INFECTION AND IMMUNITY, May 1985, p. 507-519 Vol. 48, No. 20019-9567/85/050507-13$02.00/0Copyright © 1985, American Society for Microbiology

Comparative Bacteriology of Juvenile PeriodontitisW. E. C. MOORE,'* L. V. HOLDEMAN,' E. P. CATO,' R. M. SMIBERT,V J. A. BURMEISTER,2 K. G. PALCANIS,2

AND R. R. RANNEY2Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061,and Department of Periodontics, School of Dentistry, Virginia Commonwealth University, Richmond, Virginia 232982

Received 8 November 1984/Accepted 12 February 1985

Statistical comparisons of the floras associated with juvenile periodontitis, severe periodontitis, and moderateperiodontitis indicated that differences in the bacterial compositions of affected sites in these populations werenot statistically significant. The subgingival flora of affected juvenile periodontitis sites was statisticallysignificantly different from the adjacent supragingival flora and from the subgingival floras of people withhealthy gingiva and of children with developing (experimental) gingivitis. However, the subgingival flora ofaffected juvenile periodontitis sites was not significantly different from the flora of sites with gingival indexscores of 1 or 2 in adults with developing (experimental) gingivitis. Of 357 bacterial taxa among over 18,000isolates, 54 non-treponemal species, 2 treponemal species, and mycoplasma were most associated with diseasedperiodontal sulci. These species comprised an increasing proportion of the flora during developing gingivitisand constituted over half of the cultivable flora of diseased sites.

Classical juvenile periodontitis (JP) often has been consid-ered to be a distinct periodontal disease that is distinguishedby destruction of tissue and bone associated almost exclu-sively with the first molars and incisors of periadolescentchildren (21). There are indications that the disease isfamilial (1, 3, 7, 24), and a high proportion of the patients hashigh serum antibody levels against Actinobacillus acti-nomycetemcomitans (4, 8, 22). Several authors stronglysuggest a causative role for that species (4, 8, 9, 23, 27, 30).However, studies of the total flora of JP have been limited.For a determination of the possible bacterial causes ofdestruction, the floras in periodontal pockets and gingivalsulci of patients with classical JP were analyzed and statis-tically compared with those of patients with moderate (14) orsevere (17) periodontitis, subjects with gingivitis (15, 16),and subjects with healthy gingiva.

MATERIALS AND METHODSSubjects. For the main study, using nonselective bacteri-

ological methods, samples were collected from 21 individualswith localized JP. The clinical definition of JP and the clinicalexaminations performed were as described previously (2).The age range of the subjects was 10 through 28 years(median age of 20). Seventeen were black and 4 were white;15 were female and 6 were male. Samples from 20 of thesesubjects were cultured for all bacteria; samples from onewere cultured for spirochetes only. All subjects in this groupwere examined and bacteriologically sampled in Richmondat Virginia Commonwealth University (VCU/MCV).Samples from 14 additional JP subjects were cultured only

on selective media. Eight of these subjects were sampled inBaltimore at the University of Maryland, and six weresampled in Richmond. Of these 14 subjects, 12 were black, 1was Asian, and 1 was Caucasian; 8 were male and 6 werefemale. The age range was 12 to 27 years; the mean age was19.

Samples. The teeth were isolated with cotton rolls, and thesample sites were gently dried with sterile cotton swabs. Theaccumulated supragingival plaque at the sites to be sampledwas removed as completely as possible with sterile tooth-

* Corresponding author.

picks. From the 21 persons sampled in the main study group,subgingival samples were collected from 32 affected sitesand from 8 unaffected sites. These samples were collectedfrom the depth of the sulcus or pocket with sterile, nickel-plated 00 Morse scalers with detachable tips. Residualsupragingival samples were taken from 26 affected and 7unaffected sites. Residual supragingival samples also werecollected with 00 Morse scalers and were taken before thesubgingival samples were collected. The supragingival sam-ples served both as control samples for monitoring possiblecontamination of the subgingival samples and for compara-tive purposes to determine which components of the flora

TABLE 1. Distributions of teeth with periodontal attachment lossin the main study populationAll teeth First molars and incisors

Subject No. No.-5 No. 2 2 No. No. 2 5 No. 22present mm' mmb present mm mm

0081 28 8 13 12 7 80105 31 11 17 11 9 110109 28 2 7 12 2 40113 28 2 4 12 1 20114 31 5 7 11 4 50115 26 3 5 11 2 30116 28 4 7 12 2 30122 25 7 11 9 4 60204 28 1 12 12 1 60221 28 7 8 12 5 50222 28 6 9 12 3 40243 24 0 17 11 0 80281 26 5 7 11 4 50312 30 3 19 12 3 100317 29 8 12 12 6 70338 31 2 13 11 1 80340 25 1 4 9 1 20348 27 6 15 11 4 70355 27 2 5 12 2 40368 28 11 16 12 10 120376 25 4 6 9 1 1a Loss of attachment of 5 mm or more on at least one surface.b Loss of attachment of 2 mm or more on at least one surface (includes

those 2 5 mm).

