Microbial Complexes in Subgingival Plaque - Socronsky - J Clin Perio 1998
Microbial Complexes in Subgingival Plaque - Socronsky - J Clin Perio 1998
Microbial Complexes in Subgingival Plaque - Socronsky - J Clin Perio 1998
1 Clio Pffiodorrrol
Den mark
Denmark . . righlf maned
MIEN
clinical periodontology
R. L.Kent Jr. 2
C.Smith` sled
Socronsky SS. Haffajee AD. Cunini MA, Smith C. Kent ✓r. RL: Microbial
compleses re subgingival plaque. .1 Alin Periodontal 1998: 25: 134-144.
Munksgaard. 1998,
Abstract. It has been recognized for some time that 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
sampl. and different clustering and ordination techniques. Subgingival plaque
samples were taken from the mesial aspect of each tooth in 185 subjects (mean
age 51 T 16 years) with (n=160) or without (n25) 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 saes per tooth at each visit. Similarities between pairs
of species were computed using pin 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
ns
were coistently observed using any of the analytical method. One complex
consisted of the tightly related group: Bameroider Ancrydrus. Porphyromonas
gingivalis and Treponema denticida. The led complex consisted of a tightly re-
lated core group including members of the fungracicrium nuckunim/periodonticum
subspeci., Prevalent, iniermedia. Prevotella nigresem and Peptostreptococcus
micros. Speciesassociated with this group included: Euhamerium nodanini,
Campylobamer refry, Camnylognmer shown, Sirepioroccus ragtag,. and
Campylobacter gracilis. The 3rd complex consisted of Streptococcus sanguis. S.
(ming S. mitis, S gordimii and S. in media. The 4th complex was comprised
of 3 Capnocytophaga species, Cor pyinhacter cone's.. Eikenella corroder. and
Achnobricillm aminonisceremcomiians serotype a. The 5th complex consisted of
Veillonella parvule and Arlinonigres odoniolyikm. A. actirunnyceroncomiumv sere-
type b, Selenomonas noxia and Actinomyces naesiundii genospecies 2 (A. vircosurr
were outliers with little relation to each other and the 5 major complexes. The Kelt uttCrOtbdOgyf saw.. suogingtva
tat complex related strikingly to clinical measures of periodontal disease particu- Planow evotosx ertrrePoutat
larly pocket depth and bleeding on probing. Accepted lor publication 10 May 1097
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 lade order in the micro- lining of the pocket while gram positive
ecosystem by Van Leeuwenhoek in 1683 biota that colon es gingival sulci or rods and cocci appeared to be forming
(Tal, 1980) Since that time, numerous periodontal pockets. However, examen 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).
nuc.oscopy, cultural techniques and ing degree of order in colonisation pat- Cultural, immunologic or DNA
more recently imunologic
m 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 Leenwenhoek's initial ob- rangement of morphologically distinct occur together M 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 gingimlis is almost always observed in
of bacterial species. Indeed it has been (Listgarten et al. 1975). Subgingival samples that am harboring Bacteroides
estimated that 400 or more species re- plaque was frequently characterized by firmyikes. It has teen speculated that B.
Microbial complexes 135
forrythus in some fashion precedes odontitis or acute necrothing ulcerative acetate. The released DNA was placed
colonization by P. gingival, since B gingivitis was included in the study. into the extended slots of a Minislot
forsylhus is detected more frequently by (I romunetics, Cambridge MA) and then
itself (Gmur et al. 1909). Other com- concentrated onto a nylon membrane
011111010 monitoring
plexes that have been observed include (Boehringer Mannheim) by vacuum
P gingivalis and Treponema &Nicola Subjects were screened for suitability and filed to the membrane by exposure
(Simonson et al. 1992b) and Pomba, and if accepted, were asked to sign in- to ultraviolet light followed by baking
nurianrum and Pram,. inter formed consent forms. All subjects were at 120°C for 20 mM The Minislot de.
