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Arbitrarily Primed-Polymerase ChainReaction for Identification andEpidemiologic Subtyping of Oral Isolates of Fusobacterium nucleatumMario J. Avila-Campos,* Cláudio T. Sacchi,† Anne M. Whitney,‡ Arnold G. Steigerwalt,‡and Leonard W. Mayer‡

In modern clinical microbiol-ogy, par ticularly in oralmicrobiology, detailed stud-

ies of typing or subtyping ofputative pathogens is neces-sary, especially for infectiousdiseases such as periodontaldisease, since a habitat suchas the periodontal pocket mayharbor more than 300 differ-ent bacterial species.1,2 Sev-eral methods of detectingphenotypic or genotypic vari-ation have been used to char-acterize bacterial pathogensfrom commensal strains3

where the probable etiologicagents of periodontal diseasebelong to the autochthonousmicroflora such as Fusobacte-rium nucleatum, a putativeorganism involved in develop-ment of gingivitis and perio-dontitis.

F. nucleatum is one of themost common species inhuman infections and can befound in body cavities ofhumans and other animals.Of the periodontal speciesthat are statistically associ-ated with periodontal disease,it is the most common inclinical infections of otherbody sites.4 Although manystudies examined the subgin-

Background: Fusobacterium nucleatum is the most frequently isolatedbacterium in periodontal disease and plays an important role in seriousinfections in other parts of the body. Arbitrarily primed-polymerase chainreaction (AP-PCR) was used to construct primers for specific identificationand subtyping of F. nucleatum. Subtypes may differ in virulence and,hence, are important as periodontal pathogens. Subtypes also may differin antibiotic susceptibility; therefore, knowing the subtypes may influencechoice of treatment.

Methods: We analyzed 70 DNA samples of F. nucleatum isolated frompatients with periodontal disease (PD) (N = 32) or AIDS-related PD (N = 8)and from healthy carriers (N = 30). From 90 AP-PCR primers screened,five amplification products were selected, cloned in pCR II vector, andsequenced. These sequences were used to design new pairs of specificprimers. Sequences were compared to GenBank entries with BLAST andshowed no significant matches.

Results: Three primer pairs produced bands of approximately 1 Kb(primer 5059S) or 0.5 Kb (primers FN5047 or M1211) with all F. nuclea-tum DNAs tested. PCR amplification using primer pair M8171 produced a 1 Kb band with isolates from 7 (22%) PD and 5 (63%) PD-AIDS patientsand 9 (30%) healthy controls. Using the same primer pair, 2 other bands ofapproximately 0.5 Kb and 0.4 Kb were observed with DNA from isolatesfrom 2 (6%) PD and all PD-AIDS patients, but were not observed with DNAsamples from healthy controls (P <0.0001). All the primer pairs producedno or different amplicon profiles with DNA samples from bacterial speciesother than F. nucleatum.

Conclusions: Our results suggest that PCR primer pairs 5059S, FN5047or M1211 can be used to specifically identify F. nucleatum isolates and dis-tinguish them from other bacteria. The primer pair M8171 could also be usedto differentiate F. nucleatum isolated from periodontal patients or healthyindividuals. These specific primers can be used in PCR analysis for specificidentification of F. nucleatum and to distinguish it from other bacteria associ-ated with human periodontitis. These approaches appear promising in facili-tating laboratory identification, molecular subtyping, and taxonomy ofputative periodontopathogens. J Periodontol 1999;70:1202-1208.

KEY WORDSFusobacterium nucleatum; periodontal diseases/microbiology;polymerase chain reaction/methods.

* Department of Microbiology, University of São Paulo, São Paulo, SP, Brazil.† Bacteriology Division, Adolfo Lutz Institute, São Paulo, SP, Brazil.‡ Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for

Infectious Disease, Centers for Disease Control and Prevention, Atlanta, GA.

