J Dent Hyg Sci Vol. 18, No. 2, 2018, pp.69-75 https://doi.org/10.17135/jdhs.2018.18.2.69 RESEARCH ARTICLE 치과용유니트수관에서분리한세균의부착및바이오필름형성능력 윤혜영ㆍ이시영 강릉원주대학교치과대학구강미생물학교실, 구강과학연구소 Adhesion and Biofilm Formation Abilities of Bacteria Isolated from Dental Unit Waterlines Hye Young Yoon and Si Young Lee Department of Oral Microbiology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, Gangneung 25457, Korea The purpose of our study is to compare the adhesion and biofilm formation abilities of isolates from water discharged from dental unit waterlines (DUWLs). Bacteria were isolated from a total of 15 DUWLs. Twelve isolates were selected for the experiment. To confirm the adhesion ability of the isolates, each isolate was attached to a glass coverslip using a 12-well plate. Plates were incubated at 26 o C for 7 days, and the degree of adhesion of each isolate was scored. To verify the biofilm formation ability of each isolate, biofilms were allowed to form on a 96-well polystyrene flat-bottom microtiter plate. The biofilm accumulations of all isolates formed at 26 o C for 7 days were identified and compared. A total of 56 strains were isolated from 15 water samples including 12 genera and 31 species. Of the 56 isolates, 12 isolates were selected according to the genus and used in the experiment. Sphingomonas echinoides, Methylobacterium aquaticum, and Cupriavidus pauculus had the highest adhesion ability scores of +3 among 12 isolates. Among these three isolates, the biofilm accumulation of C. pauculus was the highest and that of S. echinoides was the third-most abundant. The lowest biofilm accumulations were identified in Microbacterium testaceum and M. aquaticum. Most isolates with high adhesion ability also exhibited high biofilm formation ability. Analysis of adhesion and biofilm formation of the isolates from DUWLs can provide useful information to understand the mechanism of DUWL biofilm formation and development. Key Words: Bacterial adhesion, Biofilms, Dental infection control, Water microbiology 서론 치과용유니트수관 (dental unit waterlines, DUWL) 내물의관리는환자와환자사이, 치과종사자와환자사이의교차감염을예방하기위해필요하다 1,2). DUWL에서배출되는물에는많은미생물들이포함되어있으며, 미생물의종에대해서는이전연구들을통해밝혀졌다 3-5). DUWL에는대부분그람음성의종속영양세균이포함되어있으며, 기회병원성세균또한일부존재하는것으로알려져있다 6-8). DUWL 관리의중요성이증가하면서 DUWL 관리를위한다양한방법이사용되고있다. 이방법들중가장효과적인방법으로 DUWL 물내세균수를줄이면서 DUWL에형성된바이오필름을제거할수있는화학소독제의사용이추천되고있다 9,10). DUWL 바이오필름을완전히제거하지않는다면남아있는바이오필름에부유하고있는세균의재성장을허용하면서물내세균오염이계속되기때문에 DUWL Received: December 11, 2017, Revised: February 26, 2018, Accepted: March 5, 2018 ISSN 2233-7679 (Online) Correspondence to: Si Young Lee Department of Oral Microbiology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, 7 Jukheon-gil, Gangneung 25457, Korea Tel: +82-33-640-2455, Fax: +82-33-642-6410, E-mail: siyoung@gwnu.ac.kr, ORCID: https://orcid.org/0000-0001-8826-1413 Copyright 2018 by Journal of Dental Hygiene Science This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
