대한안과학회지 2018 년제 59 권제 8 호 J Korean Ophthalmol Soc 2018;59(8):724-729 ISSN 0378-6471 (Print) ISSN 2092-9374 (Online) https://doi.org/10.3341/jkos.2018.59.8.724 Original Article 연옥이첨가된콘택트렌즈보관용기의항녹농균효과 Anti-pseudomonal Effect of a Nephrite-containing Contact Lens Storage Case 이상민 1 정재우 1 이동현 1 박성희 2 이종헌 1 유학선 2 김윤경 3 이지은 1,4 Sang Min Lee, MD 1, Jae Woo Jung, MD 1, Dong Hyun Lee, MD 1, Sung Hee Park, MS 2, Jong Heon Lee, MD 1, Hak Sun Yu, PhD 2, Yoon Kyung Kim, PhD 3, Ji-Eun Lee, MD, PhD 1,4 부산대학교의학전문대학원양산부산대학교병원안과학교실 1, 부산대학교의학전문대학원기생충학및열대의학교실 2, 부산여자대학교안경광학과 3, 부산대학교의학전문대학원양산부산대학교병원의생명융합연구소 4 Department of Ophthalmology, Pusan National University Yangsan Hospital, Pusan National University School of Medicine 1, Yangsan, Korea Department of Parasitology and Tropical Medicine, Pusan National University School of Medicine 2, Yangsan, Korea Department of Optometry, Busan Women s College 3, Busan, Korea Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Pusan National University School of Medicine 4, Yangsan, Korea Purpose: To compare the anti-pseudomonal effect of nephrite-containing contact lens (CL) storage cases with conventional CL storage cases. Methods: The degree of proliferation and adhesion of Pseudomonas aeruginosa (P. aeruginosa) on 1st 3rd generation silicone hydrogel contact lenses (SHCLs) was compared by using both 5% nephrite-containing CL storage cases and conventional CL storage cases. The degree of adhesion of P. aeruginosa to the CL was also compared in 1% or 5% nephrite solution and multipurpose solution (MPS). Results: Nephrite-containing CL storage cases showed a significant inhibitory effect on P. aeruginosa proliferation (p = 0.03) and significantly reduced the number of adherent P. aeruginosa on all 1st 3rd generation SHCLs compared with conventional CL storage cases (p = 0.02, p = 0.03, and p < 0.01, respectively). There was no significant anti-pseudomonal effect of 1% and 5% nephrite solutions compared with MPS. Conclusions: Nephrite may be the next generation substance that reduces infectious keratitis caused by P. aeruginosa when added to CL storage cases. J Korean Ophthalmol Soc 2018;59(8):724-729 Keywords: Contact lens storage case, Infectious keratitis, Multipurpose solution, Nephrite, Pseudomonas aeruginosa Received: 2018. 2. 14. Revised: 2018. 5. 20. Accepted: 2018. 7. 