Jpn. J. Infect. Dis., 54, 2001

Laboratory and Epidemiology Communications

Clinical Study of Mutans Streptococci Using 3DSand Monoclonal Antibodies
Hiroaki Takeuchil , Kazuo Fukushimal･3, Hidenobu Senpukul , Yoshiaki Nomural ,NoboruKanekol, Akira Yanol, Eiko MoritaI , Susumu Imail, Tosiki NisizawaI ,YoshiharuKono4, Takuj i Ikemi4, Yoshihiro Toyoshima2 and Nobuhiro Hanadal *JDepartmen i of Oral Science, National Institute ofInfectious Diseases,
Toyama 1‑23‑1, Shinjuku‑ku, To砂0 162‑8640,

2Dai‑ichi Mutual Life Insurance Company Hibiya Medical Center

Tob,0 100‑8411 and3Department ofMicrobiology and 4Department of Operative Dentistry,
Nihon Universio, School ofDentisり′ at Matsudo,

Chiba 271‑8587
Communicated by Nobuhiro Hanada(Accepted March 21, 2001)

Streptococcus mutans has been implicated as the prlmary

Ma et al･ have demonstrated that passive immunization

causative orgFnism of dental caries (1 )･ S･ mutans producesuslng SPeCific antibodies agalnSt S. mutans is potentiallysurface proteln antigen (PAc) (2) for the purpose of initialeffective for protection against dental caries (8,9). Passive

attachment and produces glucosyltransferase (GTF)(3) forimmunization is safe and specific. In this study, we investi‑

the synthesis or water‑insoluble glucans (WIG). These two
substances contribute to the canogenlC Properties ofS. mutans

gated the clinical efrlCaCy Of anti‑PAc andanti‑GTF MAb ineliminating S･ mutansfrom the oral cavity. Clinical applica‑in the oral cavity (4). Recently, an anti‑PAc monoclonal anti‑tion of anti一microbial agents to tooth suぬces uslng a dental

body (KH5, MAb) recognizing an alanine‑rich repeating

drug delivery system (3DS) has been reported (10). In 3DS,region (residues 365 to 377) was identified as an antibody

an individual tray that covers the whole dentition is used as athat inhibits the attaclment of S. mutans to tooth surfaces indrug retainer. A tray that fits over the tooth surfaces is usefulrats (5,6)･ While an anti‑GTF monoclonal antibody (p‑126,MAb) inhibiting WIG synthesis by GTF‑I (7) has been
discovered. KH5 and p‑126 MAb may also be useful for

dmgs･ It is easy to manlpulate and maintains high concentra‑

for deliverlng Small but sufficient amounts of anti‑bacterialtions of MAbs in direct contact with tooth surfaces. Thus,MAbs are not diluted by saliva, and only the bacteria on theprotection against dental caries caused by S. mutans.

tooth surface, but not those on the oral mucosa, are exposed*Corresponding author: Fax: +8 1 ‑3‑5285‑ 1 I 72, E‑rhail: nhanada

to MAbs. Before the application of 3DS, dental biofilms were

@nih.go.jp

removed by means of professional mechanical tooth cleanlng

34

Jpn. J. Infect. Dis., 54, 2001person) as a trial. Saliva samples were taken before and after(PMTC). A鮎r application ofO.2% chlorhexidine (CHX), an

effective antiseptic in dentistry (1 I), the mixed MAbs werethe 3DS with CHX and then at 20, 30, 50, and 70 days a洗er

applied to the tooth surface also by 3DS. The subjects

the PBS or MAb treatment. Saliva secreted over a period of

consisted of five healthy human volunteers (age range: 47‑3 mln, While being stimulated by biting paraffin gum, was60 years old).

collected into ice‑chilled sterile bottles. The saliva was trans‑ferred via a cotton swab into the transport medium (0.4%agar, 0･ 1 5% thioglycolate/PBS) in sterile tubes (SEEDSWAB

The subjects were treated first with phosphate bufferedsaline (PBS) as a control and then with mixed MAbs (12.6 mg/

No. 1; Eiken Chemical Co., LTD, Tokyo).
The sample was further transferred with a cotton swab into
4 ml of Brain Heart Infusion (BHl) broth. The BHl broth(㌔)otxp30 1daJtSP!)07JSuZ17nW+SrJOS}tTnO3](tOTUcontainlng the saliva samples was sonicated by an ultrasonicdispersion apparatus(60 W power output) for 10 see, andpoured onto blood agar plates, and Mitis‑Salivarius agar plateswith (MSB) or without 0.02 M bacitracin (MS) in a volumeof 50 Lil using an EDDY JET spiral system (Gunze Sangyo,Inc., Tokyo). A洗er anaerobic culture for 48 h, total bacterialcolonies on the blood agar, total Streptococci colonies on theMS plates and S. mutans colonies on the MSB plates werecounted. S. mutans could be identified by their characteristic
appearance ･In three subjects, A, D and E, S. mutans was eliminated bymans of CHX treatment and failed to re‑colonize during theperiod of control and trial studies. In subjects B and C, mixed

MAbs treatment for 70 days slgnificantly reduced S. mutans
in total streptococci to 38% and 39%, respectively, of thevalues before the treatment (i.e., after treatment with PBS)

(Fig･ 1)I However, there were no significant changes in theSubject

B

Subject

number of total bacteria and total streptococci observed a洗erC

the MAb treatment (Fig. 2). These results suggest that theFig･ L Effect of the application of mixed MAbs by 3DS on S. mutans

叩plication of mixed MAbs by means of SOS minimally

population in totalstreptococci･ The figurでpresents the ratio (%) ofdisturbs the oral flora and isuseful for eliminatlng S. mutans

S･ mutans colony counts to totalstreptococcI COlony counts in subjects

in the oral cavlty.

B and C. The data for subjects A, D, and E were omitted because

S･ mutans were eliminatedvia CHX pre‑treatment.

(A)(lot)J亀tE po t [(lot)][亀tIsMZM

D5DiLiHTTTDi,sN D5伽

D6｢日日T Dd3&,sMAb or P♭sMAb or P♭s=First trial (control)◆'''''''◆''…'' second tnal (lmrTIUnizing)

Fig･ 2･ Effect of the application of mixed MAbs by 3DS onthe colony counts oftotal bacteria and streptococci.
A: Colony counts or total bactera in stimulated saliva.
B: Colony counts or total streptococci in stimulated saliva.

‑ : control study. ･･････ : trial. ‑ :period ofCHX treatment.
Saliva samples were taken before (Dl) and after (D2)the 3DS withCHX and at 20 (D3), 30 (D4), 50 (D5) and 70 (D6) days

after the PBS or MAb treatment.

35

Jpn. J. Infect. Dis., 54, 2001

7. Fukushima, K., Okada, T. and Ochiai, K. (1993): Produc‑

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36