Izumi MASHIMA

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Izumi, MASHIMA

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Phylogenetic and Ecological Study of Oral Veillonella in Biofilm Formation

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Health Sciences University of Hokkaido Academic Repository / 北海道医療大学学術リポジトリ

北海道医療大学学術リポジトリPhylogenetic and Ecological Study of OralVeillonella in Biofilm Formation著者 Izumi MASHIMAjournal orpublication title北海道医療大学歯学雑誌volume 34number 1page range 41-43year 2015-06-30URL http://id.nii.ac.jp/1145/00010334/IntroductionDental caries and periodontal diseases are caused by oralbiofilms. It is already known that oral biofilms consist ofvarious bacteria that form three−dimensional conformationsand confer resistance against immunity and antimicrobialagents.It has been suggested that oral Veillonella species haveimportant roles in the early stages of the formation of oralbiofilms (Saravanan & Kolenbrander, 2010). However, theirrole in biofilm formation has been not clarified in detail be-cause of difficulties associated with their proper handling.The aims of this study were to establish the phylogeneticrelationships between oral Veillonella species and revealtheir ecological roles in oral biofilm formation. Furthermore,this study aimed to establish a novel preventative method fordental caries and periodontal diseases by the regulation oforal biofilm formation at an early stage.1. Phylogenetic Analysis of Veillonella Species in HumanOral CavitiesThe genus Veillonella is composed of strictly anaerobicgram−negative cocci lacking flagella, spores, and capsules.The distribution and frequency of oral Veillonella species(Veillonella atypica , Veillonella denticariosi , Veillonella dis-par, Veillonella parvula , and Veillonella rogosae) in humantongue biofilms of healthy young adults have been analyzedby PCR using species−specific primer sets to clarify theconstitution of oral Veillonella during an early stage ofbiofilm formation (Mashima et al., 2011). In the process ofabove study, 12 strains that were not classified as any previ-ously described Veillonella species. A sequence analysis ofthree genes, an analysis of cellular fatty acids, and variousbiochemical tests were carried out. As a result, the 12 strainswere established as a novel Veillonella species, Veillonellatobetsuensis sp. nov., named after Tobetsu, the town whereHealth Sciences University of Hokkaido is located (Mashimaet al., 2013). The type strain of Veillonella tobetsuensis wasdeposited in the Japan Culture Microorganisms and Ameri-can Type Culture Collection.Furthermore, a species−specific primer pair for V. tobet-suensis was designed to estimate its distribution and fre-quency in the human oral cavities of healthy young adults(Mashima & Nakazawa, 2013). It was also reported that oralVeillonella might contribute to oral infectious diseases(Mashima & Nakazawa, 2013). In addition, the distributionand frequency of oral Veillonella species in periodontalpockets were revealed using PCR with 6 species−specificprimer sets (Mashima et al., 2015).2. The Ecology of Oral Veillonella in Biofilm FormationIn early ecological studies of biofilm formation, a novelmethod for biofilm assays in vitro , termed “ the wiremethod,” was developed to overcome the disadvantages ofplate assays and flow cell methods (Mashima & Nakazawa,2012). This novel method enabled a quantitative analysis atthe species level in biofilms formed by multiple bacterialspecies.To determine the roles of oral Veillonella in the earlystages of biofilm formation, biofilms formed by a combina-tion of 4 Streptococcus species as the initial colonizers and6 Veillonella species as early colonizers were analyzed. As aresult, biofilm formations composed of 24 combinations ofspecies were divided into 4 patterns, and V. tobetsuensis sig-〔学位論文〕Phylogenetic and Ecological Study of Oral Veillonella in Biofilm FormationIzumi MASHIMADepartment of Oral Microbiology, School of Dentistry, Health Sciences University of HokkaidoKey words：Oral Veillonella , Veillonella tobetsuensis , Oral Biofilm, Quorum Sensing System, Autoinducer北海道医療大学歯学雑誌 34（41－43）平成27年 41（41）第３４巻１号　　　４Ｃ１５０　１Ｃ１３３／本文　※３１‐１から組体裁変更　ＯＴＦ／０４１～０４３　学位論文　ＭＡＳＨＩＭＡ  