Oral Microbiol Immunol 1996: 11: 266-273Printed in Denmark • All rights reserved

Copyright © Munksgaard 1996

CMMtmtaology&TdImmimology

ISSN 0902-0055

Polymerase chain reactiondetection of 8 putative periodontalpathogens in subgingiva! plaqueof gingivitis and advancedperiodontitis lesionsAshimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8putative periodontal pathogens in siibgingival plaque of gingivitis and advancedperiodontitis lesions.Oral Microbiol Immunol 1996: 11: 266-273. © Munksgaard, 1996.

A 16S rRNA-based polymerase chain reaction (PCR) detection method was usedto determine the prevalence of Actinobacillus actinomycetemcomitans, Bacter-oides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingi-valis, Prevotella intermedia, Prevotella nigrescens and Treponema denticola in sub-gingival specimens of 50 advanced periodontitis, 50 adult gingivitis and 50 pedi-atric gingivitis subjects. The optimal PCR conditions were determined for eachstudy species. Agarose gel electrophoresis of PCR products from each study spe-cies revealed a single band of the predicted size. Restriction enzyme digestion ofamplicons confirmed the specificity of the amplification. PCR detection limitswere in the range of 25-100 cells. No cross-reactivity with other oral micro-organisms or nonspecific amplification was observed. The prevalence by PCR inadvanced periodontitis, adult gingivitis and pediatric gingivitis subjects was30%, 14%) and 14% for A. actinomycetemcomitans, 86%, 18% and 8%o for B.forsythus, 74%, 52% and 78% for C rectus, 80%, 70% and 66% for E. corrodens,70%, 10% and 14% for P. gingivalis, 58%, 12% and 18% for P. intermedia, 52%,20% and 22% for P. nigrescens, and 54%, 16% and 16% for T. denticola, respec-tively. The prevalence was higher in the advanced periodontitis group than inboth adult gingivitis and pediatric gingivitis for A. actinomycetemcomitans, B. for-sythus, P. gingivalis, P. intermedia, P. nigrescens and T. denticola at P<0.01, andfor E. corrodens at P<0.05. The prevalence of C. rectus was significantly higherin the advanced periodontitis group than in the adult gingivitis group at P<0.01.Matching results between PCR and culture occurred in 28% {B. forsythus) to71% {A. actinomycetemcomitans) of the samples; the major discrepancy occurredin the PCR-positive/culture-negative category. Matching results between PCR andDNA probe methods were found in 84% of the subjects {B. forsythus) and 70%{P. gingivalis). Odds ratio analysis revealed statistically significant positive as-sociations between 17 of the 28 possible combinations (P<0.01). This study dem-onstrated the utility of a 16S rRNA-based PCR detection method for identify-ing important subgingival microorganisms. The results indicated a strong associ-ation between the study species and periodontitis. Several previously unreportedsymbiotic relationships were found between the 8 species tested.

A. Ashimoto^'2, C. Chen\ I. Bakker\J. Slots^^Department of Periodontology, School ofDentistry, University of Southern California,Los Angeles, California, USA, ^Department ofOral and Maxiliofacial Surgery, Faculty ofMedicine, Tottori University, Yonago, Japan

Key words: polymerase chain reaction;diagnostics; periodontal pathogen; bacterialinteraction; periodontal disease

Jorgen Slots, University of SouthernCalifornia, School of Dentistry, MC 0641,Department of Periodontology, Los Angeles,CA 90089-0641, USA

Accepted for publication September 11,1995

Prevalence of subgingival microorganisms by PCR 267

The periodontal pocket provides anecological niche for at least 500 bac-terial taxa (19). A few of these speciesmay play a significant role in peri-odontitis. A rapid and rehable mi-crobial identification method will beuseful to associate specific organismswith periodontal disease. Current tech-niques for periodontal microbial identi-fication include culture, nucleic acidand immunologically based diagnosticmethods. Culture methods are time-consuming, expensive and may fail togrow some important organisms.Whole chromosomal DNA probes forsome organisms have problems withlow specificity, and cloned DNA probesmay show low sensitivity (17). Immuno-diagnostic methods require specificantibody reagents not readily availableand may result in false-positive resultsdue to the cross-reactivity with nontar-geted organisms.

Polymerase chain reaction (PCR) of-fers a highly sensitive and specific detec-tion method for bacteria in biologicalsamples (31). Among many potentialamplification sites, 16S rRNA genes ap-pear to be the most useful target ofPCR. 16S rRNA genes are present inevery bacterium and are highly con-served within a species (27). The genescontain signature sequences dis-tinguishable among different bacterialspecies. 16S rRNA-based PCR detec-tion method is particularly valuable fordetection of microorganisms that can-not be cultivated or easily distinguishedin culture (23, 31).

