コンクールFは、0.28%～1.12%（0.28%：50mLの水にコンクールFを5滴使用、1.12%：25mLの水にコンクールFを10滴使用）の濃度に希釈して用いるタイプの洗口液です。本論文では、希釈したコンクールFが歯周病原性細菌およびう蝕原性細菌に及ぼす影響に着目し、詳細な検討を行いました。 歯周病の主要な原因細菌としてPorphyromonas gingivalis（以下、P. gingivalis）が知られており、菌体表層に存在する線毛タンパクであるFimAが歯周組織への定着に関与することが知られています。分析には、歯周状態が健康な人のデンタルプラークから検出されることが多いFimA I型のP. gingivalisと、歯周病患者のデンタルプラークから検出されることが多いFimA Ⅱ型のP. gingivalisを用いて、コンクールFを加えた菌液を試験管内で培養して増殖能の評価を行いました。その結果、適正使用時の濃度である1%のコンクール添加時にP. gingivalisの増殖抑制が認められました（図1）。 う蝕の主要な原因細菌として、Streptococcus mutans（以下、S. mutans）とStreptococcus sobrinus（以下、S. sobrinus）が知られています。S. mutansは菌の表層に存在する血清型特異多糖抗原の構造上の違いからc、e、f、kの4種類の血清型に分類され、S. sobrinusはdとgの2種類の血清型に分類されます。これらのう蝕原性細菌をコンクールF存在下で培養したところ、適正濃度よりも低い0.1%のコンクール添加時にいずれの血清型の菌も増殖が抑制されました（図2）。 次に、コンクールFがP. gingivalisやS. mutansのバイオフィルム形成能に及ぼす影響について分析しました。この実験では、唾液成分をコーティングしたプラスチック製のプレートを歯面に見立て、コンクールFを含む口腔細菌をプレート上で培養しました。培養後に形成されたバイオフィルムは、共焦点レーザー顕微鏡を用いて観察しました。P. gingivalisにより形成されたバイオフィルムは、適正濃度である1%のコンクールFが存在することにより2分の1以下に減少しました（図3）。また、S. mutansにより形成されたバイオフィルムは、適正濃度よりも低い0.1%のコンクールFが存在することで約5分の1に減少しました（図4）。 本研究結果から、低濃度のコンクールFがP. gingivalisおよびS. mutansの増殖能とバイオフィルム形成能を抑制することが明らかになりました。このことから、適正使用時の濃度によるコンクールFを用いた洗口は、歯周病およびう蝕の予防に有効であると考えられます。『Journal of Oral Science』論文の要点 「本研究結果から、低濃度のコンクールFがP. gingivalisおよびS. mutansの増殖能とバイオフィルム形成能を抑制することが明らかになり、適正使用時の濃度によるコンクールFを用いた洗口は、歯周病およびう蝕の予防に有効であると考えられます」 『Journal of Oral Science』に掲載された論文について、野村氏に詳しく解説していただいた内容を以下に掲載する。 ウエルテック社は、ユーザーから寄せられていた「水で希釈するコンクールFは効果がない」という声に対して、実使用濃度でも有効性があることを伝えたかった。コンクールFの適正使用濃度における予防効果はどの程度なのか？ 同教室では、コンクールFの適正使用濃度での予防効果の有効性について検証すべく研究がスタートした。その研究成果が2020年3月、『Journal of Oral Science』に掲載された1）。 本研究の筆頭著者を務めた野村良太氏（同小児歯科学教室准教授）は、研究で明らかになった予防効果についてこう話す。コンクールFの予防効果は？実証するための研究がスタート 本研究結果から、低濃度のコンクーS. mutansの増殖能とバイオフィルム形成能を206Abstract: Mouth rinses are a useful supplementary tool for the preven-tion of oral infectious diseases. Although the antimicrobial effects of mouth rinses have been investigated, there are few studies focusing on the comparison of the effects among various oral bacterial species. In the pres-ent study, the inhibitory effect of a commercial mouth rinse, “ConCoolF,” and each of its major components, chlorhexidine gluconate, ethanol, and green tea extract, on multiple species of oral bacteria were investigated. Inhibition of bacterial growth was observed in all cariogenic streptococcal species with different genera, serotypes, and strains isolated from different countries when either the complete mouth rinse or chlorhexidine gluconate were used. However, no growth inhibition was observed when the bacteria were exposed to ethanol or green tea extract. Interestingly, growth inhibi-tion was greatly reduced in non-cariogenic streptococci compared with cariogenic streptococci. In addition, both the mouth rinse and chlorhexi-dine gluconate inhibited the biolms formed by both Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis), among which the inhibitory effect against S. mutans was higher than that against P. gin-givalis. These results suggest that a mouth rinse containing chlorhexidine gluconate, ethanol, and green tea extract, or chlorhexidine gluconate alone, exhibits antimicrobial activity against several oral bacteria species, having greater activity against pathogenic bacteria.Keywords; antimicrobial effect, chlorhexidine gluconate, mouth rinse, mutans streptococci, Porphyromonas gingivalisIntroductionDental caries and periodontal disease are the most common infectious diseases induced by oral pathogens [1]. Multiple agents with antimicrobial properties have been developed and combined to create mouth rinses [2]. Chlorhexidine gluconate, ethanol, and green tea extracts are the major antimicrobial agents included in mouth rinses developed for the purpose of preventing oral diseases [2,3]. Although the antimicrobial effects of these agents against well-known bacteria, such as type strains, have been well established, it is unknown whether these agents have such antimicrobial activity on uncommon oral bacteria, such as those classied into minor serotypes, those that have obtained specic virulence genes, or bacteria identied by newer molecular techniques.Oral streptococci are known to be a major component in the microbial ora of the oral cavity [4]. Among the oral streptococci, Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) are classi-ed as mutans streptococci. Mutans streptococci have been implicated as causative agents of dental caries [5]. S. mutans is serologically classied into four groups: c, e, f, and k [6], whereas S. sobrinus is serologically classied into two groups: d and g [7]. Although the distribution frequen-cies of the serotype f and k S. mutans are less