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gival plaque flora by isolation,5-7 only a few haveattempted to characterize or subtype F. nucleatum.8,9

An accurate identification of fusobaterial species istherefore of great importance not only for taxonomicreasons but also for appropriate treatment of infec-tion, since the susceptibility of different fusobacterialspecies to antibiotics varies widely.10 However, therole of this microorganism in periodontal diseases

remains undefined due to the het-erogeneity within the species.11

The detection of bacterial subtypesas common determinators of amixed anaerobic infection is ofinterest from a clinical perspective,as it may be helpful in characteriz-ing periodontitis and permittingmonitoring of the disease.12

Culture techniques used to iden-tify putative anaerobic pathogensassociated with destructive peri-odontal disease are tedious to per-form and may fail to yield some ofthe organisms. Fusobacteriumspecies have been studied byusing several methods, such asfatty acid analysis,13 compositionof the peptidoglycan layer,14 DNA-DNA hybridization,15 and oligo-nucleotide DNA probes.16

Because of its ability to accu-rately detect specific micro-organisms in mixed populations,polymerase chain reaction (PCR)is a promising method for thedirect identification of periodontalpathogens in subgingival speci-mens17 and for elucidating the roleof specific bacteria in the peri-odontal disease process. In thepresent study, we first attempted to produce a subtyping systemusing arbitrarily primed (AP)-PCR.Because of a number of problemswith poor reproducibility and diffi-culties in interlaboratory compar-isons associated with AP-PCR, wemodified our strategy and usedAP-PCR to produce products forfurther characterization. Theseproducts were cloned and se-quenced, and this information wasused to design specific oligonu-cleotide primers for identificationor typing of F. nucleatum isolatedfrom patients with three differentclinical syndromes.

MATERIALS AND METHODSBacterial Strains and Growth ConditionsThe bacterial strains used in this study are describedin Table 1. The isolates were cultivated and iden-tified according to the method of Gaetti-Jardimet al.7 Bacteria were grown in brain heart infusion

Table 1.

Bacterial Species and Reference Strains Used

Microorganism N Strains Source

Fusobacterium nucleatum 32 1 to 32 Periodontal disease (PD)

8 A1 to A8 PD with AIDS (PD-AIDS)

30 S1 to S30 Healthy individuals (H)

2 M1 & M2 Monkey*

subsp. polymorphum 1 10953T ATCC†

subsp. nucleatum 1 25586T ATCC

F. necrophorum 3 850201 CDC‡

850202 CDC

850203 CDC

F. mortiferum 3 847409 CDC

860375 CDC

890095 CDC

F. varium 2 850155 CDC

850483 CDC

Bacteroides fragilis 1 860114 CDC

B. ureolyticus 3 890013 CDC

900164 CDC

900420 CDC

Porphyromonas macacae 1 880334 CDC

Actinobacillus actinomycetemcomitans 1 29522 ATCC

1 29523 ATCC

1 33384T ATCC

1 Y4 FDC§

Eikenella corrodens 1 880187 CDC

Haemophilus aphrophilus 1 870239 CDC

Escherichia coli 1 850588 CDC

* State University of São Paulo, SP, Brazil.† American Type Culture Collection, Rockville, MD.‡ Culture collection of the Anaerobe Laboratory, Hospital Infections Program, CDC.§ Forsyth Dental Center, Boston, MA.

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broth§ (BHI) supplemented with 0.5% yeast extract.Cultures were maintained on plates of the samemedium containing 1.5% agar and 5% defibrinatedsheep blood, and were frozen in skim milk at −70°C.All cultures were grown in an anaerobic chamber at37°C. They were checked for purity by Gram stainingand conventional biochemical tests.18,19

DNA Extraction and AnalysisDNA from F. nucleatum or other bacteria wasextracted using a modification of the method ofBrenner et al.20 Briefly, bacterial cells were harvestedfrom 500 ml BHI broth and lysed at 37°C for 15 min-utes in 25 ml of 20% sucrose containing 0.2 ml of 1.4 M NaPO4 and 6 ml of 10 mg/ml lysozyme.‖ Thelysate was mixed with 1.25 ml of 0.5 M Tris-HCl, 2.5 ml of 0.5 M EDTA, 0.125 ml of 10 mg/mlpronase E,‖ and 1 ml of 25% sodium dodecyl sulfate.The DNA sample was extracted with 25 ml of phenol.After centrifugation at 5,000 rpm for 5 minutes, theaqueous layer was mixed with 3 ml of 5 M perchlo-rate and extracted with 25 ml of chloroform. Aftercentrifugation, DNA in the aqueous phase was pre-cipitated with 2 volumes of ice-cold 95% ethanol.The DNA was dissolved in sterilized water anddigested with 50 µg/ml RNase.‖ After extraction withphenol, DNA in the aqueous phase was precipitatedwith 2 volumes of ethoxyethanol¶ and dissolved in 3 ml of TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH8.0). DNA solutions were stored at 4°C. The DNAconcentration of each DNA sample was determinedspectrophotometrically.# A portion of each DNAsample was also checked for integrity on an agarosegel before use.