J Dent Hyg Sci Vol. 18, No. 2, 2018 바이오필름을완벽하게제거할수있는화학소독제를사용하는것이중요하다 11-13). DUWL 바이오필름은 DUWL 내면에 conditioning film 의형성, 세균의가역적및비가역적부착, 세균과세균사이응집으로인한군집화, 그리고세균의군집과세포외다당류 (exopolysaccharide, EPS) 의결합과정으로성숙한다 14). DUWL에부착하는세균의종에따라 DUWL 바이오필름을구성하는세균의다양성이달라질수있으며, 세균의다양성은소독제에대한바이오필름의감수성에영향을줄수있다 15,16). 바이오필름의효과적인제거및바이오필름형성방지를위해서는바이오필름을구성하는세균의종을확인하고개별세균들의바이오필름형성능력및바이오필름형성에영향을주는요인들의대한연구가필수적이다. 현재까지바이오필름을구성하는세균종에대한연구는많이진행되었지만, 각각의개별세균의바이오필름형성특성에대한연구는전무한실정이다. 개별세균의부착능력은바이오필름형성특성및소독제에대한세균의감수성에영향을주기때문에 DUWL 바이오필름을구성하는개별세균의부착및바이오필름형성능력을확인하는연구는필요하다. 실험실에서 glass coverslip 또는 well-plate는세균의부착능력및바이오필름형성능력을확인하기위해사용되고있다 17-21). Eginton 등 18) 은소독제와같은항균물질로처리후표면에대한세균의부착강도를비교하기위해 glass coverslip을사용했으며, Modesto와 Drake 19) 의연구에서는항균물질처리후세균의부착정도와바이오필름형성능력을평가하기위해 glass coverslip과 well-plate를사용하였다. 또한, 수계 (water system) 나구강등과같은환경에존재하는바이오필름을실험실에서간단히재현할수있는장치로는 well-plate가주로사용되고있다 22,23). 따라서, 본연구의목적은 DUWL에서배출되는물에서분리한균주의부착능력과바이오필름형성능력을 glass coverslip과 wellplate를사용하여확인하고두능력사이관계를확인하는것이다. 연구대상및방법 1. 시료수집및세균분리물시료 (500 ml) 는강릉시내치과 4곳에위치한치과용유니트 15대의초음파치석제거기로부터얻었다. 수집전초음파치석제거기의입구를 70% 에탄올로닦아주었다. 시료수집후, 잔류염소를중화시키기위해각물시료에 10% sodium thiosulphate (Yakuri Pure Chemicals Co., Ltd., Kyoto, Japan) 용액을 0.5 ml 씩넣어주었다. 각물시료를 0.2 μm 여과지 (Millipore, Billerica, MA, USA) 에여과시켰고, 여과지를 10 ml phosphate-buffered saline (PBS, ph 7.4) 용액에현탁시켰다. Spiral plater (IUL, Barcelona, Spain) 를사용하여현탁액의 50 μl를 R2A 고체배지 (Becton, Dickinson and Company, Sparks, MD, USA) 에도말하였고배지를 26 o C에서 7일간배양하였다. 배양후 R2A 고체배지에서성장한콜로니형태를확인하였고, 각물시료당수적으로대표적인 3 5개의콜로니를선택하였다. 선택한콜로니를 R2A 고체배지에도말하였고, 26 o C에서 7일간배양한후 R2A 액체배지 (Becton, Dickinson and Company) 로계대배양하였다. 2. 세균의동정분리된세균은중합효소연쇄반응 (polymerase chain reaction, PCR) 증폭과 16S ribosomal RNA (rrna) 유전자염기서열확인에의해동정되었다. 세균배양액의 1 ml를 13,000 g로 3분동안원심분리하고상층액은버린후 200 μl 의멸균증류수에현탁하였다. 이현탁액으로부터 G-spin TM Genomic DNA Extraction Kit (intron Biotechnology Inc., Seongnam, Korea) 를이용하여제조회사의방법대로세균의 genomic DNA를추출하였다. Universal PCR primer (27F; 5ˊ-AGA GTT TGA TCM TGG CTC AG-3ˊ, 1492R; 5ˊ-GGY TAC CTT GTT ACG ACT T-3ˊ) 와 Accupower HotStart PCR Premix (Bioneer, Deajeon, Korea) 를이용하여 GeneAmp PCR System 9700 (PerkinElmer, Waltham, MA, USA) 에서 16S rrna 유전자를증폭하였다 24). PCR stock solution은 0.1 μm forward 및 reverse primer와 100 pg의세균 genomic DNA를첨가한후최종용량이 20 μl가되도록증류수로채워제조하였다. PCR은 94 o C에서 2분간초기열처리한다음 94 o C에서 30초간변성 (denaturation), 55 o C에서 30초간결합 (annealing), 72 o C에서 1분간중합 (extension) 과정을 34회반복하여시행하였다. 증폭된 DNA는 AccuPrep R PCR Purification Kit (Bioneer) 를사용하여정제하였고코스모진텍 (Cosmogenetech, Seoul, Korea) 에의뢰하여염기서열을얻었다. 세균종동정을위하여염기서열은 Blastn (genome database of the National Center for Biotechnology Information) 을이용하여분석하였다. 3. 세균선택및준비염기서열분석을통해 12속 (genus) 과 31종 (species) 을포함한총 56균주가확인되었고그중속당 1균주씩을선택하 70