To determine the prevalence of candi-date periodontopathogens in peri-odontal disease, this study used a 16SrRNA-based PCR detection method todetermine the prevalence of Actino-bacillus actinomycetemcomitans, Bacter-oides forsythus, Campylobacter rectus,Eikenella corrodens, Porphyromonasgingivalis, Prevotella intermedia, Prevot-ella nigrescens and Treponema denticolain 50 patients with advanced peri-odontitis, 50 adults with slight gingi-vitis and 50 pediatric patients withshght gingivitis.

Material and methodsSubjects and microbial sampling

Table 1 hsts the sex and ages of the 150study subjects. Subgingival plaquesamples from advanced periodontitissubjects were submitted by extramuraldentists to the Oral Microbiology Test-ing Laboratory at the University of

Table 1, Characteristics of the study population

No. of males(range and mean age in years)

No. of females(range and mean age in years)

Advanced periodontitisAdult gingivitisPediatric gingivitis

22 (17-75; 46.4)29 (21-36; 26.0)25 (2-11; 7.4)

28 (15-71; 45.6)21 (21-33; 27.3)25(3-11; 6.8)

Southern California (USC) School ofDentistry. Adult gingivitis samples werecollected from dental students at theUSC School of Dentistry. Pediatric gin-givitis samples were obtained from thepedodontic clinic at USC School ofDentistry. Subgingival samples were ob-tained and processed as previously de-scribed (36). None of the gingivitis sub-jects had a periodontal pocket with >4mm pocket depth. The gingivitis sub-jects had not received antibiotics for thelast 3 months and were free of any sys-temic diseases. The advanced peri-odontitis samples were processed within2 days of samphng and the adult, andpediatric gingivitis samples were pro-cessed within 1 day of sampling. A

transport time of 48 h in the VMGA IIItransport system (18) does not seem tocause any appreciable loss of bacteria(16, 36). Sample sites were isolated withcotton rolls and air dried. The supra-gingival plaque was removed and paperpoints were inserted to the depth of thepockets. Advanced periodontitissamples were taken from the 3 deepestperiodontal pockets in each patient.Adult gingivitis samples were collectedfrom tooth #16 (or #17), #11 (or #12),#36 (or #37) and #44 (or #42) (toothnumbering according to the FDI two-digit system (26)). Pediatric gingivitissamples were collected from tooth #54(or #55), #51 (or #52), #74 (or #75) and#83 (or #82). The paper points were re-

Table 2. Species-specific and ubiquitous primers for PCR

Primer pairs (5'-3')Base position

(amplicon length in bp)

A, actinomvcetcmcomitansAAA CCC ATC TCT GAG TTC TTC TTCATG CCA ACT TGA CGT TAA AT

B. forsythusGCG TAT GTA ACC TGC CCG CATGC TTC AGT GTC AGT TAT ACC T

C rectusTTT CGG AGC GTA AAC TCC TTT TCTTT CTG CAA GCA GAC ACT CTT

F. corrodensCTA ATA CCG CAT ACG TCC TAA GCTA CTA AGC AAT CAA GTT GCC C

P. gingivalisAGG CAG CTT GCC ATA CTG CGACT GTT AGC AAC TAC CGA TGT

P. intermediaTTT GTT GGG GAG TAA AGC GGGTCA ACA TCT CTG TAT CCT GCG T

P. nigrescensATG AAA CAA AGG TTT TCC GGT AAGCCC ACG TCT CTG TGG GCT GCG A

T. denticolaTAA TAC CGA ATG TGC TCA TTT ACA TTCA AAG AAG CAT TCC CTC TTC TTC TTA

Ubiquitous primer*GAT TAG ATA CCC TGG TAG TCC ACCCC GGG AAC GTA TTC ACC G

478-1,034 (557)

120-760 (641)

415-1,012(598)

169-856 (688)

729-1,132 (404)

458-1,032 (575)

219-1,022 (804)

193-508 (316)

786-1,387 (602)

* Base positions of ubiquitous primers are from Fscherichia coli.

268 Ashimoto et al.

moved after 10 s, and placed into a vialcontaining VMGA III transport me-dium for microbial identification.

PCR primers

Table 2 lists the PCR primers used inthe study. Species-specific PCR primerswere designed with the help of the ribo-somal database project (RDP) pro-grams (12). The primers for B. for-sythus., C. rectus, E. corrodens., P. gingi-valis and T. denticola have beendescribed previously (31). The primersfor A. actinomycetemcomitans, P. inter-media and P. nigrescens were designedfor this study. Briefly, 16S rRNA se-quences of phylogenetically close spe-cies were aligned and the signature se-quences identified. Upstream anddownstream signature sequence primers(approximately 20-base each) were thenverified for their species-specificity bycomparing the sequences with all theavailable 16S rRNA sequences in theRDP database (12). Ubiquitousprimers that match almost all bacterial16S rRNA genes but not 18S rRNAwere used as a positive control for thePCR amplification. All primers weresynthesized on a Beckman DNA SMautomated DNA synthesizer (USCComprehensive Cancer Center, Los An-geles, CA).