AP-PCR AnalysisF. nucleatum ATCC 10953T and ATCC 25584T and 70clinical isolates were included in the AP-PCR analysis.Ninety 10-base oligonucleotide primers** were tested.PCR amplification was performed using a modificationof the method described by Williams et al.21 Volumesof 25 µl containing 1X PCR/Mg++ buffer†† (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3), 0.2 mM eachof dNTP,‡‡ 0.5 U Taq DNA polymerase,†† 0.4 µM ofprimer, and 10 ng of template. Amplification was per-formed in a DNA thermal cycler§§ programmed for94°C for 5 minutes followed by 30 cycles of 94°C for 1 minute, 34°C for 1 minute, 72°C for 2 minutes, then72°C for 5 minutes to allow the completion of DNAextension. A negative control without template DNAwas included in each AP-PCR run. Amplification prod-ucts were compared by electrophoresis in 1% agarosegel in 1X TBE buffer (1 M Tris, 0.9 M boric acid, 0.01 MEDTA, pH 8.4) stained with ethidium bromide (0.5mg/ml), and photographed on a UV light transillumina-tor. Molecular mass standard 1 Kb ladder‖‖ wasincluded.

Cloning of the AP-PCR ProductFive initial AP-PCR products randomly selected werecloned using a cloning kit¶¶ and transformed into E. coliXL1-Blue. Sizes of the inserts were verified by digestionwith EcoRI and SacI enzymes.## Plasmid DNA was puri-fied on a QIA-prep spin column*** and sequenced.

DNA Sequencing and Synthesis of Specific PrimersPlasmids containing inserted fragment were treatedwith PvuII enzyme## and purified by using micropureenzyme removers.††† DNA sequencing was performedon an automated DNA sequencer‡‡‡ using a terminatorcycle sequencing kit.§§§ Sequencing reaction productswere purified by using spin columns‖‖‖ and resolvedon a 5% acrylamide, 8 M urea gel. DNA sequenceswere aligned, edited, and consensus sequences deter-mined with the University of Wisconsin Genetics Com-puter Group (GCG) package. PCR primer sequenceswere selected using a software program.¶¶¶ The resultswere used to design and synthesize the sequence ofnew 21- to 23-mer primers (Table 2) in the Bio-technology Core Facility Branch, Centers for DiseaseControl and Prevention. Sequences were submittedusing BLAST to search for other GenBank entries(GenBank, National Center for Biotechnology Informa-tion, Bethesda, MD) with sequence similarities.22

Working Primers and PCR AmplificationThe new synthesized primer pairs (Table 2) were usedin PCR reactions with all DNA samples from F. nuclea-tum and other bacterial species. Amplification reac-tions were performed as above except the temperatureprofile was changed to 94°C for 5 minutes; followed by30 cycles at 94°C for 30 seconds; 40°C, 50°C or 55°C,respectively, for 30 seconds; 72°C for 30 seconds, fol-lowed by a 5-minute final extension at 72°C. The prod-ucts were compared by electrophoresis, stained, andphotographed as described above.

Whole Bacterial Cell and Dental Plaque PreparationsFive colonies of each F. nucleatum grown on blood agarwere boiled (100°C, 5 minutes) in 500 µl of phosphate-buffered saline (0.01 M, pH 7.2), centrifuged, and thesupernatant removed. Pellet was resuspended in 500 µlsterile water, and 10 µl was used as a template for

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§ Difco Laboratories, Detroit, MI.‖ Sigma Chemical Co., St. Louis, MO.¶ Fisher Scientific, Springfield, NJ.# DU-640, Beckman, Fullerton, CA.** Genosys Biotechnologies, Inc., The Woodlands, TX.†† Boehringer Mannheim, Indianapolis, IN.‡‡ Pharmacia Biotech, Piscataway, NJ.§§ GeneAmp PCR System 9600, Perkin Elmer, Norwalk, CT.‖‖ Gibco BRL, Life Technologies, Ltd., Bethesda, MD.¶¶ T/A pCR Script Amp SK, Strategene, La Jolla, CA.## Biolabs, Beverly, MA.*** Qiagen, Chatsworth, CA.††† Amicon Inc., Beverly, MA.‡‡‡ ABI model 373S, Applied Biosystems, Foster City, CA.§§§ PRISM Ready Reaction Dye-Deoxy, Applied Biosystems.‖‖‖ Centri-Sep, Princeton Separations, Inc., Princeton, NJ.¶¶¶ Version 5 Oligo, National Biosciences, Plymouth, MN.