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TABLE 2. Clinical values at sample sites in the main studypopulation

TypeofSubjectTooth plbProbeable Loss ofType of Subject Tofothb, p1b GI depth attachment

site no. surface' (mm)" (mm)d

Unaffected 0114 12 M 1 1 3 00115 12 M 1 0 3 00122 12 M 0 0 3 00204 29 M 1 0 3 00221 6 M 1 1 3 00243 4 M 1 2 4 10281 5 M 1 0 2 00338 4 M 1 1 3 0

Affected 0081 10 M 2 1 7 714 M 1 1 11 9

0105 7 M 3 1 9 1214 M 3 1 8 5

0109 14 M 0 0 7 519 M 1 2 9 7

0113 14 M 0 1 7 50114 14 M 2 2 9 80115 3 M 1 1 9 60116 30 M 2 1 12 140122 3 M 2 1 9 8

31 M 2 1 8 50204 30 M 1 2 7 40221 30 M 2 2 8 70222 3 M 1 0 7 50243 3 M 2 2 7 40281 14 M 2 2 9 80312 14 M 2 2 7 4

30 M 2 2 6 30317 8 D 1 1 8 9

14 M 2 2 9 70338 30 D 2 2 8 60340 28 M 2 1 7 4

30 M 2 1 8 50348 3 D 2 2 5 7

14 D 2 2 6 80355 14 M 2 2 6 5

30 D 2 2 9 70368 8 D 2 2 7 9

14 M 2 1 7 50376 29 M 1 1 8 5

30 D 1 2 12 10

a Teeth are numbered 1 to 32 from maxillary right to mandibular right; M,mesial; D, distal.

b PI, Plaque index.'Distance from gingival margin to base of pocket.d Distance from cemento-enamel junction to base of pocket.

were most specifically associated with the depth of theperiodontal pocket or site of tissue destruction.

Subgingival samples from the second group of 14 subjects,to be cultured only on selective media, were collected onlyfrom affected sites. Each of the 18 samples from the eightpatients sampled at the University of Maryland was col-lected"with one paper point inserted to the depth of thesulcus for 10 s. Each of the 13 sites from six patients atVCU/MCV was first sampled by paper point and thenimmediately by scaler. In all cases, microscopic counts,dilutions, and inoculation of media were performed immedi-ately after sampling, before incubated transport of culturesto Virginia Polytechnic Institute and State University forisolation and identification.

Culture procedures. Methods for bacterial isolation andidentification and statistical analyses were identical to thosedescribed earlier (16, 17). Briefly, immediately after sam-

pling, the tip with the sample or the paper point was placedin prereduced, anaerobically sterilized broth under oxygen-free CO2. Samples were dispersed by shaking with 100-,um-diameter glass beads, diluted in prereduced salts solution,and cultured in roll tubes of BHIA-D4 medium containingrabbit serum and on reduced anaerobic blood agar plates(BHIA-D4 basal medium with 5% [vol/vol] rabbit blood)(16). Portions of the same dilutions also were inoculated tomedia (16) for the isolation of treponemes and mycoplasma.After 5 days of incubation, bacterial colonies were pickedwithout selection, in a randomized pattern, to obtain arepresentative cross section of the flora. Usually, 15 colo-nies were picked from roll tubes and 15 were picked fromplates. All resulting cultures were streaked and repicked. Ifmore than one morphotype or colony type was present, eachwas isolated and identified. Identification was made byelectrophoretic analysis of the soluble cellular proteins (with-out sodium dodecyl sulfate) (12) and by biochemical andchromatographic procedures (6), supplemented with appro-priate tests as required for facultative or nutritionally de-manding taxa. Undescribed species were assigned letter andnumber designations.Actinomyces species were serotyped with fluorescent-an-

tibody conjugates. Strains of actinomycetes that failed toreact with available monovalent fluorescent-antibody conju-gates, denoted by (-) below, were assigned to the speciesthat they resembled phenotypically. Spirochetes were enu-merated from the highest dilutions that produced growth

TABLE 3. Clincial values at sample sites studiedbacteriologically with selective media only"

Place of Tooth PI GI Probeable Loss ofstudy Subject surface depth attachment

U. Md.b A 3 M 0 2 6 830 M 1 1 7 8

B 3M 1 1 5 314 M 1 1 10 719 M 2 1 11 830 M 1 1 6 3

C 30 M 1 2 6 3D 14 D 2 2 5 3E 14 M 1 1 6 3

19 M 1 1 5 330 M 0 1 9 7

F 3D 1 1 5 614 D 1 2 10 7

G 3D 2 2 7 83M 2 1 8 7

14 M 2 2 7 6H 14 D 1 1 8 5

19 M 2 1 7 5

VCU/MCV' 0464 3 M 1 1 6 314 M 1 2 8 519 M 0 2 8 6

0465 3 M 2 2 7 414 M 2 2 7 2

0493 3 D 2 3 9 519 M 1 2 6 5

0505 7 M 2 3 5 314 M 2 2 6 3

0520 3 M 3 2 4 530 M 2 2 4 6

0534 3 D 2 2 6 430 D 2 1 6 4

a Explanations of column headings are the same as for Table 2.b Sampled in Baltimore at the University of Maryland.' Sampled in Richmond at Virginia Commonwealth University.

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TABLE 5. Taxa that are in higher proportion in disease than in TABLE 5-Continuedother categories (as a percentage of the floras)' Species Disease' Other Ratio

Species Disease" Other Ratio Bacterioides D14 0.13 0.05 2.6Fusobacterium nucleatum 8.15 3.81 2.1 Bacteroides D10 0.11 0.02 5.5Eubacterium nodatum 4.07 0.13 31.1 Actinomyces WVA 963 ()0.10 0.09 1.1Peptostreptococcus micros 4.07 1.55 2.6 Centipeda periodontii 0.10 0.00Strains not surviving through identifi- 4.13 3.57 1.2 Lactobacillus D12 0.10 0.02 5.0