Table 3. Species used to study the specificityof PCR primers for detection of putativeperiodontal pathogens

A ctinobacillus actinomycetemcomitansBacteroides forsythusCampylobacter rectusEikenella corrodensEnteric gram-negative rodsFacultative and anaerobic Actinomyces

speciesFusobacterium speciesHaemophilus aphrophilusMitsuokella dentalisNeisseria speciesPeptostreptococcus microsPorphyromonas gingivalisPrevotella corporisPrevotella denticolaPrevotella intermediaPrevotella melaninogenicaPrevotella nigrescensPropionibacterium speciesPseudomonas aeruginosaStaphylococcus aureusStaphylococcus epidermidisViridans and beta-hemolytic Streptococcus

speciesCandida species

PCR detection

The samples in VMGA III werewarmed to 37°C for 10 min and mixedwell on a Vortex mixer; 0.2 ml of themicrobial suspension was washed 3times with distilled water. The bacterialpellets were resuspended in 0.1 ml ofdistilled water, boiled for 10 min andplaced on ice. After centrifugation toremove cell debris, the supernatant wasused for PCR analysis.

PCR was performed as described bySaiki et al. (25). Briefly, 5 ]x\ of samplewas added to 45 jil of reaction mixturecontaining 5 |il lOXPCR buffer (Pro-mega, Madison, WI), 1.25 unit TaqDNA polymerase (Promega), and 0.2mM of each of deoxyribonucleotides(Pharmacia LKB, Piscataway, NJ). In-itial experiments found optimal MgCl2concentrations in the mixture to be 1.0mM for A. actinomycetemcomitans, P.intermedia and P. nigrescens and 1.5mM for B. forsythus, C. rectus, E.corrodens, P. gingivalis and T. denticola.PCR amplification was performed in aDNA thermal cycler (PTC-100, MJ re-search, Boston, MA). The PCR tem-perature profile for B. forsythus, C. rec-tus, E. corrodens, P. gingivalis and T.denticola included an initial denatura-tion step at 95 °C for 2 min, followed by36 cycles of a denaturation step at 95 °Cfor 30 s, a primer annealing step at60 °C for 1 min and an extension step at72 °C for 1 min and a final step of 72 °Cfor 2 min. The temperature profile forA. actinomycetemcomitans, P. interme-dia and P. nigrescens included an initialstep of 95°C/2 min, followed by 36'cycles of 94°C/30 s, 55°C/1 min and72°C/2 min, and a final step of 72°C/10min.

PCR products were analyzed by 1.5%agarose gel electrophoresis performedat 4 V/cm in Tris-acetate EDTA buffer.The gel was stained with 0.5 fig/ml ethi-dium bromide and photographed under300-nm ultraviolet light. A 1-kb DNAladder digest (Life Technologies, Gai-thersburg, MD) served as the molecularweight marker.

Type strains and common referencestrains of the target organisms wereused to determine the specificity andthe detection levels of the PCRmethod. These included A. actino-mycetemcomitans strains ATCC 29522,29523 and 29524, B. forsythus strainATCC 43037, P. gingivalis strainATCC 33277, P. intermedia strainATCC 25611 and P. nigrescens strain

ATCC 33563, and clinical isolates ofspecies listed in Table 3.

Each PCR primer pair was testedagainst samples containing the targetorganism alone or the organism mixedwith the species listed in Table 3. Thelack of cross-reactivity of the PCRmethod was also verified using genetic-ally close species such as A. actinomyce-temcomitans and Haemophilus aphro-philus. To determine the PCR detectionlevels, PCR was performed on sampleswith a variable amount of the target or-ganism mixed with other bacterial spe-cies. The detection level was expressedas the lowest number of target cells de-tected by PCR.

Selected PCR products from typestrains and 1 or 2 clinical samples weresubject to restriction enzyme digest toconfirm the specificity of the amplicons.The restriction enzymes were selectedbased on the 16S rRNA gene sequenceof the target sites; these included Sma Ifor A. actinomycetemcomitans, E. corro-dens, P. gingivalis and T. denticola, DraI for B. forsythua, P. intermedia and P.nigrescens, Cla I for B. forsythus and T.denticola, and Hind III and Xba I forC. rectus. Approximately 3~5 \ig ofDNA was digested to completion in atotal volume of 50 jil according to themanufacturer's recommendations. Therestricted DNA fragments were separ-ated by 2.0% agarose electrophoresis inTris-boric EDTA buffer containing 0.5Jig/ml ethidium bromide and visualizedunder ultraviolet illumination as de-scribed above.

1 2 3 4 5 6 7 8 910

(kbp)

2.01.61.00.5

Fig. 1. Electrophoresis results of PCR ampli-fication. A single DNA band of the predictedsize was obtained by PCR using specificprimer pair against the target organism.Lane 1, 1 kb ladder; Lane 2-10, A. actino-mycetemcomitans, B. forsythus, C. rectus, E.corrodens, P. gingivalis, P. intermedia, P. nig-rescens, T. denticola and ubiquitous primersrespectively.