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PCR.12 Colonies from control bacterial species werealso used in the PCR reactions. In addition, pools ofsupra- and subgingival plaque from 5 individuals withno evidence of periodontal disease were taken andboiled as described above and 10 µl of the supernatantwas used as a template.

RESULTSAP-PCRDNA samples from F. nucleatum isolated from patientswith the previously defined syndromes (PD, PD-AIDS,or healthy; Table 1) or monkeys were screened withninety 10-mer primers of arbitrary sequence. Mostprimers produced no or very faint bands. Primers 50-47and 50-59 produced amplification products of 0.7 Kband 1.5 Kb with all F. nucleatum DNAs, but results

were not consistent between experiments(data not shown). This can be explainedbecause of the size of arbitrary primersused (10-mer) and the low annealing tem-perature in the PCR reaction. Three AP-PCRproducts obtained with primer 50-47 (CCAACC TGT A) using DNA from strains 27, A2,and A4, and 2 obtained with 50-59 (GACAGT AGC A) from strains 26 and ATCC25586T were selected for further characteri-zation, cloned and sequenced. Table 3 pro-vides designations and further description ofthe clones. Sequences were compared withGenBank entries with BLAST and showedno significant sequence similarities. Thesesequences were used to design 5 new spe-cific primer pairs (Table 2), and PCR prod-ucts obtained with all DNA samples werecompared.

Specific Primer PCR ReactionsFive specific primer pairs were evaluated forsensitivity and specificity of amplificationwith DNA from F. nucleatum. Amplification

reactions of the primer pairs FN5047, FN24, 5059S,and M1211 were obtained and their products com-pared. Few differences in amplicon profiles amongDNA samples from F. nucleatum isolated from PDpatients or healthy individuals were observed. For 3 ofthese primer pairs, only bands of approximately 1 Kb(5059S) or 0.5 Kb (FN5047 or M1211) were observedin all DNA samples from F. nucleatum isolates (Fig. 1).The primer pair FN24 produced several weaker butdetectable bands of different sizes, but the patterns didnot correlate with the bacterial source, i.e., from PD,healthy, PD-AIDS, or monkey.

PCR amplification using the primer pair M8171produced a variety of patterns including amplificationproduct of approximately 1 Kb from 7 (22%) isolates

from PD patients, 5 (63%) isolatesfrom PD-AIDS patients, and 9(30%) isolates from healthy individ-uals. Also, this primer pair produced2 additional bands of approxi-mately 0.5 Kb and 0.4 Kb in 2 PDpatient isolates and in all 8 PD-AIDSpatient isolates, but these 2 bandswere not observed when DNA sam-ples from healthy controls wereused (P <0.0001) (Fig. 2). These 3bands (1 Kb, 0.5 Kb, and 0.4 Kb)were also observed with DNA fromthe monkey isolates.

All the synthesized primer pairsproduced no or amplicon patternsdifferent from F. nucleatum with DNA

Table 2.

Nomenclature, Composition, and AnnealingTemperature of the Species-Specific Primer Pairs

Primer Oligonucleotide Sequence 5′ → 3′ Annealing Temperature (°C)

FN5047 CAA ATG CTT GTG TCA ATA ATA CT

TTT AGA AGA AAT GGT AGA ATA AT 40

FN24 AGT AGC ACA AGG GAG ATG TAT G

CAA GAA CTA CAA TAG AAC CTG A 40

5059S ATT GTG GCT AAA AAT TAT AGT T

ACC CTC ACT TTG AGG ATT ATA G 40

M1211 AGA AGA ACA AGC CAA TAA GGA

TGA ACC CAG ACA CAA CAA AGA 50

M8171 GAA AAG TGC GGT GAA AAA CCA

CGC CGG AAG TAC CTT ATG TGC 55

Table 3.

Derivation and Designation of the Clones

Clone DNA Template Arbitrary Size (base pairs) Sequence Used(strain) Primer From Sequencing to Design

Primer Pairs

P5047 27 50-47 820 FN5047

A5047C1 A2 50-47 637 FN24

A5047A4 A4 50-47 468 M8171

P505926 26 50-59 913 M1211

P5059 ATCC 25586 50-59 ND* 5059S

* Not determined.