윤혜영ㆍ이시영 : 치과용유니트수관분리균의바이오필름형성능력 여총 12 균주가부착실험을위해사용되었다. 12 균주는 R2A 액체배지에계대되었고 26 o C 에서 7 일동안배양하였다. 4. 분리균주의부착양상확인분리균주의부착능력을확인하기위해각균주가 glass coverslip에부착하는양상을확인했다. 각균주의부착양상은 Park 등 17) 이보고한방법을변형하여확인하였다. R2A 액체배지에서배양된세균은 0.5 McFarland standard (1 10 8 colony-forming unit [CFU]/ml) 와동등한농도의세균현탁액을얻기위해 R2A 액체배지에희석되었다. 12-well plates (SPL, Pocheon, Korea) 의각 well에멸균된 glass coverslip (round, 12 mm diameter) 을넣고 R2A 액체배지를 4 ml씩첨가하였다. 그후, 세균현탁액을각 well에 0.4 ml씩첨가하고 26 o C 배양기에서 7일동안배양하였다. 배양후, 바이오필름이형성된 glass coverslip은멸균된 PBS (ph 7.4) 로약하게씻어 glass coverslip에부착하지않은세균을제거하였다. 각세균을부착하여나타난 glass coverslip의혼탁정도를비교하였고혼탁도를 1 3점으로점수화하였다. 5. 분리균주의바이오필름축적량확인분리균주의바이오필름형성능력을확인하기위해이전연구에서설명한방법을변형하여수행하였다 21,25). 각균주의바이오필름을 7일동안형성시키고그축적량을확인하였다. 96-well polystyrene flat-bottom microtiter plate (SPL) 에 R2A 액체배지를 180 μl 넣고 1 10 8 CFU/ml로농 Table 1. List of All the Species Isolated in the 15 Water Samples from Different Dental Unit Waterlines Phylum Genus Species Total Proteobacteria Acidovorax Acidovorax delafieldii a 1 Acidovorax soli 1 Brevundimonas Brevundimonas subvibrioides a 1 Cupriavidus Cupriavidus pauculus a 1 Methylobacterium Methylobacterium aquaticum a 1 Methylobacterium fujisawaense 4 Methylobacterium populi 1 Methylobacterium radiotolerans 1 Novosphingobium Novosphingobium aromaticivorans 1 Novosphingobium capsulatum 2 Novosphingobium fuchskuhlense a 4 Novosphingobium nitrogenifigens 3 Novosphingobium resinovorum 1 Novosphingobium sediminicola 1 Novosphingobium stygium 1 Novosphingobium subterraneum 1 Pelomonas Pelomonas puraquae a 2 Polaromonas Polaromonas aquatica a 5 Sphingobium Sphingobium limneticum 1 Sphingobium xenophagum a 4 Sphingobium yanoikuyae 1 Sphingomonas Sphingomonas echinoides a 7 Sphingomonas ginsenosidimutans 2 Sphingomonas hunanensis 1 Sphingomonas oligophenolica 1 Sphingomonas paucimobilis 1 Sphingomonas starnbergensis 1 Sphingomonas wittichii 1 Sphingopyxis Sphingopyxis panaciterrae a 2 Bacteroidetes Sediminibacterium Sediminibacterium salmoneum a 1 Actinobacteria Microbacterium Microbacterium testaceum a 1 Total 56 a The selected strains used for susceptibility tests. 71