media (Ali at 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 ata month intervals post therapy. Plaque "lanes" on a single 15X15 cm nylon
anderstanding the biology of subgingi- accumulation (OM, overt gingivitis(Oro, membrane as well as 2 control lanes
val plaque and in planning strategies bleeding on probing (0/1), suppuration containing 10 or 10 4 cells of each test
for its control. Knowledge of the eco- (0/1). probing pocket depth and probing specim. The membrane with fixed DNA
logical relationships among bacterial attachmem level were measured at 6 sites vats placed in a Miniblotter 45 (Im-
species can direct and focus investi- per tooth (mesiobmcal, buccal, disto- mundics, 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"
Technological developments permit lars et each visit. The baseline clinical pattern was produced with 5 of the
the evaluation of large numbers of bac- features of the 185 subjects are presented probe lanes remaining empty to permit
terial species in large numbers of plaque in Table I. accurate localization. Each channel was
samples from a wide range of subjects. used as a hybridization chamber for
Such procedures provide a data base in Microbiological asammment
separate DNA probes. Signals were do
which associations among bacterial tatted by chemiluminescence.
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-DIVA 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- 42°C for 1 hr M 50% formamide, 5 xSSC
with mom levels of each specks. Cluster cies wore determined in each plaque (1 XSSC=150 mM NaCl, 15 mM Na
analysis has been useful in describing sample using the checkerboard DNA- citrate. pH 7.0), I% casein (Sigma, St.
closely related species when more than DNA hybridization technique (Socran- Louis MO), 5 X Denhardfs reagent, 25
a few pairs of species are examined. sky et al. I994). After the removal of mM sodium phosphate (pH 6.5) and 0.5
Community ordination is a procedure supragingival plaque, subgingival men' yeast RNA (Boehringer Mann-
that attempts to indicate closely related plaque samples were taken with individ- heim). Digoxigenin-labeled, whole chro-
species within a community and then ual sterile Gracey curdles 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 (Fein-
feilm communities of species within the were placed in separate Eppendorf berg & Vogelsilin 1983). The probes and
ecosystem of imerest. The purpose of tubes containing BD ml TE (10 mM hybridization buffer were placed in indi-
the present investigation was to use Tris-HCI, 1 mM EDTA. pH 7.61. 0.15 vidual lanes of the Miniblotter and the
cluster analysis and community ordi- ml of 0.5M NaOH was added to each whole apparatus placed in a sealed plas-
nation techniques to examine relatMil sample and the sample boiled M a water tic bag. Membranes were hybridized
ships among bacterial species in subgin- bath for 5 mM. The samples were neu- overnight at 429C 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 I. Baseline clinical char bjeci OmnP14-1851
Material and Methods Mean (4513) Range
nonahnlon
age (Years/ 5,16 20-87
185 subjects ranging in age from 20-87 no. mng teeth 3.1±3.1 o-8
years who were considered to be peri- ma. 43
dentally healthy M=25) or with evi- mean pocket depth 1 ,11m) .7-6.6
dence of prior attachment lam (n=160) moan attachment level (nm) 0.8-6.8
were selected for study. All subjects had sites wiih:
plaque 6,430 o-tou
at least 20 teeth. Exclusion criteria in- and 72,0 o-tou
cluded pregnancy, periodontal therapy BOP 49,7 0-10o
or antibiotics in the previous 3 months, suppuration 0.49
any systemic condition which might pocket depth <4 mm 74,0 11-100
have affected the progression or treat- pocket depth 4-6 ram 2,17 0.76
ment of periodontitis and the need for Pocket depth >6 nim ,46 0.57
pm-medication for monitoring or ther- attachment level mm 72,4 -100
apy. No subject with localized juvenile allaclmmut level 0.6 mm 2,111 7
attachment level >6 mm 0.6.4
periodontitis, rapidly progressive pert-
136 St
4
2
5x SSC. I NDenhardt's reagent, 20 Will SDS, 20 min, twice) in a Disk Wisk ap- I:25.000 dilution of anti-digoxigenin
Na phosphate (pH 6.51. 0.2 mg/m1 yeast paratus (Schleicher and Schuell. Keene antibody conjugated with alkaline
RNA. 20 ng/ml of labeled probe. ION NH). phosphatase using the modification de-
dextran sulfate and I% casein. Mem- scribed by Engler-Blum at aL (1993).
bra nes were washed at low stringency to Defection and enumeration After washing, the membranes were in-
remove loosely bound probe and then at To detect hybrids, membranes were cubated in Lumiphos 530 (Lumigen,
high stringency (68°C, 0.IxSSC. al % blocked and then incubated with a Southfield. MI1 for 45 min at 37°C.