cation Bacteroides melaninogenicus 9343 0.09 0.08 1.1Eubacterium timidum 2.84 0.59 4.8 gr.Wolinella recta 2.62 0.75 3.5 Fusobacterium D07 0.09 0.00Lactobacillus minutus 2.48 0.21 11.8 Facultative positive rod D09 0.09 0.00Eubacterium D08 2.18 0.18 12.1 Streptococc-us D37 0.09 0.08 1.1Bacteroides intermedius 4197 1.95 0.32 6.1 Streptococcus intermedius II 0.09 0.01 9.0Lactobacillus D02 1.94 0.85 2.3 Bacteroides D41 0.08 0.02 4.0Bacteroides gingivalis 1.74 0.07 24.9 Eubacterium D12 0.08 0.00Eubacterium brachy 1.63 0.16 10.2 Eubacterium D15 0.08 0.00Bacteroides denticola 1.36 0.64 2.1 Eubacterium D23 0.08 0.05 1.6Eubacterium Dll 1.36 0.02 68.0 Streptococcus DO5 0.08 0.05 1.6Bacteroides intermedius 8944 1.34 0.81 1.7 Bacteroides D10C 0.06 0.04 1.5Propionibacterium acnes 1.22 1.03 1.2 Fusobacterium DO5 0.06 0.00Bacteroides oris 1.04 0.52 2.0 Fusobacterium D13 0.06 0.00Peptostreptococcus anaerobius 1.04 0.45 2.3 Streptococcus D50 0.06 0.01 6.0Eubacterium D06 1.02 0.27 3.8 Bacteroides D12 0.05 0.01 5.0Bacteroides gracilis 0.99 0.90 1.1 Bifidobacterium D01 0.05 0.03 1.7Selenomonas sputigena 0.96 0.73 1.3 Coccus D18 0.05 0.00Actinomyces israelii I 0.78 0.53 1.5 Eubacterium D18 0.05 0.00Actinomyces meyeri -)0.75 0.34 2.2 Facultative negative coccus D02 0.05 0.01 5.0Wolinella X 0.66 0.45 1.5 Facultative positive coccus D40 0.05 0.04 1.2Bacteroides pneumosintes 0.60 0.10 6.0 Staphylococcus hominis 0.05 0.02 2.5Eubacterium alactolyticum 0.57 0.01 57.0 Acinetobacter calcoaceticus 0.04 0.01 4.0Fusobacterium RD 0.57 0.04 14.2 Actinomyces D06 0.04 0.01 4.0Streptococcus mutans 0.52 0.14 3.5 Bacillus D01 0.04 0.00Bacteroides D28 0.50 0.16 3.1 Bacteroides bivius 0.04 0.03 1.3Wolinella curva 0.47 0.35 1.3 Bacteroides D45 0.04 0.00Fusobacterium D09 0.42 0.02 21.0 Bacteroides D49 0.04 0.03 1.3Actinomyces meyeri 0.42 0.23 1.8 Bacteroides D50 0.04 0.00Bacteroides D25 0.39 0.09 4.3 Coccus D49 0.04 0.01 4.0Fusobacterium D02 0.38 0.10 3.8 Facultative negative rod D04 0.04 0.00Bacteroides buccae 0.37 0.16 2.3 Facultative positive rod DO5 0.04 0.03 1.3Bacteroides loescheii 0.37 0.24 1.5 Lactobacillus brevis 0.04 0.03 1.3Bacteroides oralis 0.37 0.18 2.1 Pasteurella ureae 0.04 0.00Eubacterium D13 0.37 0.03 12.3 Peptostreptococcus D03 0.04 0.00Arachnia propionica 0.36 0.15 2.4 Pseudomonas alcaligenes 0.04 0.02 2.0Selenomonas D06 0.33 0.17 1.9 Staphylococcus capitis 0.04 0.01 4.0Campylobacter concisus 0.32 0.28 1.1 Bacteroides D04 0.03 0.01 3.0Streptococcus sanguis III 0.32 0.18 1.8 Bacteroides DO5 0.03 0.00Peptostreptococcus AN2 0.28 0.07 4.0 Bifidobacterium DO5 0.03 0.00Bacteroides D23 0.27 0.00 Centipeda A 0.03 0.00Bifidobacterium dentium 0.27 0.03 9.0 Coccus D46 0.03 0.00Actinobacillus actinomycetemcomi- 0.24 0.04 6.0 Corynebacterium D02 0.03 0.01 3.0

tans Eubacterium D17 0.03 0.00Streptococcus sobrinuis 0.24 0.21 1.1 Facultative negative rod D15 0.03 0.00Bacteroides D42 0.23 0.02 11.5 Facultative positive rod D06 0.03 0.00Bacteroides melaninogenicus 0.23 0.10 2.3 Facultative positive rod D12 0.03 0.00Eubacterium D04 0.23 0.05 4.6 Lactobacillus D07 0.03 0.01 3.0Bacteroides Ml 0.22 0.06 3.7 Peptostreptococcus magnus 0.03 0.01 3.0Propionibacteriumn avidum 0.22 0.00 Peptostreptococcus prevotii 0.03 0.01 3.0Streptococcus intermedius IV 0.22 0.21 1.0 Peptostreptococcus D43 0.03 0.00Bacteroides capillosus 0.19 0.13 1.5 Propionibacterium D02 0.03 0.00Lactobacillus D10 0.19 0.06 3.2 Staphylococcus saprophyticus 0.03 0.01 3.0Selenomonas D15 0.19 0.07 2.7 Selenomonas D07 0.03 0.01 3.0Bacteroides zoogleoformans 0.18 0.00 Selenomonas D13 0.03 0.02 1.5Lactobacillus D08 0.18 0.05 3.6 Total isolates 7,853 10,069Selenomonas D14 0.18 0.05 3.6Bacteroides D10B 0.15 0.00 aAdditional species that were 0.01% in disease and were not isolated fromBacteroides D19 0.15 0.06 2.5 other sites: Actinomyces D18; Bacillus coagulans; Bac-illus D02, D04, D05,Bacteroides veroralis 0.15 0.08 1.9 007; Bacteroides D38, D40, D46, D52, D53; Bifidobacterium cantenulatum;Eubacterium D24 0.14 0.01 14.0 Bifidobacterium D03, D04; CDC JK group; Clostridium D01. D02; coccus

D22; Corynebacterium D01; Eubac-terium D05, D19, D20. D22. D25, D28;Fusobacterium D01 0.14 0.13 1.1 Fusobacterium D14; facultative negative rod D08, D18; facultative positiveStreptococcus D13 0.14 0.02 7.0 rod D03, D07, D08, D16, D17; Kiebsiella ozaenae; Lac-tobacillus acidophilus;Streptococcus morbilloruim 0.14 0.13 1.1 Micrococcus varians; Peptostreptococcus asaccharolytic-us; Peptostrepto-Bacteroides D32 0.13 0.02 6.5 coccus sacc-harolyticus; Peptostreptococcus D47; Selenomonas D17. D18;Lactobacillus catenaforme 0.13 0.02 2.6 Streptococcus D36. D38, and D44.

bSubgingival severe, moderate, juvenile periodontitis and sites with a GIContinued score of 2 in adults.