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samples from non-target microbialspecies (Table 1). The synthesizedprimers produced no or ampliconpatterns different from F. nucleatumwith boiled colonies of non-targetbacteria (data not shown). Boiledsamples of dental plaque yieldedsimilar PCR products when usingthe 5 synthesized primer pairs.Since F. nucleatum is an inhabitantof autochthonous microbiota, it wasdetected from plaque samples thatwere taken from individuals with noevidence of periodontal disease.

DISCUSSIONThe significance of Fusobacteriumnucleatum in the development ofperiodontal disease, as well as infec-tions in other organs, has generatedinterest because of its pathogenicpotential, its frequency in periodon-tal lesions,4 its production of tissueirritants,23 its synergism in mixedinfections,24 and its ability to formaggregates with other suspectpathogens in periodontal diseaseand thus act as a bridge betweenearly and late colonizers on thetooth surface.25

Nucleic acid detection methods have been developedfor various medical and periodontal pathogens.26,27 InAP-PCR analysis, the genomic DNA is amplified usingsingle oligonucleotides of arbitrari ly chosensequences and non-stringent annealing temperatures,resulting in a series of PCR amplification products withdifferent sizes that provide a characteristic electro-phoretic fingerprint.28

AP-PCR has been used for fingerprinting individualstrains of various microbial species,29-31 and bandingpatterns have been used to distinguish between micro-bial species.32 In our study, the AP-PCR method showed a great polymorphism among F. nucleatumspecies. However, a poor degree of technical repro-ducibility and discriminatory power was achieved by thistyping method in accordance with George et al.11

Several parameters can alter AP-PCR profiles and make standardization very difficult to obtain repro-ducible results. This may be related to the relatively lowannealing and extension temperature required for theshort 10-base primers.

In this study, 5 AP-PCR products were selected andcharacterized to synthesize 5 specific primer pairs,which reacted specifically with F. nucleatum typestrains and 70 clinical isolates. These specific primers

can be used in PCR analysis for specific identificationof F. nucleatum and to distinguish it from other bacte-ria associated with periodontitis such as Actinobacillusactinomycetemcomitans, Eikenella corrodens, orHaemophilus aphrophilus. They should, therefore, behelpful for species-specific detection of F. nucleatumin clinical samples from periodontal patients.

Primer pairs 5059S produced a single amplicon of1 Kb with all F. nucleatum DNAs tested. Primer pairsFN5047 and M1211 each produced a single band of0.5 Kb with all F. nucleatum DNAs. These primersproduced several amplicons using DNA samples fromother bacterial species, but the patterns observedwere different from the F. nucleatum DNA samples.

Amplifications when supernatant from colonies orboiled dental plaque was used as a template witheach primer pair were similar to those with F. nuclea-tum DNAs and show the usefulness for rapid identifi-cation of this organism. The production of bands insubgingival plaque with primers FN5047, 5059S, andM1211 suggests that hemoglobin or other compoundsin subgingival specimens do not significantly inhibitthe amplification reaction.

Two reference strains of F. nucleatum of differentsubspecies were used: ATCC 10953, subsp. polymor-phum, and ATCC 25586, subsp. nucleatum. These

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Figure 1.Ten µl DNA from F. nucleatum per well amplified with FN5047 or M1211 primer (A) and 5059Sprimer (B) Lanes 1 and 18, 1-Kb DNA ladder; lanes 2 to 7 and 10 to 15, F. nucleatum isolates;lanes 8 and 16, F. nucleatum ATCC 10953; lanes 9 and 17, F. nucleatum ATCC 25586.

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they are simply markers of patho-genic strains.

The use of PCR allied with con-ventional cloning technology and the development of appropriategenetic exchange systems repre-sent the dawn of an exciting new era for the genetic and molecularcharacterization of anaerobes. These approaches appear promis-ing in facilitating laboratory identifi-cation and taxonomic classification of putative periodontopathogens.The clinical utility of these tools in diagnosis and management ofbacterial diseases remains to bedetermined.

ACKNOWLEDGMENTSThe authors thank Dr. Michael M. Miller for his collaboration and Mr. David Whaley and Mrs. LoisWiggs from Anaerobe Laboratory,Hospital Infections Program, and the Biotechnology Core FacilityBranch, Scientific ResourcesProgram, NCID, CDC for their technical assistance. This study

was supported in part by Fundação de Amparo àPesquisa do Estado de São Paulo, SP, Brazil, grant96/3115-6.