Mirrnhial complexes 137
Stetielicel [manna
Fig. I presents a dendrogram of the re- g Area plots demoting M e geme, nt of saes c Ionised by members of the orange and
red c The data are based on exams at o 13,221 subgingival Mabee an. The
sults of the cluster analysis of all 13,261
samples using 3 coefficients and an av-
88 Lad ;LI indicate Me percentage of s tes cola teed by 0, I 4, 5-8 and a-1,1 members
of the orange complex. The right hand pan I press Is the same data, but overlaid with the
erage unwmghted linkage sort. 5 clus- percentage of sites in each of those mtegor es that shreued I, 2 or 3 members of the red
ters were formed with >6064 similarity complex.
and tn.-eluded 29 of the 32 taxa evalu-
ated. The red cluster consisted of P. gin-
;tool's. B. Forsyth. and F denacola.
The orange cluster consisted of 6' nu-
electrum subspecies. P. in media and
nigrescens. Peptostreprococcus micros
and Campy/abode, rectos. Cumpylo-
heeler shoos?, Campylobacter gracilis, 64 27
E nodaturn and S. roustellat. The 3
Cognocyrophagu species, Cennpylo-
barter coml.,. Einenella corroder. and
Actinoboollusacanornyceterneornitans
WORTS a formed the green cluster,
while a group of streptococci made up
the yellow cluster. Streptococcus mi.,
Streptococcus usuguis and Streptococ-
cos oxalis were most closely related
I.
within this group. Aram/ogees odonto- Pn' T1=a*rler=e : "
lyre r. and Veillonella parvule formed Ft, P. Area plots depicting thss of Atm colonTed by members of the red and eran.
the purple cluster. 2t 0,5100- coin kg.. T. left hand panel indrea es the 1, of des ...zed by 0, 1.2 or 3 membersof
dii genospecies 2 (Actinomyces she red complex. The right hand...Texnts the same data, but overlaid with the perm.,
«scosus), Selenomonas rt oxia and A. of sites in each of those categories that exhibited 0,1-4, 5-8 and 9-12 members of the orange
actinornyceterneornitans serotype b did coot rex
not cluster with other species.
onst ated the relationships among the and A naeslundn genospecies 2 (A.
Amerelations among N.M.l mere. ea
Cap ocgtophogo species, most of the visrosus) was evident (Figs. 2 .3).
determined by community ordination
Streptococcus species. E. corroders and Fig, 4, 5 present principal compon-
The results of community ordination C conc.. These species were closely ents analysis of the same data and indi-
using correspondence analysis are pre- relat d to each other and somewhat re- cate similar groupings of species This
t
tot P. ginglanas K nun. se vincontll
0 Doi
harbored 1, 2 or 3 members of the red
0 2437..eho complex. It is readily apparent that
% sites colonized
Relationship between bacterial spades
tee 7 P gingivalis F. nuc. as vincenal S. sang&
and GIME. parameters
0 TO B Pg SI TO Pg ■ Td BI•Pg All
x
Counts
of
found that P intermedia. C. radar and On observing Fig. 14, it is tempting Further. other studies have suggested
P micros were significantly ekvated in to speculate that the relationships ob- that different therapies affect different
saliva samples from subjects with ad- served mimic to some extent the segments of the microbiota and the use
vanced periodontitis compared with crobial succession patterns that may an therapy may be ineffective
samples from subjects with initial or no exist in developing plaque. It seems powering the target species and might
in lowering
periodontitis. Further, treatment that likely that A. weekend; genospecies 2 adversely affect other members of the
included systemically administered me- vireos.) and the streptococcal spa. microbial community (Han). et al.
tronidreole drereased levels of these cies are early colonreen followed by 1996/.
species and improved periodontal sta- Capnorpophaga sp, C. cantina. and E The data of the present investigation
tus. Members of the orange complex corradenr. A. odontaipicus and V par- suggest the nature of some of the mi-
have been associated with infections in rots may be bridging species leading to crobial complexes in subgingival
nomperiodontal sites. Sundqvist (1992) the or 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 specm. of species evaluated was rearenably ex-
cluded F. nucleation, P. intermedia, P. lation and in part based on data about tensive. the investigation represents an
mirror. anaerabius. Euharterhart the composition of plaques in various initial attempt at evaluating in
and C rector. Strong associations were clinical conditions (Moore & Moore among subgingival speci..