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TABLE 6. Species that are not increased in proportion in diseasesites (as a percentage of the floras)a

Species Diseaseb Other Ratio

Actinomyces WVU 963 0.64 3.50 0.2Actinomyces WVU 963 sero. cross 0.00 0.04 0Actinomyces D01 0.01 0.01 1Actinomyces D07 0.01 0.01 1Actinomyces D08 0.10 0.10 1Actinomyces D14 0.00 0.02 0Actinomyces israelii (-) 1.09 1.61 0.6Actinomyces israelii II 0.19 1.30 0.1Actinomyces israelii sero. cross 0.00 0.06 0Actinomyces naeslundii (-) 1.49 3.81 0.3Actinomyces naeslundii I 2.39 7.05 0.3Actinomyces naeslundii II 0.01 0.08 0.1Actinomyces naeslundii III 1.83 2.07 0.9Actinomyces naeslundii sero. cross 0.00 0.06 0Actinomyces NVC 2.04 3.72 0.5Actinomyces odontolyticus (-) 0.88 1.23 0.7Actinomyces odontolyticus I 1.20 2.00 0.6Actinomyces odontolyticus II 0.08 0.08 1Actinomyces viscosus (-) 0.23 0.32 0.7Actinomyces viscosus II 1.31 1.80 0.7Arachnia P1 0.00 0.03 0Bacillus circulans 0.01 0.24 0.04Bacterionema matruchotii 0.04 0.18 0.2Bacteroides buccalis 0.20 0.20 1Bacteroides corporis 0.03 0.03 1Bacteroides D19E 0.00 0.07 0Bacteroides loescheii D1C20 group 0.25 0.29 0.9Bacteroides D22 0.04 0.04 1Bacteroides D26 0.06 0.25 0.2Bacteroides D29 0.01 0.01 1Bacteroides D31 0.06 0.19 0.3Bacteroides D33 0.01 0.03 0.3Bacteroides D24 0.11 0.14 0.8Bacteroides D39 0.01 0.03 0.3Bacteroides D43 0.00 0.03 0Bacteroides disiens 0.04 0.04 1Bifidobacterium D02 0.00 0.07 0Capnocytophaga gingivalis 0.45 1.94 0.2Capnocytophaga ochracea 1.44 2.81 0.5Capnocytophaga sputigena 0.24 0.57 0.4Capnocytophaga X 0.01 0.09 0.1Clostridium putrificum 0.00 0.02 0Clostridium subterminale 0.00 0.02 0Coccus D19 0.00 0.02 0Coccus D29 0.01 0.02 0.5Coccus D30 0.11 0.19 0.6Coccus SM1 0.50 0.94 0.5Coccus SM2 0.00 0.06 0Coccus SM4 0.01 0.3 0.3Eikenella corrodens 0.18 0.22 0.8Eubacterium saburreum 0.28 0.63 0.4Eubacterium D14 0.01 0.01 1Eubacterium D21 0.01 0.01 1Eubacterium D26 0.00 0.02 0Eubacterium D27 0.00 0.02 0Fusobacterium D03 0.03 0.08 0.4Fusobacterium D04 0.05 0.13 0.4Fusobacterium D06 0.03 0.07 0.4Fusobacterium D08 0.00 0.02 0Fusobacterium D10 0.03 0.03 1Fusobacterium naviforme 0.08 0.23 0.3Facultative negative coccus D03 0.04 0.06 0.7Facultative negative rod D09 0.01 0.17 0.1Facultative negative rod D10 0.00 0.02 0Facultative negative rod D13 0.03 0.03 1Facultative negative rod D27 0.00 0.03 0Facultative negative rod D28 0.00 0.02 0Facultative positive coccus D25 0.04 0.27 0.1Facultative positive coccus D41 0.01 0.07 0.1Facultative positive coccus D42 0.01 0.01 1Facultative positive coccus D51 0.00 0.02 0

TABLE 6

Species

Haemophilus aphrophilusHaemophilus parainfluenzaeHaemophilus paraphrophilusHaemophilus segnisLactobacillus D09Lactobacillus D14Leptotrichia buccalisLeptotrichia D16Leptotrichia D22Leptotrichia D35Leptotrichia D37Leptotrichia D48Neisseria flavaNeisseria mucosaNeisseria subflavaPropionibacterium granulosumPropionibacterium D03Rothia dentocariosaStaphylococcus aureus

Staphylococcus epidermidusStaphylococcus haemolyticusStaphylococcus warneriStreptococcus anginosusStreptococcus bovisStreptococcus D02Streptococcus D06Streptococcus D07Streptococcus DllStreptococcus D15Streptococcus D16Streptococcus D21Streptococcus D22Streptococcus D24Streptococcus D31Streptococcus D39Streptococcus D45Streptococcus mitisStreptococcus mitis IStreptococcus mitis IVStreptococcus mitis VIStreptococcus parvulusStreptococcus SAStreptococcus salivariusStreptococcus sanguis I