REFERENCES1. Moore WEC. Microbiology of periodontal disease.

J Periodont Res 1987;22:335-341.2. Bolstad AI, Jensen HB. Polymerase chain reaction-

amplified nonradioactive probes for identification of Fuso-bacterium nucleatum. J Clin Microbiol 1993;31:528-532.

3. Eisenstein BI. New molecular techniques for microbialepidemiology and the diagnosis of infectious diseases.J Infect Dis 1990;161:595-602.

4. Moore WEC, Moore LVH. The bacteria of periodontaldiseases. Periodontol 2000 1994;5:66-77.

5. Dzink JL, Tanner ACR, Haffajee AD, Socransky SS.Gram-negative species associated with destructiveperiodontal lesions. J Clin Microbiol 1985;12:648-659.

6. Könönen E, Jousemies-Somer H, Asikainen S.Relationship between oral Gram-negative anaerobicbacteria in saliva of the mother and the colonization ofher edentulous infants. Oral Microbiol Immunol1992;7:273-276.

7. Gaetti-Jardim E Jr., Zelante F, Avila-Campos MJ. Oralspecies of Fusobacterium from human and environ-mental samples. J Dent 1996;24:345-348.

8. Dix K, Watanabe SM, McArdle S, et al. Species-specificoligodeoxynucleotide probes for the identification ofperiodontal bacteria. J Clin Microbiol 1990;28:319-323.

9. Morris ML, Andrews RH, Rogers AH. Investigations ofthe taxonomy and systematics of Fusobacteriumnucleatum using allozyme electrophoresis. Int J SystBacteriol 1997;47:103-110.

subspecies may have different roles in the etiology ofperiodontitis.33 Both subspecies produced similar pat-terns with all primers. On the other hand, our clinicalisolates were not identified at the subspecies level.There are many difficulties in the search for the etio-logic agents of destructive periodontal diseases, includ-ing technical problems and difficulties in determiningthe state of activity of periodontal disease.34 Anotherlevel of complexity exists because strains withinspecies differ in virulence, as has been suggested for F.nucleatum subspecies.35

Although the synthesized primers FN5047, FN24,5059S, and M1211 were able to specifically identify F. nucleatum, they were not capable of subtyping thestrains with respect to the different patient syndromesor specimen sources, i.e., PD, PD-AIDS, healthy ormonkeys. Primer M8171 produced unique bands (1 Kband 0.4 Kb) in F. nucleatum isolated from some of thePD patients, all of the PD-AIDS patients, and both ofthe monkeys. These bands were not observed in sam-ples from healthy isolates. The relation of these bandsin these isolates and the nature or function of the prod-ucts encoded by these fragments remain unclear. Thissuggests that the M8171 primer pair could be used todifferentiate F. nucleatum isolated from PD patients orhealthy individuals, or from different specimen sources.More studies are necessary to determine if these smallfragments might be related to any virulence factor or if

Figure 2.DNA from F. nucleatum from different sources amplified with M8171 primer. Lanes 1 to 4, PD isolates;lane 5, F. nucleatum ATCC 10953; lane 6, F. nucleatum ATCC 25586; lanes 7 to 14, PD-AIDS isolates;lanes 15 and 16, monkey isolates; lanes 17 to 19, healthy isolates; lane 20, 1 Kb. DNA ladder.

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10. George WL, Kirby BD, Sutter VL, et al. Gram-negativeanaerobic bacilli: Their role in infection and patterns ofsusceptibility to antimicrobial agents. II. Little knownFusobacterium species and miscellaneous genera. RevInfect Dis 1981;3:599-626.

11. George KS, Reynolds MA, Falkler WA Jr. Arbitrarilyprimed polymerase chain reaction fingerprinting andclonal analysis of oral Fusobacterium nucleatumisolates. Oral Microbiol Immunol 1997;12:219-226.

12. Furcht C, Eschrich K, Merte K. Detection of Eikenellacorrodens and Actinobacillus actinomycetemcomitansby use of the polymerase chain reaction (PCR) in vitroand in subgingival plaque. J Clin Periodontol 1996;23:891-897.

13. Jantzen E, Hofstad T. Fatty acids of Fusobacteriumspecies: Taxonomic implications. J Gen Microbiol 1981;123:163-171.

14. Vasstrand EN, Jensen HB, Miron T, Hofstad T. Com-position of peptidoglycans in Bacteroidaceae: Deter-mination and distribution of lanthionine. Infect Immun1982;36:114-122.