found between F nuclea and P. 1994). It ateresting to note that a While some of the associations depicted
micros, F. nuelestum and C r eelle and
tum treatment such as SRP has a profound may not be completely accurate, certain
P. intermedin and P micros. Summanen effect on the species of the red complex associations were seen repeatedly using
et al l (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 increare in accord with data in the literature.
ous abscesses of intravenous drug users in A. traestundii genrepeci. 2 (A. Further. certain complexes, and mem-
were F nucleation. P micros. P. in viscosuffand some of the streptococcal bers within the complexes. related
media.nigreseens, A. adantolyticur species (Haifa* nal. 1997). strongly to clinical parameters of in-
and V permute while F. nucleation, P. The red complex showed the strong- flammation and periodontal destruc.
intermedia, P. nigrescens, P. mirror and est relationship with the clinical par. non. The data provide a framework for
Pubacterium species were most com- ameters considered most meaningful in understanding the complex ecology ob-
monly detected in 46 cases of anaerobic periodontal diagnosis. For example. the served in plaque and could be used to
empyema (Circa et al. 19951. P in individual species in the complex as well guide approaches to diagnosis and ther-
has been shown to stimulate the as the complex itself related very apy of periodontal diseases.
growth of S. constellatur in vitro and the strongly with pocket depth and bleed-
combination of these sp.ies often pro- ing on probing. As described earlier in
d uced fatal pulmonary infections in a the Discussion, other investigators have
Acknowledgments
mouse model system (Shinzato & Saito noted the relationship between pocket
19945 One possible mechanism for co- depth and members of the red complex This work was supported in part by re-
aggregation between E. iodation and (Gmur et al. 1989. Kigure et al. 1995. search grants DE-0488I and DE-I0977
strains of F. nreleanren was described by Simonson et al. 1992b). The orange from the National Institute of Dental
George and Falkler 119921. The wag- complex also 'elated to packet depth. Research
gregation was thought to invalve a pro- although this relationship and the re-
tein receptor on F. nucleation and a lationship to other clinical parameters
heat-stable protein or polysaccharide was less striking. Zusammenfassung
component on E. in/datum. Knowledge of the assaiations be- .1rISnAs.d. Komplex.. in der subsingivolen
While species within complexes were tween subgingival species maybe used Pimple
closely associated. the complexes them- to iterpret
n and guide periodontal then- Es wurde sen einiger Zeit erkannt, dell bak-
selves seem to have specific relation- apy. The recognition that an inter-re- lerielle in der subging.len Plaque
ships with one another (Fig. 12). Spe- lated complex of species such as the red in Komplexen existieren. Der Zurek der vor-
cies such as A. naeslundii genospecies 2 complex exists suggests that therapies liegenden Untersuchung war es. die Defini -
(A. vireos.). and members of the yel- that affect I of there species may influ- tion solcher Gemeinsehaften tamer Verwen-
dung der Oaten einer groBen Aneahl von
low and green complexes were less com- ence the colon sea of the other spe-
Ylaqueprohen sow verschiedener Ouster-
reonly associated with members of the cies in this group. In additio n.eit might an iken re versuchen Bei d
red and orange compkres than with be speculated that altering th orange INS Person= I Altersdurchshont 1,11a,
each other. The 2 species M the purple complex might prevent subsequent re) wurden von der MesiaMache eines *lot
compkx were strongly related to each colonization of species in the red com- Zahn. min tn=1601 der okre Parodontitis
other and to a lesser extent to members plex providing the therapist with a sec- subgingivek zlaintsond/sn 'Room.