Streptococcus sanguis II

Streptococcus SMSelenomonas DO1Selenomonas D02Selenomonas D03Selenomonas D04Selenomonas D05Selenomonas D09Selenomonas D10Selenomonas DllSelenomonas D12AVeillonella atypicaVeilionella disparVeillonella parvula

5-ContinuedDisease'0.150.030.060.010.030.000.200.090.040.010.010.000.040.030.010.010.000.090.010.360.080.013.090.010.040.130.360.000.000.170.040.040.010.090.380.000.590.050.000.000.010.090.001.381.570.520.050.330.060.930.000.010.080.130.010.170.223.62

Other Ratio

0.15 10.10 0.30.15 0.40.10 0.10.08 0.40.02 00.48 0.40.19 0.50.15 0.30.11 0.10.01 10.07 00.05 0.80.21 0.10.26 0.040.02 0.50.03 00.49 0.20.02 0.50.36 10.08 10.03 0.34.23 0.70.01 10.38 0.10.23 0.62.55 0.10.08 00.02 01.85 0.010.08 0.50.09 0.40.04 0.20.10 0.90.54 0.70.05 01.15 0.50.15 0.30.03 00.02 00.02 0.50.36 0.20.09 03.63 0.43.59 0.40.92 0.60.37 0.10.35 0.90.31 0.11.53 0.60.02 00.02 0.50.13 0.60.33 0.30.03 0.30.37 0.50.55 0.48.73 0.4

Total isolates 7,853 10,069

a Additional species that were not isolated from disease sites but were

0.01% of the flora in other sites were: Acinetobacter twoffii; Actinomycesodontolyticus (cross); Bacillus D03; Bacteroides putredinis; Bacteroides D36.D44, D51; Clostridium sporogenes; coccus D32, SM3; Fusobacterium D12;facultative motile rod D20; facultative negative rod D07, D17, D22, D24, D26;facultative positive coccus D52; facultative positive rod Dll, D14; Gemellahaemolysans; Lactobacillusjensenii; Lactobacillus plantarum; LactobacillusD22; Propionibacterium A2, D01, D04; Staphylococcus simulans; Streptococ-cus D10, D12, D25, D34, D35, D40, D48; Streptococcus mitis VII; andSelenomonas D08.

b Subgingival severe, moderate, juvenile periodontitis, and sites with GIscores of 2 in adults.

Actinomyces NV is a uniform group of strains that react with bothActinomyces naeslundii I and Actinomyces viscosus II antisera.

Facultative positive rod D10 0.00 0.02 0

Continued

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a-ft-er incubation for 5 to 21 days. Ten single colonies ofspirochetes were isolated, purified, and characterized fromeach sample that produced growth (16). The selective mediaof Mandell and Socransky (10) and of Slots (26) were used toestimate the incidence of Actinobacillus actinomycet-emcomitans.For statistical analyses by the lambda test of Good (5),

which was used to compare the variation between subjectswithin groups with differences in flora composition betweengroups, all 30 subgingival isolates from each affected s'ite inthe same patient or subject were combined into a single 30-,60-, or 90-isolate sample to represent the subgingival flora ofaffected sites in that subject. Supragingival isolates weresimilarly combined to represent the supragingival flora.These floras were compared with those of clinical groupsreported previously (14-17) by lambda analyses (5) of meanminimum percent similarities of subjects' floras within andbetween subject groups.

RESULTS AND DISCUSSIONClinical mneasurements. The numbers of teeth with attach-

ment loss (main study population) are shown in Table 1.These data confirm the localized distribution of advancedperiodontal destruction. The maximum number of severelyaffected teeth in any subject was 11. In all subjects, the mostseverely affected teeth were first molars and incisors. Lessattachment loss was present on some additional teeth, mostof which also were first molars and incisors. One subject hadno severely involved teeth (by the 5-mm attachment losscriterion) but was included because of attachment loss of upto 4 mm with 5 to 7 mm probeable depth on eight first molarsand incisors at age 10.

Clinical values reflecting amounts of plaque, gingivalinflammation, probeable depth, and attachment loss at thesites sampled for complete bacteriological study are shownin Table 2. Unaffected sites sampled had little or no loss ofattachment and shallow sulci, but half had detectable gingi-val inflammation. Affected sites had 5- to 12-mm pocketswith corresponding loss of attachment. All but two werevisibly inflamed, and half bled after gentle stroking of thecoronal portion of the pocket with a probe. All Unaffectedsample sites were premolar except for one, which wascanine. All but three samples from affected sites were fromfirst molars or incisors.Attachment loss patterns and sample sites in subjects

sampled for culturing only on selective media were compa-rable to those in the main study population. Clinical valuesin these sites are listed in Table 3.

Bacteriological data. The composition of the supragingivalfloras of sites affected with juvenile periodontitis (JP1) wasnot statistically significantly different from the supragingivalfloras of affected sites in populations with severe (SP1) ormoderate (MP1) periodontitis, or from the supragingivalfloras of subjects with healthy gingiva (HP1), healthy sites inthe patients with juvenile periodontitis (HJ1), or from thoseof sites with gingival index (GI) scores of 0, 1, or 2, in adults(AGO, AG1, AG2) and in children (CGO, CG1, CG2) in theexperimental gingivitis model (15, 16). Similarly, the subgin-gival floras of affected sites in patients with juvenile (JP2),severe (SP2), or moderate (MP2) periodontitis or sites withGI scores of 2 in adults were not statistically significantlydifferent from each other by lambda analysis (Table 4).These data indicate that (i) the subgingival floras of each ofthe disease categories share many species in common, (ii)the subgingival floras of diseased sites differ significantlyfrom their corresponding supragingival floras, and (iii) the

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514 MOORE ET AL.