Prevalence of subgingival microorganisms by PCR 269

Species •

\/ ////// /A

1 ^y''yXyyyX/j^

^^^^^^^^^^^^^

—

[H pediatric gingivitis(n=50)

Q adult gingivits (n=50)

• advanced periodontitis (n=50)

yZZ/ZZZ/ZZ/ZZZZZZZZZZZZZZZZZ/ZZA

\/zzzy/^yyyyy/^ '^yzZ/Z/^z/zz//zz//zzzzzz/zzzzzz/1 1

L , ,.-,., : 1Vz^z^^^^

^^z/^^zz^

-

1 [ 1 [

1 1

A. actinomycetemcomitans

B. forsythus

C. rectus

E. corrodens

P. gingivalis

P. intermedia

P. nigrescens

T. denticola

0 10 20 30 40 50 60 70

Fig. 2. Prevalence of subgingival microorganisms detected by PCR.

80 90 100 (%)

Culture and identification

Culture and species identification of sub-gingival organisms were performed ac-cording to established methods (31).Briefly, samples diluted to 1:10^ to 1:10^were plated onto 4.3% Brucella agarbase (BBL Microbiology Systems,Cockeysville, MD) supplemented with0.3% Bacto-agar, 5% defibrinated sheepblood, 0.2% hemolyzed sheep red bloodcells, 0.2% yeast extract, 0.0005% heminand 0.00005%) menadione for total vi-able counts and for identification of B.forsythus, C. rectus, P. gingivalis and P.intermedia/P. nigrescens. Aliquots fromundiluted and 1:10 diluted samples werealso plated onto TSBV medium selectivefor A. actinomycetemcomitans (29). Pri-mary incubation of the Brucella bloodagar medium took place at 35°C for 8-10 days in a Coy anaerobic chamber(Coy Laboratory Products, Ann Arbor,MI) containing 85% N2-10% H2-5%CO2. The TSBV medium was incubatedat 35°C for 3 days in 10% CO2-90% air.The organisms were presumptively iden-tified using rapid identification methods(30). Proportional recovery of individualspecies was determined by comparingtheir colony counts with the total viablecounts on the nonselective Brucella agarmedium.

DNA probe detection

A digoxigenin-labeled whole chromoso-mal DNA probe for B. forsythus and P.gingivalis was developed using the Gen-

Nonradioactive DNA Labelingand Detection Kit (Boehringer Mann-heim Biochemicals, Indianapolis, IN).The procedures for DNA probe detect-ion were as previously described (16,36).

Statistical analysis

One-factor analysis of variance was usedto determine the statistical significancein the differences of the prevalence andthe number of detected species among 3subject groups. The odds ratio calcu-lation was applied to analyze the re-lationship between two bacterial species.The significant of the association was de-termined by the chi-square test.

ResuitsSpecificity and sensitivity of PCR

The specificity of PCR primers wasconfirmed against phylogeneticallyclose species and against common oralspecies (Table 3). No cross-reaction wasobserved for all tested primers. The de-tection sensitivity was 25-100 colony-forming units (data not shown). Allprimers amplified a band of the pre-dicted size and no amplification of dif-ferent sized bands was observed (Fig.1). The amplicons showed correct re-striction digested fragments of the pre-dicted size (data not shown).

Prevalence by PCR

The prevalence of the organisms in ad-vanced periodontitis, adult gingivitis

and pediatric gingivitis subjects was30%, 14% and 14% for A. actinomyce-temcomitans, 86%, 18% and 8% for B.forsythus, 1A%, 52% and 78% for C.rectus, 80%, 70% and 66% for E. corro-dens, WA, 10% and 14% for P. gingi-valis, 58%, 12% and 18% for P. interme-dia, 52%, 20% and 22% for P. nigres-cens, and 54%, 16% and 16% for T.denticola, respectively (Fig. 2). Theprevalence was higher in advanced peri-odontitis group than in both adult andpediatric gingivitis for A. actinomyce-temcomitans, B. forsythus, P. gingivalis,P. intermedia, P. nigrescens and T.denticola at P<0.01, and for E. corro-dens at /*<0.05. The prevalence of C.rectus was higher in advanced peri-odontitis group than in adult gingivitisgroup (i'<0.01). There was no signifi-cant differences between adult gingivitisand pediatric gingivitis groups exceptfor a higher prevalence of C. rectus inpediatric gingivitis than in adult gingi-vitis (P<0.01).

Fig. 3 shows the distribution of sub-jects according to number of study spe-cies identified. The mean number ofspecies was 5.00 for advanced peri-odontitis (significantly higher than inthe other 2 subject groups (P<0.01)),2.13 for adult gingivitis and 2.36 forpediatric gingivitis. Forty advancedperiodontitis patients (80%) were colon-ized by 4 or more species. In contrast,39 pediatric gingivitis subjects (78%)and 41 adult gingivitis subjects (82%)harbored less than 4 species.