of the orange, green and yellow com- ond route to control this group of or en. Dater yerwendang von Gane- Genon,
plexes. The reasons for these relation- ganisms. A actinomyreternearnirons D NA-Sullen tad der Schachbrett-DNA-
D NA-Hybridnierung wurden rias Vorkom•
ships among complexes is unclear but serotype b related poorly to members of
men und der Anteil von 40 subging.len
it might be speculated that antagonistic the red and orange complexes sug-
Taxa bei 13261 Plaqueproben bestimmt. Zu
relationships (Greater 19961 may exist gesting that therapies effective against jedem Termin warden an 6 Mellen pro Zahn
or thatenvironments selective for one one set of pathogens n5ay no nec.s- klinischen Mesa ungen durchgernhrt Ahn-
sec arily be effective against others. This in- hchkeite ewi.hen Speeiespuaren wurden un-
ond group of organisms. deed appears to be the case for SRP Mr Vemendung von Phi-Koeffieienten en
Microbial complexes 103
realm( and die Speziesclusterung ed.& denticala. La second complete emit modal magmegation actio ren between
umcr Verwendung einer ungewichteten constime par un noyatt apparen- Porphyromonas gingivalis and Gm/mom
Durchschniworiemienen Serung. Mit al- te comprenant des membres des sous-espbees demicoa O ierobiology and
kn analmischen Method. warden 5 Haug- Fusobacterium nuelernperiodon ' • P 1 I1 280-284,
komplexe bst8ndig beobachtet. Ein Kom- orella inrermedia. Prevotella nformiono Grenier. D (19925) Nutritional interactions
plex lowland acts der eng verbundenen Grup- Peprovrepoeoceus meow, Les splens asso- between two suspected periodontopathog-
Pw Bact,oblei. forstibm, Porphibonionat cims avec co groom compremimM &barb, ens, Trepone. dem., and Porpbyra.
gingoalis and Treponema dentia. Der rium Campylolwrier wen, C., giovalis. Inferlion and Immunity
zweite Komplex lowland acts einer eng vet- lobar,' Winn... Streploeneno owl.. et 60, 529,5301.
bundenen Rentralgruppe mit den Subspezies CompyWarier gracilis. Le troisieme Grenier, D. (1996) Antagonistic effect Moral
von Fusoberforium nuckanimbierbionlictun. Clad compose de Strrplecurnor
plate Clair bacteria town. Treponema denfirola.
Prey.. LI MM. Arte. misn,,,e.. mins. 5. ganknii aS. inierme- Journal of Clinical Microbiology 34. (249-
und Peptostrepiocoma niimn Spe:ies. die ALT. Lc quatritme complexe constme .252.
mil dieser
Eu Gruppe ass.. warm sin, fob Par 5 esPhm. , almb0,baiga. Campy, Haffajee. A. 1,, Cugini, M. A., Dibart, S.
genus, imam, Con,lobarter tinder .f.i.., Edirne. comas et Act, Smith, C_, Kcal, R L... Socransky S.S.
recrus, Cum,bobacier sbamie_bereprococesu noliaedias aoinomyeeternemnans semi, a. (1997) The effect of SRP on the clinical
sections and Cornbarre. grarilis. Der cinspneme ample. era, Lampe. per and microbiological parameters of peri-
drine Komplex hem.. aus Srrepralveria Vernon, parrula et Arrinomyeei iblantablb odontal diseases. Journal of Clinical Per,
.binguis, oral.. S. mlris. gordonii und tvs Ala oriphtrie, A. atiintimyteienurn adonio(ogylt 32,334.
inierineditis Der vier, Xamplev .14, die Haffajee, A. IX & Socransky, S. S. (1994) Mi-
3 Capno0ophaga Sperles. ((mlobimer rionbm naeslundli genospecies 2 (A. tokru- crobial etiological agents of destructive
roncisus. Zikenella earroderis used Aerio, uid, avaient Ms peu de relation entre eux et periodontal diseases. Socransky, S.S
calm; aciinoimernromiums Mr,. a. Der atree les 5 comple.s principaux. Le pruner Haffajee, A. LI. ed. Microbiology and Inc
UMW Komplex bemand aus G./5964. compleite avait une relation frappante avec munology of periodontal diseases Period-
and Actinomyees odonioblicus. A. at., les mcsurcs chniques de la maladie parodon. ontology 20130 5. 18 111.
nomycelemcomibm Scrotyp Selenomonas tale, en particulier la profondeur des pmhes Homka. Y., Saito. A., Nakagawa. T. Saida,
noxia und Acibiontyces naeslunda Genospe- et le saignement au sondage. K. Yamada.. & Okuda, K.119941 Erect
■
zies 2 (A. viscorms) arn aufierhalb 4er ClE) of initial thaupg M dynamic's 0,51.191°)
der mit geringur VerbiWung weinander und globulin G levels to some periodontopath-
zu den 5 Haumkomplexen. Der erste Kom- C bacteria in serum and gingival crevicular
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