TABLE 8. Incidence of predominant species that increase in diseased sites (percent of samples positive for each species)"Periodontitis Experimental gingivitis

Species Juvenile Severe Moderate Healthy Healthy Adults Childrenpersons sites, JP

GI GI GI GI GI 6I AllSup. Sub. Sup. Sub. Sup. Sub. Sup. Sub. Sup. Sub. = = 2 = = 2

Fusobacterium nucleatum 58 68Eubacterium D08 4 32Eubacterium nodatum 39Wolinella recta 12 36Eubacteriumn timidum 27 58Bacteroides oris 4 23Lactobacillus minutus 8 26Peptostreptococcus micros 19 45Eubacterium D06 12 29Bacteroides denticola 35 32Bacteroides Ml 8 6Lactobacillus D02 11 26Actinobacillus 6

actinomycetemcomitansActinomyces meyeri (-) 11 6Peptostreptococcus 11 23

anaerobiusEubacterium Dll 16Bacteroides 11 23

intermedius 4197Eubacterium brachy 8 19Fusobacterium RD 8 16Bacteroides gingivalis 4 6Bacteroides oralis 12 19Bacteroides pneumosintes 13Bacteroides D23 6Eubacterium alactolyticum 6Bacteroides D25 10Eubacterium D24 3Fusobacterium D02 4 13Lactobacillus D08 3Eubacterium D13 10Fusobacterium D09 13Lactobacillus D10 3Selenomonas D14 4 10Peptostreptococcus AN2 8 10Selenomonas D15 4 10Bacteroides D14 8 6Bacteroides D19 3Bacteroides 8 6

melaninogenicusLactobacillus 3

catenaformeArachnia propionica 3Bacteroides buccae 12 3Bacteroides D28 3Eubacterium D04 3Lactobacillu.s D12 3Bacteroides D10Bacteroides D10BBacteroides D32Bacteroides D42Bacteroides

zoogleoformansBifidobacterium

dentiumCentipeda periodontiiPropionibacteriumavidum

Streptococcus D13Streptococcus mutans 8

65 85 68 78 34 44 43 50 56 50 50 41 56 62 615 37 5 23

11 50 14 385 20 23 48

24 70 18 3032 15 18 814 50 18 2830 48 32 503 28 14 15

27 22 18 17S

27 33 32 185 2

5 427 22

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3 249

3 411 11

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5 115 4

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55 149 9

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22 43 384 14 12

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5 8 1115

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2 2 7 23 82 2 12

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No. of samples 26 31

Sup., Supragingival; sub., subgingival.

37 46 22 125 32 27 7 8 18 42 36 46 43 13 559

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516 MOORE ET AL.

TABLE 10. Comparison of the treponema floras (by species concentrations) of sites with different states of periodontal health expressedas probability' on the basis of L analyses of minimum similarities

Periodontitis Healthy Healthy Experimental gingivitis

Category Juvenile Severe Moderate persons sites, JP Adults Children

Sup. Sub. Sup. Sub. Sup. Sub. Sup. Sub. Sup. Sub. GI = 0 GI = 1 GI = 2 GI =O 0G = 1 GI = 2

JP1 ** 0.56 ** 0.24 ** * * 0.07 0.05 0.29 0.87 0.78 0.71 0.31 0.83JP2 ** ** 0.92 ** 0.22 *** * ** * * 0.06 0.06 0.05 * *SPi 0.56 ** ** 0.31 ** *** ** * * 0.17 0.73 0.60 0.53 0.21 0.61SP2 ** 0.92 ** *** 0.24 *** * * * ** * * * ** **MP1 0.24 ** 0.31 * * * 0.09 ** ** 0.22 0.90 0.47 0.67 0.25 0.91MP2 ** 0.22 ** 0.24 * *** *** *** *** ** 0.23 0.14 0.09 * 0.12HP1 * *** *** *** * * 0.41 0.70 0.53 0.99 0.97 0.82 0.98 0.88 0.99HP2 * * ** * 0.09 * 0.41 0.40 0.19 0.75 0.92 0.65 0.92 0.75 0.99HJ1 0.07 ** * *** ** * 0.70 0.40 0.99 0.85 0.46 0.44 0.63 0.88 0.25HJ2 0.05 * * *** ** * 0.53 0.19 0.99 0.66 0.18 0.47 0.54 0.69 0.23AGO 0.29 * 0.17 ** 0.22 ** 0.99 0.75 0.85 0.66 0.54 0.23 0.83 0.99 0.42AG1 0.87 0.06 0.73 * 0.90 0.23 0.97 0.92 0.46 0.18 0.54 0.65 0.99 0.52 0.49AG2 0.78 0.06 0.60 * 0.47 0.14 0.82 0.65 0.44 0.47 0.23 0.65 0.48 0.38 0.16CGO 0.71 0.05 0.53 * 0.67 0.09 0.98 0.92 0.63 0.54 0.83 0.99 0.48 0.83 0.65CG1 0.31 * 0.21 ** 0.25 * 0.88 0.75 0.88 0.69 0.99 0.52 0.38 0.83 0.67CG2 0.83 * 0.61 ** 0.91 0.12 0.99 0.99 0.25 0.23 0.42 0.49 0.16 0.65 0.67

No.o fpeople 17 21 17 20 15 27 20 20 7 8 4 4 4 4 4 4No. of samples 26 32 34 42 22 134 34 34 7 8 18 42 36 46 39 17

a Probability of null hypothesis: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

subgingival floras of diseased sites differ significantly fromsubgingival floras of healthy sites.Because there was not a significant difference in the

subgingival floras of juvenile, severe, and moderate peri-odontitis and adult gingivitis sites with GI scores of 2, thesefloras were combined to represent the "disease" flora. Thisflora was compared with the combined floras of all othersites and categories, which represented the "nondisease"flora. There were 171 species that were found to occur ingreater proportions in the disease subgingival flora than inthe nondisease flora (Table 5), and 167 species that weredetected in equal or greater proportions in the nondiseaseflora (Table 6).