Comparison between PCR and culture orDNA probe detection

Culture data were available for 49 of the50 advanced periodontitis subjects.Table 4 compares the detection of indi-vidual organisms by PCR and by cul-ture. Matching results between PCRand culture occurred in 28% of the sub-jects for B. forsythus, to 71% of the sub-jects for A. actinomycetemcomitans. Themajor discrepancy between PCR andculture were several PCR-positive/cul-ture-negative findings, ranging from16% for A. actinomycetemcomitans to69% for B. forsythus. PCR negative/cul-ture-positive results were found in lessthan 6% of the subjects for P. gingivalis,B. forsythus and C. rectus, and in 12and 16% of the subjects for A. actino-mycetemcomitans and P. intermedia/P.nigrescens, respectively.

A comparison between PCR andDNA probe detection v^as performed in

270 Ashimoto et al.

44 samples (Table 5). Matching resultswere found in 70% of the subjects forP. gingivalis and in 84% for B. for-sythus. The discrepancy between thetwo methods for detection of B. for-sythus appeared to be evenly divided be-tween the PCR-positive/DNA probe-negative (7%) and the PCR-negative/DNA probe-positive (9%) category. The

discrepancy in detection of P. gingivaliswas due mainly to a large proportion(23%) of PCR-negative/DNA probe-positive results.

Association between bacterial species

Odds ratio analysis revealed a positiveassociation for 17 of the 28 bacterial

H pediatric gingivitis (n=50)

adult gingivitis (n=50)

advanced periodontitis (n=50)

3 4 5Number of detected species

Fig. 3. Number of species detected by PCR in each patient.

Table 4. Comparison of detection by PCR and culture in 49 periodontitis patients

culture + culture- Total culture + culture— Total

A. actinomycetemcomitansPCR+ 5 (10.2) 8 (16.3) 13PCR- 6(12.2) 30(61.2) 36

B. forsythusPCR+ 8 (16.3)PCR- 1 (2.0)

34 (69.4)6 (12.2)

427

Total 11 38 49 Total 40 49

C. rectus P. gingivalisPCR+ 12(24.5) 24(49.0) 36 PCR+ 9(18.4) 25(51.0) 34PCR- 3(6.1) 10(20.4) 13 PCR- 0(0.0) 15(30.6) 15

Total 15 34 49 Total 40 49

P. intermedia/P. nigrescensPCR+ 25(51.0) 12(24.5) 37PCR- 8(16.3) 4(8.2) 12

Total 33 16 49

Table 5. Comparison of detection by PCR and DNA probe in 44 periodontitis patients

PCR+PCR-

Total

DNAprobe+

35 (79.5)4(9.1)

39

B. forsythus

DNAprobe—

3 (6.8)2 (4.5)

5

Total

386

44

PCR+PCR-

Total

DNAprobe+

29 (65.9)10 (22.7)

39

P. gingivalis

DNAprobe-

3 (6.8)2 (4.5)

5

Total

3212

44

combinations tested (chi-square test;P<0.01) (Table 6). The highest oddsratio occurred for C. rectus and T.denticola at 32.90. Forty-two of the 43T. denticola-posiiiye subjects were alsocolonized by C. rectus. Other statisti-cally significant odds ratio included B.forsythus/T. denticola (14.60), B. for-sythus/1 . gingivalis (13.58), P. interme-dia/T. dentiola (7.56), C. rectus/E. corro-dens (6.35), P. gingivalis/P. intermedia(5.85), B. forsythus/P. intermedia (5.66),A. actinomycetemcomitans/P. nigrescens(5.19), B. forsythus/P. nigrescens (4.53),B. forsythus/C. rectus (4.44), C rectus/P. nigrescens (3.78), A. actinomycetem-comitans/B. forsythus (3.57), A. actino-mycetemcomitans/P. gingivalis (3.57), P.gingivalis/T. denticola (3.44), C. rectus/P. intermedia (3.34), A. actinomycetem-comitans/T. denticola (2.96), and P. gin-givalis/P. nigrescens (2.75). No negativeassociation (odds ratio <0.5) was foundbetween any two test species.

Discussion

This study reported the use of 16SrRNA-based PCR detection of 8 majorputative periodontal pathogens. Al-though 16S rRNA genes from differentbacterial species share a high degree ofsimilarity, we were able to identifyunique regions suitable for PCR appli-cation. The PCR method will be valu-able for identification of organisms ex-hibiting high degree of genetic andphenotypic similarity. For example, A.actinomycetemcomitans is geneticallyclosely related to H. aphrophilus; bothspecies are found in subgingival plaqueand exhibit similar colony morphology.P. intermedia and P. nigrescens show94% similarity and 6.6% difference in16S rRNA genes (21); both organismsare frequent inhabitants of periodontalpockets and are difficult to distinguishby conventional culture identification.Nevertheless, the 16S rRNA-base PCRdetection was able to identify each ofthese organisms without evidence ofcross-reactivity.