J Dent Hyg Sci Vol. 18, No. 2, 2018 도조정된세균현탁액 20 μl를추가했다. Plate는 static 상태로 26 o C에서 7일동안배양하였다. 배양후, plate 내배지를제거하고비부착세균을제거하기위해 150 μl PBS로 2 번세척했다. 새로운 R2A 액체배지 100 μl는추가되었고, 바이오필름은파이펫팅으로 plate에서분리및현탁되었다. 세균현탁액은 10배수로희석되었고희석액은 R2A 고체배지에 spiral plater를이용하여도말하였다. 배지는 26 o C에서 7일배양되었고세균의집락을 colony counter (IUL) 로계수한후 CFU/ml로계산하였다. Table 2. Adhesion Score of Each Isolate on Glass Slip Isolate Score Acidovorax delafieldii ++ Brevundimonas subvibrioides ++ Cupriavidus pauculus +++ Methylobacterium aquaticum +++ Microbacterium testaceum ++ Novosphingobium fuchskuhlense + Pelomonas puraquae ++ Polaromonas aquatica ++ Sediminibacterium salmoneum ++ Sphingobium xenophagum + Sphingomonas echinoides +++ Sphingopyxis panaciterrae ++ 결과 1. 분리균주 15개의물시료로부터총 56균주가분리되었다 (Table 1). 56균주는 12속과 31종을포함하였다. 바이오필름형성능력과부착능력의평가를위해, 각속당 1균주씩선택되었다. 실험에사용된균주는 Sphingomonas echinoides, Polaromonas aquatica, Novosphingobium fuchskuhlense, Sphingobium xenophagum, Pelomonas puraquae, Sphingopyxis panaciterrae, Acidovorax delafieldii, Brevundimonas subvibrioides, Cupriavidus pauculus, Methylobacterium aquaticum, Microbacterium testaceum, Sediminibacterium salmoneum과같다. 2. 분리균주의부착양상실험에사용된 12균주의부착점수는 Table 2에나타냈다. C. pauculus, M. aquaticum과 S. echinoides의부착점수는가장높은 +3으로관찰되었다. N. fuchskuhlense와 S. xenophagum 의부착점수가 +1 로가장낮았고나머지 7 균주는모두 +2 의부착점수를나타냈다. 3. 분리균주의바이오필름축적량 7일동안배양후 microtiter plate에축적된바이오필름양은 Fig. 1에나타냈다. C. pauculus가 8.06 log CFU/ml로가장많은바이오필름축적량을보여주었고, P. aquatica와 S. echinoides가그다음으로바이오필름축적량이많았다. M. testaceum의바이오필름축적량은 4.69 log CFU/ml로 Fig. 1. The mean biofilm accumulation of each isolate formed in 12-well plate for 7 days. The error bars indicate standard deviations of the mean for three repeated experiments in triplicate. CFU: colony-forming unit. 72
윤혜영ㆍ이시영 : 치과용유니트수관분리균의바이오필름형성능력 가장적었다. 이와유사하게 M. aquaticum의바이오필름축적량은 4.72 log CFU/ml로적었다. 고찰 본연구에서는 DUWL에서분리한균주의바이오필름능력및부착능력을확인하였다. 본실험에사용된 12균주들중 Sphingomonas spp. 는배지배양법및 pyrosequencing 분석을사용하여 DUWL 내다양성을확인한이전연구들의결과에서공통적으로많이발견되는세균이다 3,4,8,26). Methylobacterium spp., Novosphingobium spp., Sphingobium spp. 또한 DUWL 내세균의다양성을확인한대부분의연구에서빈번하게발견되었으며그외나머지균주들도 DUWL 내에서검출되는세균에포함된다 3,8,26). 배양배지법으로 DUWL 내세균을동정한연구의경우배지에배양이되지않는세균들은제외한후대표적인세균들을확인하였고 3,26), 분자생물학적방법인 pyrosequencing 분석으로 DUWL 내세균의다양성을확인한연구에서는세균을종수준까지정확하게확인할수없었다 8). 지금까지의연구결과들로 DUWL 내발견되는대표적인세균들을결정할수없기때문에본연구에서는분리된총 56균주중속당 1균주씩선택하여실험에사용하였다. 단단한표면에세균이부착하는것은바이오필름이형성되고성숙하는과정에서첫단계에해당하기때문에중요하다 27). 우리의연구에사용된 12균주중에서높은부착능력을보여준 S. echinoides, M. aquaticum, C. pauculus는초기 DUWL 바이오필름형성과정에영향을주었을가능성이있다. Simoes 등 27) 은음용수에서분리한세균의 polystyrene에대한부착능력과바이오필름형성능력사이관계를확인했고, 그결과부착능력이낮은균주보다높은균주가바이오필름형성능력이더높은것을보여주었다. 이와유사하게우리의연구에서부착능력이높은 S. echinoides와 C. pauculus는높은바이오필름형성능력을보여주었다. M. aquaticum의경우부착능력은높았지만, 바이오필름형성능력은낮았다. 