It is reasonable to assume that species that play animportant role in periodontal destruction should occur ingreater proportions in the flora of diseased sites than in thatof other sites, and that they should make up at least a modestfraction of the flora. The 53 non-spirochetal species thateach constituted at least 0.1% of the disease flora and thatwere found in at least twofold greater proportions in dis-eased sites as compared with other sites are listed in Tables7 and 8. Most of these species were present in sites affectedwith juvenile, moderate, or severe periodontitis; they repre-sented 52% of the total cultivable flora in sites affected withmoderate periodontitis and 60 to 61% of the flora in sitesaffected with juvenile and severe periodonitis (Table 7).With the exception of unaffected subgingival sites in patientswith JP, where they were 31% of the flora, their next greatestconcentration was in adult gingivitis sites with GI scores of2. Several of these species increased during the developmentof experimental gingivitis, and in those trials some of thesespecies were not detected until gingivitis had reached GIscores of 2 (16).The distribution of frequency of occurrence of these

species (Table 8) is reasonably similar to the distribution oftheir concentrations in the flora, indicating that for mostspecies, single subjects did not greatly affect the observedproportions of the flora.

Until it can be established whether the composition of the

flora changes during active progression of the lesion orwhether the patient resistance changes to allow periodicprogression of destruction by floras that are not destructiveat other times (studies in progress), we cannot determinewhether those species that occurred infrequently in highproportions of the flora indicate (i) "the only active sitesobserved" (ii) less usual etiology, or (iii) simply occasionalcommensalism. Different sites with comparable destructionin the same patient often harbor floras of statistically signif-icantly different compositions (13). Therefore, during activedestruction, either change in flora composition or change inpatient resistance without change in flora composition isequally possible. Because we have included all species thatrepresent as little as 0.1% of the disease flora, and we haveevidence from sequential clinical measurements that at leasta few of the sites were active sites, species that are agents ofdestruction almost certainly are included in the suspect list.Analyses similar to those above were made with

mycoplasma and treponemal isolates. These data weretreated separately because they are based on results fromselective media and are not directly comparable. The ob-served incidence is from 104 to 106 cells in each samplerather than 30 colonies taken at random to represent thesample. The incidence of mycoplasma and treponemes ineach of the subject categories is shown in Table 9. Thedistribution of several of these species is similar to that ofthe 53 non-treponemal species that are specifically associ-ated with diseased sites.Lambda analyses of the mycoplasma and spirochete data

were based upon the logarithm base 10 dilution in which thespecies were observed (Table 10). Again the subgingivalfloras of persons with juvenile, moderate, and severe peri-odontitis were not significantly different from each other butwere statistically different from most other sites. Therefore,the floras of the diseased sites were combined and comparedwith the combined floras of all other sites (Table 11). Thedata indicate that Treponema denticola and the "largetreponeme" are most specifically associated with diseasedsites.

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JUVENILE PERIODONTITIS 517

TABLE 11. Incidence of mycoplasma and treponemes in diseasesand other sites (as a percentage of samples)Species Disease Other Ratio

Increased in diseaseCulturalTreponema socranskii 60.1 30.0 2.0

subsp. buccalisa 1.9 0.6 3.2subsp. socranskiia 4.8 0.3 16.0

Mycoplasma 31.2 8.5 3.7Treponema denticola 20.2 1.1 18.4Treponema D 11.1 2.8 4.0Treponema pectinovorum 10.6 3.0 3.5Treponema F 4.8 1.4 3.4Treponema G 2.9 0.8 3.6Treponema vincentii 2.9 0.0Treponema E 1.4 0.3 4.7Treponema T 1.4 0.0Treponema U 0.5 0.0

Microscopic"Large treponeme" 31.2 2.5 12.6Treponemes observed 76.4 12.1 6.3

Decreased in diseaseCulturalTreponema socranskii

subsp. paredisa 3.4 3.9 0.9Treponema L 1.0 1.1 0.9Treponema N 0.5 0.6 0.8Treponema S 0.5 0.6 0.8Treponema C 0.0 1.4 0Treponema H 0.0 0.3 0Treponema M 0.0 0.3 0Treponema P 0.0 0.3 0Treponema R 0.0 0.3 0

No. of samples 208 363 590a Preliminary data; all isolates have not yet been identified to subspecies.

Strong associations between Capnocytophaga species andthe subgingival flora of juvenile periodontitis have beenreported (18, 19, 23, 25). Data from the present and relatedstudies do not support this specific association. All threerecognized species and total Capnocytophaga isolates were

found with greater frequency in floras of healthy gingiva, andin supragingival floras of periodontitis conditions, than in thesubgingival JP flora. Incidence and proportions of Capno-cytophaga species also were greater in experimental gingivi-tis in children than in JP subjects (Table 7) (L. V. Holdeman,W. E. C. Moore, E. P. Cato, J. A. Burmeister, K. G.Palcanis, and R. R. Ranney, submitted for publication). Ourresults are in agreement with those of Slots and Rosling (28),who also found no association between Capnocytophagaand juvenile periodontitis.

It has been suggested that Actinobacillus actinomycet-emcomitans is the primary agent of juvenile periodontitis(28). On the routine nonselective BHIA-D4 medium, Acti-nobacillus actinomycetemcomitans was found to be 21% ofthe subgingival flora of one site in one of our JP patients. Itis reasonable to assume that this species had a significantimpact upon the ecology and perhaps the pathology of thatsite. Actinobacillus actinomycetemcomitans also was de-tected with nonselective medium in lower proportions inanother subgingival site in that patient, in subgingival sites ofthree patients with moderate periodontitis, and in one adultand one child in sites with GI socres of 0.Twelve of our JP subjects were under 21 years of age and

9 were 21 or older. The compositions of the floras of thesetwo subgroups were not significantly different (P = 0.88).The only patient with detectable levels of Actinobacillusactinomycetemcomitans was 15 years of age.