The annealing temperatures and themagnesium concentrations are criticalfactors in PCR detection. High an-nealing temperatures and low mag-nesium concentrations generally im-prove the distinction of microbial spe-cies, but may result in low detectionsensitivity. In contrast, lower annealingtemperatures and higher concentrationsof magnesium increase detection sensi-tivity but may result in cross-reactivity

Prevalence of subgingival microorganisms by PCR 271

Table 6. The odds ratios of associations among tested species from 150 subjects

A. actinomy-cetemcomitans

B. forsythusC. rectusE. corrodensP. gingivalisP. intermediaP. nigrescensT. denticola

A. actinomy-cetemcomitans

_3.57*1.941.033.57*1.965.19*2.96*

B.forsythus

-4.44*2.01

13.58*5.66*4.53*

14.60*

Crectus

—6.35*1.833.33*3.78*

32.90*

E.corrodens

—

1.670.901.991.41

P.gingivalis

—

5.85*2.75*3.44*

P.intermedia

—

2.127.56*

P.nigrescens

—

2.56

T.denticola

Chi-square test; P<0.01.

and undesirable nonspecific amplifi-cation bands. The optimal PCR con-ditions should be verified for each indi-vidual species. We observed a lack ofamplification for P. intermedia if thePCR annealing temperature was raisedfrom 55°C to 60°C. Cross-reactivity be-tween P. intermedia and P. nigrescenswas noted if magnesium concentrationwas increased from 1 to 1.5 mM.

In the present study, individualsamples were divided into small ali-quots for use in repeated experimentsto detect the 8 study species and tocompare PCR, culture and DNA probeidentification. This allowed only 0.5%volume of each samples to be applied ineach PCR test. Since the PCR detectionlimits were in the range of 25-100 cells,samples containing less than 5000-20,000 cells of the individual speciesmight not reveal the organisms. There-fore, the bacterial prevalence in the sub-jects studied may have been underesti-mated.

Comparison between PCR and cul-ture detection of A. actinomycetem-comitans, B. forsythus, C. rectus, P. gin-givalis and P. intermedia/P. nigrescensrevealed a discrepancy ranging from28% {A. actinomycetemcomitans) to71% {B. forsythus), with most discre-pancies occurring in the PCR-positive/culture-negative category. Since cultureidentification of A. actinomycetem-comitans was performed on selectivemedium, which greatly increases the de-tection sensitivity, the PCR and cultureresults were more alike for this species.The other 5 species were cultivated onnonselective medium, which hampersthe identification of small number ofthe organism in the presence of a largenumber of background cells. Some spe-cies such as B. forsythus may also bedifficult to grow in culture even whenoccurring in large proportions. Detec-tion of dead bacteria by PCR may

further contribute to the higher detec-tion rate of PCR.

The PCR and DNA probe detectionmethods were more compatible anddemonstrated matching results in 84%of the samples for B. forsythus and in70% for P. gingivalis. Neither nucleicacid-based identification method re-quires the growth of viable bacteria.Cross-reactivity for the whole DNAprobe (17) may explain the high numberof PCR-negative/DNA probe-positiveresults for P. gingivalis. Alternatively,different aliquots of samples may con-tain variable number of target organ-isms and may contribute to the discre-pancies between detection methods.

The prevalence for each of the speciesA. actinomycetemcomitans, B. forsythus,P. gingivalis, P. intermedia, P. nigres-cens and T. denticola was 22% or less inadult gingivitis and pediatric gingivitissubjects, suggesting that these organ-isms are not major components of thesubgingival plaque in nonperiodontitispatients. The prevalence of these 6 spe-cies was significantly higher in ad-vanced periodontitis subjects ( JP<0.01) ,

ranging from 30% for A. actinomyce-temcomitans to more than 50% for theother 5 species. In comparison, a sub-gingival nonperiodontopathic organismlike Bacteroides heparinolyticus appearsonly in 6% of periodontitis patients,using PCR detection (3). These datasuggest a strong association betweenthe 6 study organisms and peri-odontitis.

The prevalence of C. rectus and E.corrodens in adult gingivitis and pedi-atric gingivitis subjects was greater than50%, indicating that these two speciesmay be part of the normal oral flora.However, C rectus and E. corrodenstended to occur at higher prevalence inadvanced periodontitis. These two spe-cies may be considered endogenouspathogens that occasionally contribute

to the development of periodontitis.Our findings also revealed a lowerprevalence of C. rectus in adult gingi-vitis than in pediatric gingivitis, sug-gesting a negative relationship betweenthis organism and age. C rectus and E.corrodens may need to exceed relativelyhigh critical threshold values in the sub-gingival flora to produce progressiveperiodontal disease.

Previous studies have determined theprevalence of subgingival periodontalpathogens in healthy or diseased sub-jects by culture, DNA probe and immu-nofluorescence identification methods(1, 4-16, 20, 22, 32, 33, 35, 36). It isdifficult to compare the results fromthis and other studies, since the subjectsand the detection methods used weredifferent. In this study, PCR detectionappeared to detect a higher prevalenceof the test organisms than in some ofthe previous culture studies. In ad-dition, we have shown that the difficult-to-grow T. denticola inhabits more than50% of advanced peridontitis lesions.Overall, the results of our study are ingeneral agreement with previousstudies.

