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치과용 유니트 수관에서 분리한 세균의 부착 및 바이오필름 형성 능력

Adhesion and Biofilm Formation Abilities of Bacteria Isolated from Dental Unit Waterlines

  • 윤혜영 (강릉원주대학교 치과대학 구강미생물학교실, 구강과학연구소) ;
  • 이시영 (강릉원주대학교 치과대학 구강미생물학교실, 구강과학연구소)
  • Yoon, Hye Young (Department of Oral Microbiology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University) ;
  • Lee, Si Young (Department of Oral Microbiology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University)
  • 투고 : 2017.12.11
  • 심사 : 2018.03.05
  • 발행 : 2018.04.30

초록

우리 연구의 목적은 DUWL에서 배출되는 물에서 분리한 균주의 부착 능력과 바이오필름 형성 능력을 확인하고 두 능력 사이 관계를 확인하는 것이다. DUWL로부터 분리한 12균주를 실험에 사용하였다. 각 균주의 부착 능력을 확인하기 위해, 12-well plates의 각 well에 멸균된 glass coverslip, R2A 액체 배지, 그리고 $1{\times}10^8CFU/ml$의 농도로 조정된 세균 현탁액을 넣고 $26^{\circ}C$ 배양기에서 7일 동안 배양하였다. 배양 후, glass coverslip에 부착한 정도에 따라 1~3점으로 점수를 부여하였다. 분리 균주의 바이오필름 형성 능력을 확인하기 위해, 96-well polystyrene flat-bottom microtiter plate에 R2A 액체 배지와 세균 현탁액을 넣고 $26^{\circ}C$에서 7일 동안 배양하였다. 배양 후, plate에 형성된 바이오필름은 R2A 액체 배지에 현탁했고, 현탁액을 R2A 고체 배지에 도말하였다. $26^{\circ}C$에서 7일 배양한 후 세균의 집락을 계수하고 CFU/ml를 계산하였다. DUWL로부터 총 56균주가 분리되었으며, 12속과 31종을 포함하였다. 실험에는 속당 1균주씩 선택하여 총 12균주가 사용되었다. 12균주 중에 S. echinoides, M. aquaticum, C. pauculus의 부착 능력 점수는 +3으로 가장 높았다. 바이오필름 축적량은 C. pauculus가 가장 많았고, M. testaceum이 가장 적었다. 대부분의 부착 능력이 높은 균주는 바이오필름 형성 능력 또한 높았다. 본 연구의 결과는 DUWL 바이오필름의 형성 기전을 파악하는데 도움을 주며 나아가 바이오필름 형성을 억제하는 방법의 개발에 기본적인 정보를 제공할 수 있을 것이다.

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 $26^{\circ}C$ 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 $26^{\circ}C$ 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.

키워드

참고문헌

  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. Szymanska 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. Simoes LC, Simoes 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: Changes in the strength of attachment of micro-organisms to surfaces following treatment with disinfectants and cleansing agents. Lett Appl Microbiol 27: 101-105, 1998. https://doi.org/10.1046/j.1472-765X.1998.00390.x
  19. Modesto A, Drake DR: Multiple exposures to chlorhexidine and xylitol: adhesion and biofilm formation by Streptococcus mutans. Curr Microbiol 52: 418-423, 2006. https://doi.org/10.1007/s00284-005-0104-0
  20. Bodenmiller D, Toh E, Brun YV: Development of surface adhesion in Caulobacter crescentus. J Bacteriol 186: 1438-1447, 2004. https://doi.org/10.1128/JB.186.5.1438-1447.2004
  21. Antunes AL, Bonfanti JW, Perez LR, et al.: High vancomycin resistance among biofilms produced by Staphylococcus species isolated from central venous catheters. Mem Inst Oswaldo Cruz 106: 51-55, 2011. https://doi.org/10.1590/S0074-02762011000100008
  22. Yoon HY, Lee SY: Establishment of a dental unit biofilm model using well-plate. J Dent Hyg Sci 17: 283-289, 2017. https://doi.org/10.17135/jdhs.2017.17.4.283
  23. Walker C, Sedlacek MJ: An in vitro biofilm model of subgingival plaque. Oral Microbiol Immunol 22: 152-161, 2007. https://doi.org/10.1111/j.1399-302X.2007.00336.x
  24. de Lillo A, Ashley FP, Palmer RM, et al.: Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets. Oral Microbiol Immunol 21: 61-68, 2006. https://doi.org/10.1111/j.1399-302X.2005.00255.x
  25. Stepanovic S, Vukovic D, Hola V, et al.: Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS 115: 891-899, 2007. https://doi.org/10.1111/j.1600-0463.2007.apm_630.x
  26. Yabune T, Imazato S, Ebisu S: Assessment of inhibitory effects of fluoride-coated tubes on biofilm formation by using the in vitro dental unit waterline biofilm model. Appl Environ Microbiol 74: 5958-5964, 2008. https://doi.org/10.1128/AEM.00610-08
  27. Simoes LC, Simoes M, Vieira MJ: Adhesion and biofilm formation on polystyrene by drinking water-isolated bacteria. Antonie Van Leeuwenhoek 98: 317-329, 2010. https://doi.org/10.1007/s10482-010-9444-2
  28. Azeredo J, Oliveira R: The role of exopolymers produced by Sphingomonas paucimobilis in biofilm formation and composition. Biofouling 16: 17-27, 2000. https://doi.org/10.1080/08927010009378427
  29. Rickard AH, McBain AJ, Ledder RG, Handley PS, Gilbert P: Coaggregation between freshwater bacteria within biofilm and planktonic communities. FEMS Microbiol Lett 220: 133-140, 2003. https://doi.org/10.1016/S0378-1097(03)00094-6
  30. Yabune T, Imazato S, Ebisu S: Inhibitory effect of PVDF tubes on biofilm formation in dental unit waterlines. Dent Mater 21: 780-786, 2005. https://doi.org/10.1016/j.dental.2005.01.016

피인용 문헌

  1. 교내 실습실의 유니트체어의 수관관리에 관한 융합연구 vol.10, pp.10, 2019, https://doi.org/10.15207/jkcs.2019.10.10.027