Although many of our JP subjects (and some patients withgeneralized severe periodontitis) had high levels of serum

antibody to Actinobacillus actinomycetemcomitans (22), thisspecies was not detected as frequently in the JP flora as hasbeen reported by some other authors (10, 19, 27, 30).Therefore, we used the selective media of Mandell andSocransky (10) and of Slots (26) to survey JP patients inRichmond, Va. and in Baltimore, Md. Representative colo-nies of all morphotypes were characterized by conventionaltests and by electrophoretic patterns of the cellular proteins.The results from selective media were compared with thetotal cultural counts from the same samples on plates ofnonselective BHIA-D4 medium (16) (Table 12).The survey studies indicated that our use of scaler sam-

ples was not the reason for our failure to detect highconcentrations and incidence of Actinobacillus actinomyce-temcomitans in the JP patients in the main study. In theRichmond survey, each affected pocket was first sampledwith a paper point and then resampled with a scaler. Thetotal counts from the two samples in each pair were quitesimilar. The survey sample in which Actinobacillus acti-nomycetemcomitans comprised nearly all of the flora dem-onstrates that, at least in some sites, Actinobacillus acti-nomycetemcomitans might be an etiological agent of JP.Although our survey sample population was small, there isan indication that many patients with signs of classical JP donot carry culturally detectable levels of Actinobacillus acti-nomycetemcomitans, and in many other patients it is presentin insignificant numbers. Of equal interest is the indicationthat the prevalence of Actinobacillus actinomycetemcomit-ans may differ in different geographic areas. This compari-son might explain other reports in which Actinobacillusactinomycetemcomitans was not found to be associated withJP (20, 29).

TABLE 12. Detection of Actinobacillus actinomycetemcomitans in subgingival sites affected with JP

No. of Actinobacilflus-positive samples

Source Medium Method people No. of sitesSource Medium Method ~~~~~sampled sampled Mean % of total(% positive) (% positive) nonselective Range

medium count

Richmond areaMain study Nonselective Scalers 20 (5) 31 (6) 12 3.0-21Survey Selective Paper points 6" (17) 13" (23) 3 0.6-7

Scalers 6" (33) 13" (23) 1 i 0.3-2Baltimore areaSurvey Selective Paper points 8 (50) 18 (61) 16 0.1-114ba The same site was first sampled by paper point, then by a scaler.b In this sample (114%), Actinobacillus actinomycete,ncomitans also was the predominant species on the nonselective medium.

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518 MOORE ET AL.

Bacteriodes Ml, an undescribed species that producesblack colonies on anaerobic BHIA-D4 blood agar, was evenmore specifically associated with JP than was Actinobacillusactinomycetemcomitans (Table 7). Bacteroides Ml repre-sented 47% of the sulcus flora of an affected site in onejuvenile patient and 9% of the flora of a site in anotherjuvenile patient. It represented 12 and 3%, respectively, ofthe residual supragingival flora of these two sites and wasdetected at 3% of the supragingival flora of only one othersite (moderate periodontitis) among 559 samples from 95subjects.The data are consistent with a hypothesis of multiple

possible etiologies of periodontal destruction based on aselect group of species that occurs specifically or in greatlyincreased concentrations or frequency in diseased sites.Several of these species are frequent pathogens of otherbody tissues. For example, Peptostreptococcus anaerobiusis among the 10 most common anaerobic pathogens inclinical infections of other body sites. Among Actinomycesspecies referred to us from clincal laboratories, Actinomycesmeyeri is the most common species in brain abscesses.Peptostreptococcus micros, Lactobacillus minutus, Eubac-terium alactolyticum, Bacteroides bivius, Bacteroides inter-medius, Bacteroides oralis, Bacteroides melaninogenicus,Bacteroides pneumosintes, and Fusobacterium nucleatumare frequently predominant species in other clinical infec-tions (11). Other species in Table 7 have been reported fromnon-oral clinical infections, but their infrequent occurrencesuggests they may represent occasional contaminants ofclinical specimens. Still others of these species may occur innon-oral infections but not be recognized because they arenot described or have been described only recently.

It would be surprising if several of these species did notcontribute to periodontal destruction in JP, MP, and SPpopulations, just as there is no single cause of pneumonia,osteomyelitis, or vaginitis where complex floras also are

found. Inspection of the current data suggests that any of thespecies in Table 7 may predominate in diseased sites, some

with greater frequency than others. With one or two excep-tions, there is no indication in the present work that tissuedestruction in JP is caused by different species from those insevere or moderate chronic periodontitis. However, it isapparent that JP patients have a distinct susceptibility totissue destruction and this condition is well correlated withhigh levels of antibody to Actinobacillus actinomycet-emcomitans. The correlation does not, in itself, indicate thatActinobacillus actinomycetemcomitans is the predominantor only cause of periodontal destruction in JP patients.

ACKNOWLEDGMENTSWe thank J. B. Suzuki, University of Maryland College of Dental

Surgery, and his staff for selecting and scheduling the JP patientsfrom the Baltimore area and for making the necessary laboratoryfacilities available. We thank the California State Department ofHealth Services for cooperative studies with the actinomyces andfor the monovalent fluorescent anti-actinomyces conjugates. Wealso thank Ella W. Beaver, Polly H. Cooper, Jane L. Hungate, AnnC. Ridpath, Debra B. Singsabaugh, Sue C. Smith, Margaret L.Vaught, and Dianne M. Wall for technical assistance, and KimHardison and Felicia Caine for assistance in subject management.The JP study was financed by Public Health Service grant

DE05139 from the National Institute of Dental Research and byproject 2025790 from the Commonwealth of Virginia.

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