15. Potts TV, Holdeman LV, Slots J. Relationship among theoral fusobacteria assessed by DNA-DNA hybridization.J Dent Res 1983;62:702-705.

16. Bolstad AI, Skaug N, Jensen HB. Use of syntheticoligonucleotide DNA probes for the identification ofdifferent strains of Fusobacterium nucleatum. J Perio-dont Res 1991;26:519-526.

17. Slots J, Ashimoto A, Flynn MJ, Li G, Chen C. Detectionof putative pathogens in subgingival specimens by 16Sribosomal DNA amplification with the polymerasechain reaction. Clin Infect Dis 1995;20:304-307.

18. Holdeman LV, Cato EP, Moore WEC. AnaerobicLaboratory Manual, 4th ed. Blacksburg, VA: VirginiaPolytechnic and State University; 1977:132.

19. Sutter VL, Citron DM, Finegold SM, Bricknell K.Wadsworth Anaerobic Bacteriology Manual, 2nd ed. St. Louis: Mosby; 1980:131.

20. Brenner DJ, McWhor ter AC, Leete-Knutson JK,Steigerwalt AG. Escherichia vulneris: A new species ofEnterobacteriaceae associated with human wounds. J Clin Microbiol 1982;15:1103-1140.

21. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA,Tingey SV. DNA polymorphisms amplified by arbitraryprimers are useful as genetic markers. Nucleic AcidsRes 1990;18:6531-6535.

22. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ.Basic local alignment search tool. J Mol Biol 1990;215:403-410.

23. Bartold PM, Gully NJ, Zilm PS, Rogers AH. Identificationof components in Fusobacterium nucleatum chemostat-culture supernatants that are potent inhibitors of humangingival fibroblast proliferation. J Periodont Res 1991;26:314-322.

24. Feuille FL, Kesavalu L, Steffen MJ, Holt SC, Ebersole JL.Synergistic tissue destruction induced by Porphyromonasgingivalis and Fusobacterium nucleatum. J Dent Res1994;73:159-161.

25. Kolenbrander PE, London J. Adhere today, heretomorrow: Oral bacterial adherence. J Bacteriol 1993;175:3247-3252.

26. Tenover F. DNA probes for infectious diseases. ClinMicrobiol Newslett 1985;7:105-112.

27. Gersdord H, Meissner A, Pelz K, Krekeler G, Gobel UB.Identification of Bacteroides forsythus in subgingivalplaque from patients with advanced periodontitis. J ClinMicrobiol 1993;31:941-946.

28. Welsh J, McClelland M. Fingerprinting genomes usingPCR with arbitrary primers. Nucleic Acids Res 1990;18:7213-7218.

29. Ménard C, Brousseau R, Mouton C. Application ofpolymerase chain reaction with arbitrary primer (AP-PCR) to strain identif ication of Porphyromonas(Bacteroides) gingivalis. FEMS Microbiol Lett 1992;95:163-168.

30. Slots J, Chen C. Detection of Porphyromonas gingivalisassociated with human periodontitis by DNA methods.Clin Infect Dis 1993;16:317-318.

31. Lotufo RFM, Flynn J, Chen C, Slots J. Moleculardetection of Bacteroides forsythus in human perio-dontitis. Oral Microbiol Immunol 1994;9:154-160.

32. Myers LE, Silva SVPS, Procunier JD, Little PB. Genomicfingerprinting of Haemophilus sommus isolates by usinga random-amplified polymorphic DNA assay. J ClinMicrobiol 1993;31:512-517.

33. Gharbia SE, Shah HN, Lawson PA, Haapasalo M. Thedistribution and frequency of Fusobacterium nucleatumsubspecies in the oral cavity. Oral Microbiol Immunol1990;5:324-327.

34. Socransky SS, Haffajee AD, Smith LF, Dzink JL.Difficulties encountered in the search for the etiologicagents of destructive periodontal disease. J Clin Perio-dontol 1987;14:588-593.

35. Genco RJ, Loos BG. The use of genomic DNAfingerprinting in studies of the epidemiology of bacteriain periodontitis. J Clin Periodontol 1991;18:396-405.

Send reprint requests to: Dr. Mario Julio Avila-Campos,Department of Microbiology, ICB, USP, 05508-900, SãoPaulo, SP, Brazil.

Accepted for publication February 16, 1999.

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