2015.07.22 18.54.00  Page 41 nificantly stimulated the formation of biofilms with Strepto-coccus gordonii (Mashima & Nakazawa, 2014). Based onthe results of these studies, the combination of V. tobetsuen-sis and S. gordonii is considered a model for biofilm forma-tion at an early stage, and has been used in subsequent stud-ies.Although co−aggregation is one of the important factorsin biofilm formation, it was not observed for V. tobetsuensisand S. gordonii . These results suggest that molecular factorsproduced by V. tobetsuensis stimulated biofilm formationwith S. gordonii . Therefore, an autoinducer−mediated quo-rum sensing system in V. tobetsuensis was the focus of thisstudy. Autoinducer−1 (AI−1) and autoinducer−2 (AI−2)from V. tobetsuensis were detected and partially purified(Mashima & Nakazawa, 2015). Furthermore, the effects ofAI−2 derived from V. tobetsuensis on S. gordonii biofilmformation were examined.The highest AI−1 activity was observed during the lateexponential phase. When the crude AI−1 obtained at the lateexponential phase was analyzed by two−dimensional thin−layer chromatography, two spots were observed at a positionsimilar to C8−HSL. The highest V. tobetsuensis AI−2 activ-ity was observed during the middle exponential phase. Thepartially purified AI−2 showed 10 times more AI−2 activitythan the control strain. This result indicated that the partialpurification of V. tobetsuensis AI−2 was successful.Furthermore, the partially purified AI−2 from the superna-tant of V. tobetsuensis significantly inhibited S. gordoniibiofilm formation.Many previous reports regarding AI−2, especially thoserelated to oral bacteria, have indicated that it promotesbiofilm formation. These results indicate that V. tobetsuensisAI−2 might be a new type of AI.Further studies including a structural analysis of these AIsderived from V. tobetsuensis and a biofilm assay to examinetheir roles in oral biofilm formation are required.ReferencesSaravanan P, Kolenbrander PE. Central role of the earlycolonizer Veillonella sp. in establishing multispeciesbiofilm communities with initial, middle, and late coloniz-ers of enamel. J Bacteriol 192 : 2965−2972, 2010.Mashima I, Kamaguchi A, Nakazawa F. The distributionand frequency of oral Veillonella spp. in the tonguebiofilm of healthy young adults. Curr Microbiol 63 : 403−407, 2011.Mashima I, Nakazawa F. The wire method for generatingexperimental biofilms formed by oral Streptococcus andVeillonella species, Dent J Heal Sci Univ Hokkaido 31 :73−80, 2012.Mashima I, Nakazawa F. Identification of Veillonella tobet-suensis in Tongue Biofilm by Using a Species−SpecificPrimer Pair. Anaerobe 22 : 77−81, 2013.Mashima I, Nakazawa F. A review on the characterizationof a novel oral Veillonella species, V. tobetsuensis , and itsrole in oral biofilm formation, J Oral Biosci, 55 : 184−190, 2013.Mashima I, Kamaguchi A, Miyakawa H, Nakazawa F.Veillonella tobetsuensis sp. nov. , an anaerobic, Gram −negative coccus isolated from human tongue biofilms. IntJ Syst Evol Microbiol 63 : 1443−1449, 2013.Mashima I, Nakazawa F. The influence of oral Veilloenllaspecies on biofilms formed by Streptococcus species. An-aerobe 28 : 54−61, 2014.Mashima I, Fujita M, Nakatsuka Y, Kado T, Furuichi Y,Herastuti S, Nakazawa F. The distribution and frequencyof Veillonella spp. associated with chronic periodontaldiseases. IJCMAS, 4 : 150−160, 2015.Mashima I, Nakazawa F. Signaling Molecules from Veil-lonella tobetsuensis in Biofilm Formation with Strepto-coccus gordonii . J Bacteriol, 197 : 2104-2111, 2015.42 Izumi MASHIMA／Phylogenetic and Ecological Study of Oral Veillonella in Biofilm Formation（42）第３４巻１号　　　４Ｃ１５０　１Ｃ１３３／本文　※３１‐１から組体裁変更　ＯＴＦ／０４１～０４３　学位論文　ＭＡＳＨＩＭＡ  2015.07.07 18.50.29  Page 42 眞島 いづみ平成13年3月 富士見丘高等学校 卒業平成16年4月 北海道医療大学歯学部歯学科 入学平成22年3月 北海道医療大学歯学部歯学科 卒業平成23年4月 北海道医療大学大学院歯学研究科 入学平成27年3月 北海道医療大学大学院歯学研究科 修了平成27年4月 日本学術振興会特別研究員PD，北海道医療大学学外研究員43The Dental Journal of Health Sciences University of Hokkaido 34 2015（43）第３４巻１号　　　４Ｃ１５０　１Ｃ１３３／本文　※３１‐１から組体裁変更　ＯＴＦ／０４１～０４３　学位論文　ＭＡＳＨＩＭＡ  2015.07.07 18.50.29  Page 43 

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Phylogenetic and Ecological Study of Oral Veillonella in Biofilm Formation

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