이는 M. aquaticum의바이오필름형성과정에부착능력보다다른요소의영향을더크게받았기때문으로추측된다. 바이오필름형성에는세균의부착능력외에응집및 EPS 생산능력등과같은요소들도영향을줄수있다 14,28,29). 따라서분리균주들의응집및 EPS 생산능력과같은다른특성들을확인하는추가적인연구가필요할것이다. Yabune 등 30) 은불소코팅을한 DUWL의바이오필름형 성억제효과를확인했다. 불소코팅을하지않은 DUWL보다불소코팅한 DUWL을연결한치과용유니트에서배출되는물내세균의수가더낮았고, 주사전자현미경으로 DUWL 내면을관찰했을때불소코팅한 DUWL의표면에부착한세균이거의없었다. 이현상은불소코팅한 DUWL 의사용후 6개월까지지속되었다. Yabune 등 30) 의연구결과는불소코팅 DUWL의세균부착을억제하는효과가바이오필름의형성을예방할수있다는것을보여주었다. 이연구와같이표면으로부터세균의부착을억제하는방법은바이오필름의형성을예방하고억제하는데효과적일수있다. 세균의부착을억제하는방법의주요타깃은바이오필름형성과정에서초기에관여하는세균들이기때문에이세균들에대한정보는 DUWL 바이오필름형성을억제시키기위해중요할수있다. 세균의부착은부착하는표면의물질에영향을받을수있다 27,30). 본연구에서는실험실에서세균의부착능력및바이오필름형성능력을확인하는기본적인실험방법에따라세균이부착하는표면으로 glass나 polystyrene을사용했다. 하지만 DUWL의재질로는보통 polyurethane이나 polyvinylchloride가사용되기때문에 14), 이재질에대한분리균주의부착및바이오필름형성특성에대한연구는추가적으로수행되어야할것이다. 본연구의결과는 DUWL 바이오필름의형성기전을파악하는데도움을주며나아가바이오필름형성을억제하는방법의개발에기본적인정보를제공할수있을것이다. 요약 우리연구의목적은 DUWL에서배출되는물에서분리한균주의부착능력과바이오필름형성능력을확인하고두능력사이관계를확인하는것이다. DUWL로부터분리한 12균주를실험에사용하였다. 각균주의부착능력을확인하기위해, 12-well plates의각 well에멸균된 glass coverslip, R2A 액체배지, 그리고 1 10 8 CFU/ml의농도로조정된세균현탁액을넣고 26 C 배양기에서 7일동안배양하였다. 배양후, glass coverslip에부착한정도에따라 1 3점으로점수를부여하였다. 분리균주의바이오필름형성능력을확인하기위해, 96-well polystyrene flat-bottom microtiter plate에 R2A 액체배지와세균현탁액을넣고 26 C에서 7일동안배양하였다. 배양후, plate에형성된바이오필름은 R2A 액체배지에현탁했고, 현탁액을 R2A 고체배지에도말하였다. 26 C에서 7일배양한후세균의집락을계수하고 CFU/ml를계산하였다. DUWL로부터총 56 73
J Dent Hyg Sci Vol. 18, No. 2, 2018 균주가분리되었으며, 12속과 31종을포함하였다. 실험에는속당 1균주씩선택하여총 12균주가사용되었다. 12 균주중에 S. echinoides, M. aquaticum, C. pauculus의부착능력점수는 +3으로가장높았다. 바이오필름축적량은 C. pauculus가가장많았고, M. testaceum이가장적었다. 대부분의부착능력이높은균주는바이오필름형성능력또한높았다. 본연구의결과는 DUWL 바이오필름의형성기전을파악하는데도움을주며나아가바이오필름형성을억제하는방법의개발에기본적인정보를제공할수있을것이다. 감사의글 이논문은 2015년도정부 ( 교육부 ) 의재원으로한국연구재단의지원을받아수행된기초연구사업임 (2015R1D1A 1A01057790). References 1. Walker JT, Bradshaw DJ, Finney M, et al.: Microbiological evaluation of dental unit water systems in general dental practice in Europe. Eur J Oral Sci 112: 412-418, 2004. https://doi.org/10.1111/j.1600-0722.2004.00151.x 2. O'Donnell MJ, Boyle MA, Russell RJ, Coleman DC: Management of dental unit waterline biofilms in the 21st century. Future Microbiol 6: 1209-1226, 2011. https://doi.org/10.2217/fmb.11.104 3. Barbeau J, Tanguay R, Faucher E, et al.: Multiparametric analysis of waterline contamination in dental units. Appl Environ Microbiol 62: 3954-3959, 1996. 4. Singh R, Stine OC, Smith DL, Spitznagel JK Jr, Labib ME, Williams HN: Microbial diversity of biofilms in dental unit water systems. Appl Environ Microbiol 69: 3412-3420, 2003. https://doi.org/10.1128/aem.69.6.3412-3420.2003 5. Szymańska J, Sitkowska J, Dutkiewicz J: Microbial contamination of dental unit waterlines. Ann Agric Environ Med 15: 173-179, 2008. 