycetemcomitans DNA-Sonden und der Schacbbrett-DNA-
ships among complexes is unclear but serotype b related poorly to members of DNA-Hybridisierung wurden das Vorkom-
it might be speculated that antagonistic the red and orange complexes sug- men und der Anteil von 40 subgmgivalen
Taxa bei 13261 Plaqueproben bestimmt. Zu
relationships (Grenier 1996) may exist gesting that therapies effective against jedem Termin wurden an 6 Stellen pro Zahn
or that environments selective for one one set of pathogens may not necess- klinischen Messungen durchgefuhrt. Ahn-
group may be less hospitable to a sec- arily be effective against others. This in- lichkeite zwischen Speziespaaren wurden un-
ond group of organisms. deed appears to be the case for SRP ter Verwendung von Phi-Koeffizienten er-
rechnet und die Speziesclusterung erfolgte nemu denlieola. La second complexe etait modal coaggregation reaction between
unter Verwendung einer ungewichtcten constitue par un noyau elroitement apparen- Porphyromonas gingivalis and Treponi-ma
Durchscbnitt-ortentierten Sortierung, Mit al- te comprenant des membres des sous-especes dcnlieota. Oral Microbiology and Immu-
ien analytischen Methoden wurden 5 Haupt- Fusobacterium nucleatum/periodonticum, Pre- nology 7. 280-284,
komplexe bestandig beobachlet. Ein Kom- voiella intermedia, Prevotella nigrescens et Grenier. D, (1992b) Nutritional interactions
plex bestand aus der eng verbundenen Grup- Peptostreplococcus micros. Les especes asso- between two suspected periodontopathog-
pe: Bacieroldes forsythus, Porphyrtmionox ciees avec ce groupe comprenaient: Eubacie- ens, Treponemu dentieola and Porphyro-
gingivalis und Treponema denticolu. Der rium nodatuni, Campylobacter recttts. Campy- monas gingivalis. Infection and Immunity
zweite Komplex bestand aus etner eng ver- lobaeier showae. Strepioeoccus eonsleltalus et 60.5298-5301,
bundenen Zentralgruppe tiiit den Subspezies Campylobacter graeilis. Le troisieme com- Grenier, D, (1996) Antagonistic effect of oral
von Fuxobucleriwn nuelealitm/periotlurUicum, plexe etait compose de Slreptocoecus sanguis, bacteria towards Treponema dentieola.
Prevolt'llu intermediu, Prevotella niigrescens S. oralis, S. mitis, S. gordonii et S. interme- Journal of Clinical Mierobiology 34. 1249-
und Peptostreptococctts micros. Spt-zies. die dius. Le quatrieme cotiiplexe etait constitue 1252,
mit dieser Gruppe assoziiert waren, sind fol- par 3 especes de Capnoeytophuga, Canipylo- Haffajee, A, D . Cugini. M, A.. Dibart. S..
gende: Eubacterium nodatuni, Campylobacler baeter eoncisus, Eikenella eorrodens et Aeti- Smith, C . Kent. R, L, Jr, & Socransky S.S,
rectus. Campyhbucter shonue, Sireptococeus nobacillus aetinomycetemeomitans serotype a. (1997) The effect of SRP on the clinical
constellalus und Catnpylohaeter graeilis. Der Le cinquieme compiexe erait compose par and microbiological parameters of peri-
dritte Komplex bestand aus Streptococcus Veillonella parvula et Avtinomyces odoniolyli- odontal diseases. Journal of Clinical Peri-
sanguis. S. oralis, S. mitis, S. gordonii und 5, cus. A la peripherie, A. actinomycetemcomi- odonlology 24. 324-334,
intermedius. Der vicne Komplex umfajiie die lans serotype b, Selenomonas noxia et Aeti-
3 Capnocytophaga Spezies, Campylobacter nomyces naeslundii genospecies 2 (A. visco- Haffajee. A. D. & Socransky, S, S. (1994) Mi-
coneisus. Eikenella corrodens und Aeiinoba- sus), avaient Ires peu de relation entre eux et crobia! etiological agents of destructive
eillus actinomyeelemcomitans Serotyp a, Der avec les 5 complexes principaux, Le premier periodontal diseases. In: Socransky. S,S &
funfte lComplex bestand aus I'eillonelta par- complexe avait une relation frappante avec Haffajee. A, D . ed. Microbiology and Im-
vula und Actinomyces odontolyticus. A. acti- les mesures cliniques de la maladie parodon- munology of periodontal diseases, Period-
nomycetemccmitans Serotyp b. Selenomonas tale. en particulier la profondeur des poches ontology 2000. 5. 78-111,
noxia und Actinomyees naeslundii Gcnospe- et !e saignement au sondage. Hosaka, Y. Saito. A.. Nakagawa. T. Seida.
zies 2 {A. viscosus) lagen auCerhalb der Clu- K,. Yamada. S, & Okuda. K, (1994) Effect
ster mit geringer Verbindung zueinandcr und of initial therapy on dynamics of immuno-
zu den 5 Hauptkomplexen, Der crste Kom- globulin G levels to some periodontopath-
ptex hatte eine auffallende Verbindung zu ic bacteria in serum and gingival crevicular
den klinischen Parametern der Parodontaler-
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1998 - Socransky - Microbial Complexes in Subgingival Plaque

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,/ Clin Periodontol IWS: 25: 134-144 Copyright © Munksguard 1998

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Microbial complexes in S.S.Socransky\ A.D.Haffaiee\

subgingival plaque Departments of, 'Periodontology, ^Biostalistics,

Table L Baseline elinieal eharacteristies of subject group («= 185)

F/^, I. Dendrogram of a cluster analysis of 32 subgingival taxa. The

present. Signals were evaluated visually

Community ordination was per-

aging data for a chosen parameter Proportion of Sites exhibiting

onstrated the relationships among the and A. naeslundii genospecies 2 {_A.

% sites colonized the same data together with the percen-

Fi^. ti. Bar chart of the mean (±SEM) % of species

Fig. 14. Diagrammatic representation of the relation-

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