The relationship between any twobacterial species in all subjects was in-vestigated by odds ratio analysis. Wefound a statistically significant positiveassociation in 17 of the 28 possiblecombinations. Previous positive re-lationships between periodontopatho-gens have been observed for T. dentico-la/P. gingivalis (24, 28), B. forsythus/C.rectus, P. gingivalis/P. intermedia, P. in-termedia/C. rectus, and E. corrodens/C.rectus (34), and among P. gingivalis, P.intermedia and B. forsythus (2, 16). Thisstudy noted additional synergistic re-lationships between 9 species pairs,including positive associations betweenA. actinomycetemcomitans/B. forsythusand E. corrodens/P. gingivalis, whichwere previously reported to exhibit

272 Ashimoto et al.

negative associations (34). The use ofdifferent patient populations and detec-tion methods may explain the discrep-ancy between the studies. The remain-ing 7 positive associations were not in-cluded in previous studies, partly due tothe new species status of P. nigrescens.

High odds ratio between organismsmay indicate a symbiotic relationship inperiodontal pockets. A pathogen maymore readily colonize subgingival sitesalready occupied by other organisms,due to gingival inflammation or growthfactors produced by other organisms.However, some organisms may occurtogether in periodontitis lesions merelybecause they both produce destructivedisease without interacting with eachother. A therapeutic regimen leading toconcomitant suppression or eliminationof symbiotic microorganisms mayachieve particularly great clinical bene-fits.

In conclusion, the present study dem-onstrated the usefulness of 16S rRNA-based PCR detection for periodontalorganisms in subgingival samples. ThePCR detection method was more sensi-tive than culture, and seemed to haveless cross-reactivity than DNA probedetection. PCR detection may befurther simplified when multiplex PCRmethod for detection of different peri-odontal organisms in a single step am-plification is developed. Our datashowed that A. actinomycetemcomitans,B. forsythus, P. gingivalis, P. interme-dia, P. nigrescens and T. denticola wereassociated with advanced periodontitis.C. rectus and E. corrodens appeared tobe frequent colonizers of subgingivalpockets and may also be associatedwith periodontitis.

Acknowiedgements

We thank M. Jane Flynn for assistancein microbial culture identification andSumalee Sangsurasak for collectingpediatric gingivitis specimens.

References

1. Ali RW, Bakken Y, Nilsen R, Skaug N.Comparative detection frequency of 6putative periodontal pathogens in Sud-anese and Norwegian adult periodontitispatients. J Periodontol 1994: 65: 1046-1052.

2. Ali RW, Skaug N, Nilsen R, Bakken YMicrobial associations of 4 putativeperiodontal pathogens in Sudanese adultperiodontitis patients determined by

DNA probe analysis. J Periodontol1994: 65:1053-1057.

3. Ashimoto A, Flynn MJ, Slots J. Molecu-lar genetic detection of Bacteroides hep-arinolyticus in adult periodontitis. OralMicrobiol Immunol 1995: 10: 284-287.

4. Bretz WA, Lopatin DE, Loesche WJ.Benzoyl-arginine naphthylamide(BANA) hydrolysis by Treponemadenticola and/or Bacteroides gingivalis inperiodontal plaques. Oral Microbiol Im-munol 1990: 5: 275-279.

5. Chen CKC, Dunford RG, Reynolds HS,Zambon JJ. Eikenella corrodens in thehuman oral cavity. J Periodontol 1989:60: 611-616.

6. Collins JG, Offenbacher S, Arnold RR.Effects of a combination therapy toeliminate Porphyromonas gingivalis in re-fractory periodontitis. J Periodontol1993: 64: 998-1007.

7. Dahlen G, Wikstrom M. Occurrence ofenteric rods, staphylococci and Candidain subgingival samples. Oral MicrobiolImmunol 1995: 10: 42^6.

8. Dzink JL, Socransky SS, Haffajee AD.The predominant cultivable microbiotaof active and inactive lesions of destruc-tive periodontal diseases. J Clin Peri-odontol 1988: 15: 316-323.

9. Frisken KW, Tagg JR, Laws AJ, OrrMB. Suspected periodontopathic micro-organisms and their oral habitats inyoung children. Oral Microbiol Immun-ol 1987: 2: 60-64.

10. Jones AA, Kornman KS, Newbold DA,Manwell MA. Clinical and microbiologi-cal effects of controUed-release locally de-livered minocycline in periodontitis. JPeriodontol 1994: 65: 1058-1066.

11. Lai C-H, Oshima H, Slots J, ListgartenMA. Wo line Ha recta in adult gingivitisand periodontitis. J Periodont Res 1992:27: 8-14.