6. Dutil S, Tessier S, Veillette M, et al.: Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines. J Appl Microbiol 100: 955-963, 2006. https://doi.org/10.1111/j.1365-2672.2006.02845.x 7. Szymanska J, Sitkowska J: Opportunistic bacteria in dental unit waterlines: assessment and characteristics. Future Microbiol 8: 681-689, 2013. https://doi.org/10.2217/fmb.13.33 8. Costa D, Mercier A, Gravouil K, et al.: Pyrosequencing analysis of bacterial diversity in dental unit waterlines. Water Res 81: 223-231, 2015. https://doi.org/10.1016/j.watres.2015.05.065 9. Cobb CM, Martel CR, McKnight SA 3rd, Pasley-Mowry C, Ferguson BL, Williams K: How does time-dependent dental unit waterline flushing affect planktonic bacteria levels? J Dent Educ 66: 549-555, 2002. 10. Walker JT, Bradshaw DJ, Fulford MR, Marsh PD: Microbiological evaluation of a range of disinfectant products to control mixed-species biofilm contamination in a laboratory model of a dental unit water system. Appl Environ Microbiol 69: 3327-3332, 2003. https://doi.org/10.1128/aem.69.6.3327-3332.2003 11. Whitehouse RL, Peters E, Lizotte J, Lilge C: Influence of biofilms on microbial contamination in dental unit water. J Dent 19: 290-295, 1991. https://doi.org/10.1016/0300-5712(91)90075-a 12. Meiller TF, Kelley JI, Baqui AA, DePaola LG: Laboratory evaluation of anti-biofilm agents for use in dental unit waterlines. J Clin Dent 12: 97-103, 2001. 13. Meiller TF, Depaola LG, Kelley JI, Baqui AA, Turng BF, Falkler WA: Dental unit waterlines: biofilms, disinfection and recurrence. J Am Dent Assoc 130: 65-72, 1999. https://doi.org/10.14219/jada.archive.1999.0030 14. Walker JT, Marsh PD: Microbial biofilm formation in DUWS and their control using disinfectants. J Dent 35: 721-730, 2007. https://doi.org/10.1016/j.jdent.2007.07.005 15. Liu Y, Zhang W, Sileika T, Warta R, Cianciotto NP, Packman A: Role of bacterial adhesion in the microbial ecology of biofilms in cooling tower systems. Biofouling 25: 241-253, 2009. https://doi.org/10.1080/08927010802713414 16. Simões LC, Simões M, Vieira MJ: Influence of the diversity of bacterial isolates from drinking water on resistance of biofilms to disinfection. Appl Environ Microbiol 76: 6673-6679, 2010. https://doi.org/10.1128/aem.00872-10 17.Park JH, Lee JK, Um HS, Chang BS, Lee SY: A periodontitis-associated multispecies model of an oral biofilm. J Periodontal Implant Sci 44: 79-84, 2014. https://doi.org/10.5051/jpis.2014.44.2.79 18. Eginton PJ, Holah J, Allison DG, Handley PS, Gilbert P: 74
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