than 3% in the oral cavity, these strains are considered to be associated with cardiovascular disease, such as infective endocarditis and intracerebral hemorrhage [8,9]. Other oral streptococci such as Streptococcus oralis (S. oralis), Streptococcus gordonii (S. gordonii), Streptococcus mitis (S. mitis), and Streptococcus salivarius (S. salivarius) have low cariogenicity, and several of these are regarded as early colonizers of tooth surfaces [10,11].Periodontal disease is induced by infection with bacterial species asso-ciated with periodontitis [12]. Porphyromonas gingivalis (P. gingivalis) is an important periodontal pathogen and is also associated with the develop-ment of systemic diseases [13]. Many virulence factors have been reported for P. gingivalis and the virulence of P. gingivalis is highly dependent on its mA genotype [14]. The efcacy of mouth rinses on P. gingivalis strains with different mA genotypes has not yet been reported.In the present study, the ability of a mouth rinse formulated with chlorhexidine gluconate, ethanol, and green tea extract, as well as each individual component, to inhibit the growth of major oral pathogenic bacterial species with different serotypes or genotypes and commensal bacteria with low virulence, were analyzed. In addition, the antimicrobial effects of the mouth rinse or chlorhexidine gluconate on bacterial biolm formation by two species, S. mutans and P. gingivalis, were compared.Materials and MethodsPreparation of mouth rinse and antimicrobial agentsMouth rinse formulated with chlorhexidine gluconate, ethanol, and green tea extract (ConCoolF) was provided by Weltec Corporation (Osaka, Japan). In addition to the antimicrobial agents, the mouth rinse was composed of monoammonium glycyrrhizinate, l-menthol, propylene glycol, polyoxy-ethylene hardened castor oil and avoring agent. The concentrations of the antimicrobial agents were as follows: chlorhexidine gluconate = 0.05%, ethanol = 14.7%, and green tea extract = 0.40%. ConCoolF mouth rinse was diluted into each bacterial broth and tested at concentrations of 0%, 0.001%, 0.01%, 0.1%, 1%, and 10% for use in bacterial growth and bio-lm studies. Chlorhexidine gluconate, ethanol, and green tea extract were diluted individually into each broth and tested at concentrations of 0%, 0.001%, 0.01%, 0.1%, 1%, and 10%. A concentration of 100% for each mouth rinse component was dened as the percentage of each agent in ConCoolF; for example, a nal concentration of 14.7% ethanol in the broth culture was considered 100% ethanol.Bacterial strains and growth conditionsTable 1 lists the bacterial strains used in this study. A total of six S. mutans strains with different serotypes (two each of serotypes c and k, one each of serotypes e and f) and isolated from different locations in the body (ve strains isolated from the oral cavity and one strain isolated from blood) were selected from the laboratory stock [6,15-17]. Among the S. mutans strains, four (TLJ1-1, TLJ10-5, TLJ34-4, and TLJ11-2) were isolated in Thailand and two (MT8148 and TW295) were isolated in Japan. Two S. sobrinus strains from the laboratory stock were used, one each of serotypes d and g [18]. The non-cariogenic streptococci used in this study were S. oralis (ATCC 10557), S. gordonii (ATCC 10558), S. mitis (ATCC 49456) and S. salivarius (HHT), selected from the laboratory stock [18,19]. S. mutans and S. sobrinus strains were cultured on Mitis Salivarius (MS) agar (Difco Laboratories, Detroit, MI, USA) plates containing bacitracin (0.2 U/mL; Sigma-Aldrich Co., St. Louis, MO, USA) and 15% (w/v) sucrose and other Journal of Oral Science, Vol. 62, No. 2, 206-211, 2020Original articleInhibitory effect of a mouth rinse formulated with chlorhexidine gluconate, ethanol, and green tea extract against major oral bacterial speciesRyota Nomura1), Hiroaki Inaba2), Saaya Matayoshi1), Sho Yoshida2), Yuki Matsumi2), Michiyo Matsumoto-Nakano2), and Kazuhiko Nakano1)1) Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Suita, Japan2) Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan(Received April 4, 2019; Accepted August 16, 2019)Correspondence to Dr. Ryota Nomura, Department of Pediatric Dentistry, Division of Oral Infections and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamada-oka, Suita, Osaka 565-0871, JapanFax: +81-6-6879-2965 E-mail: rnomura@dent.osaka-u.ac.jpJ-STAGE Advance Publication: March 11, 2020Color gures can be viewed in the online issue at J-STAGE.doi.org/10.2334/josnusd.18-0483DN/JST.JSTAGE/josnusd/18-0483野村良太准教授QUINT ORAL INFORMATIONクイント・オーラル・インフォメーションP R5
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