12. Larsen N, Olsen GJ, Maidak BL et al.The ribosomal database project. NucleicAcids Res 1993: 21: suppl 3021-3023.

13. Lippke JA, Peros WJ, Keville MW, Sav-itt ED, French CK. DNA probe detec-tion of Eikenella corrodens, Wolinellarecta and Fusobacterium nucleatum insubgingival plaque. Oral Microbiol Im-munol 1991:6: 81-87.

14. Listgarten MA, Lai CH, Young V. Mi-crobial composition and pattern of anti-biotic resistance in subgingival microbialsamples from patients with refractoryperiodontitis. J Periodontol 1993: 64:155-161.

15. Loesche WJ, Lopatin DE, StoU J, vanPoperin N, Hujoel PP. Comparison ofvarious detection methods for peri-odontopathic bacteria: can culture beconsidered the primary reference stan-dard? J Clin Microbiol 1992: 30: 418-426.

16. Lotufo RFM, Flynn J, Chen C, Slots J.Molecular detection of Bacteroides for-sythus in human periodontitis. OralMicrobiol Immunol 1994: 9: 154-160.

17. Melvin WL, Assad DA, Miller GA,Gher ME, Simonson L, York AK. Com-parison of DNA probe and ELISAanalysis methods and their associationwith adult periodontitis. J Periodontol1994: 65: 576-582.

18. Moller A JR. Microbiological examin-ation of root canals and periapicaltissues of human teeth. Odontol Tidskr1966: 74: 1-380.

19. Moore WEC, Moore LVH. The bacteriaof periodontal diseases. Periodontol2000 1994: 5: 66-77.

20. Okuda K, Fukumoto Y, Takazoe I.Enumeration of cultivable black-pig-mented Bacteroides species in humansubgingival dental plaque and fecalsamples. Oral Microbiol Immunol 1988:3: 28-31.

21. Paster BJ, Dewhirst FE, Olsen 1, FraserG. Phylogeny of Bacteroides, Prevotella,and Porphyromonas spp. and relatedbacteria. J Bacteriol 1994: 176: 725-732.

22. Rams TE, Feik D, Slots J. Campylo-bacter rectus in human periodontitis.Oral Microbiol Immunol 1993: 8: 230-235.

23. Relman DA. The identification of uncul-tured microbial pathogens. J Infect Dis1993: 168: 1-8.

24. Rivere GR, Smith KS, Carranza N Jr etal. Association between Porphyromonasgingivalis and oral treponema in subgin-gival plaque. Oral Microbiol Immunol1996: 11: 150-155.

25. Saiki RK, Geldand DH, Stoffel S et al.Primer-mediated enzymatic amplifi-cation of DNA with a thermostableDNA polymerase. Science 1988: 239:487^91.

26. Sandham JA. The FDI two-digit systemof designating teeth. Int Dent J 1983: 33:390-392.

27. Schmidt T, Relman DA. Phylogeneticidentification of uncultured pathogensusing ribosomal RNA sequences.Methods Enzymol 1994: 235: 205-222.

28. Simonson LG, McMahon KT, ChildersDW, Morton HE. Bacterial synergy ofTreponema denticola and Porphyromon-as gingivalis in a multinational popula-tion. Oral Microbiol Immunol 1992: 7:111-112.

29. Slots J. Selective medium for isolation ofActinobacillus actinomycetemcomitans. JClin Microbiol 1982: 15: 606-609.

30. Slots J. Rapid identification of import-ant periodontal microorganisms by cul-tivation. Oral Microbiol Immunol 1986:1: 48-55.

31. Slots J, Ashimoto A, Flynn MJ, Li G,Chen C. Detection of putative peri-odontal pathogens in subgingival speci-mens by 16S ribosomal DNA amplifi-cation with the polymerase chain reac-tion. Clin Infect Dis 1995: 20 (suppl 2):S304-S307.

32. Slots J, Rams TE, Feik D, Taveras HD,Gillespie GM. Subgingival microfiora ofadvanced periodontitis in the Domin-

Prevalence of subgingival microorganisms by PCR 273

ican Republic. J Periodontol 1991: 62:543-547.

33. Slots J, Zambon JJ, Rosling BG, Reyn-olds HS. Actinobacillus actinomycetem-comitans in human periodontal disease.Association, serology, leukotoxicity, andtreatment. J Periodont Res 1982: 17:447^48.

34. Socransky SS, Haffajee AD, Dzink JL,Hillman JD. Associations between mi-crobial species in subgingival plaquesamples. Oral Microbiol Immunol 1988:3: 1-7.

35. Tanner ACM, Listgarten MA, EbersoleJL, Strzempko MN. Bacteroides for-sythus sp. nov., a slow-growing fusiform

Bacteroides sp. from the human oral cav-ity. Int J Syst Bacteriol 1986: 36: 213-221.

36. Tay F, Liu YB, Flynn MJ, Slots J. Evalu-ation of a non-radioactive DNA probefor detecting Porphyromonas gingivalisin subgingival specimens. Oral Micro-biol Immunol 1992: 7: 344-348.