20. Address reprint requests to Ji-Eun Lee, MD, PhD Department of Ophthalmology, Pusan National University Yangsan Hospital, #20 Geumo-ro, Mulgeum-eup, Yangsan 50612, Korea Tel: 82-55-360-2590, Fax: 82-55-360-2161 E-mail: jiel75@hanmail.net * This article has been supported by the National Research Foundation of Korea (Grant No. NRF-2017R1D1A1B03032153). * Conflicts of Interest: The authors have no conflicts to disclose. 감염각막염은영구적실명을초래할수있는중요한질환으로, 콘택트렌즈사용으로인해그발생빈도가점차증가하고있다. 1,2 국내에서콘택트렌즈착용과관련된감염각막염의가장흔한원인균은녹농균으로알려져있으며, 3 세균에서분비되는독소와단백분해효소로인해각막조직의파괴가급속하게진행하여 24시간이내각막천공이발생할수있다. 4-6 현재까지도진행이빠르고치료후에도심한시력손상을남길위험이큰녹농균에의해발생한콘택트렌즈관련감염각막염사례가다수보고되고있는실정이다. 7,8 c2018 The Korean Ophthalmological Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 724
- 이상민외 : 콘택트렌즈보관에서연옥의항균효과 - 콘택트렌즈와연관된감염각막염의발생은콘택트렌즈착용은물론, 보관및세척과같은관리용품의상태, 착용자의잘못된습관에의해서도증가될수있다. 따라서최근에는콘택트렌즈자체의항균효과를높이는동시에, 렌즈보관용기, 세척액등의관리제품들도항균효과를개선시키는등콘택트렌즈관련합병증을줄이기위한다방면의연구들이이루어지고있다. 9-11 국외에서는콘택트렌즈, 다목적용액, 렌즈와관리용품에새로운물질을첨가하여항균효과를높이는개발이이루어지고있는반면, 12-14 국내에서는이와같은연구가전무한실정으로, Jung et al 15 이연옥 (nephrite) 을첨가한콘택트렌즈보관용기와다목적용액에서항아메바효과가증대됨을보고한바있어, 콘택트렌즈관리체계에서항균효과를증대시키기위한첨가물질로옥의가능성을제시한바있다. 이에본연구에서는콘택트렌즈와관련된감염각막염에서가장흔한원인균으로알려진녹농균에대해연옥이콘택트렌즈보관용기와관리용액의첨가물질로서항균효과가있는지알아보고자하였다. 대상과방법 연옥이첨가된보관용기의항녹농균효과를알아보기위해폴리에틸렌재질의전통적인콘택트렌즈보관용기의전체무게 5% 에해당하는연옥을용기에균일하게포함되도록하여새로운보관용기를제작하였다. 본연구는양산부산대학교병원의학연구윤리심의위원회 (institutional review board, IRB) 의승인면제하에진행되었다. 세대별콘택트렌즈는 1세대 Air Optix (Alcon, Fort Worth, TX, USA), 2세대 Acuvue Oasys (Johnson & Johnson, Limerick, Ireland) 및 3세대 Biofinity (Cooper vision, Southampton, UK) 로상용화되어있는실리콘하이드로겔렌즈를구분하여준비하였다. 소프트콘택트렌즈용다목적용액은 Opti-Free Express (Alcon, Fort Worth, TX, USA) 를사용하였고, 연옥수는나노크기의미세입자로가공한연옥분을 phosphate buffer saline (PBS) 에 100 mg/ml 농도로준비하여 ph 7.2로보정한후 24시간동안보관하였다. 이후 2,500 rpm에서 5분간원심분리하여상층액을취한후 1% 및 5% 로각각희석된용액을만들었다. 녹농균 (Pseudomonas aeruginosa) 은양산부산대학교병원병원체자원은행에서제공한병원체자원 (P04011) 을이용하여수행되었으며, 녹농균을 PBS에희석하여볼텍스혼합기를이용하여잘섞은후트립틱소이배지 (Tryptic soy agar plate) 에도말하여 37 C에서 24시간배양하였다. 배양후 Luria-Bertani (LB) 한천배지에 1개의집락 (colony) 을접종 하여 37 C에서 10-18시간배양하였다. 이후분광광도계 (Spectronic 21D, Milton Roy Co., Ivyland, PA, USA) 를이용하여 optical density (OD) 600의값이 0.1이되도록준비하였다. 기존의콘택트렌즈보관용기와연옥이첨가된보관용기에서녹농균의증식정도를비교하기위해 PBS 용액 2 ml 가채워진각보관용기에녹농균 0.1 ml (2 10 7 /ml) 를접종하였다. 접종된보관용기들을실온에서 24시간동안배양시킨후 0.1 ml 표본을 LB 한천배지에접종하였다. 접종된용기를 33 에서 24시간배양후배양된균의집락수를집락형성단위 (colony-forming unit, CFU) 로측정하였다. 실험은각각 5회씩반복수행하여그결과를평균하여비교하였다. 전통적인콘택트렌즈보관용기와 5% 의연옥이포함된콘택트렌즈보관용기에실제보관된콘택트렌즈에녹농균이부착되는정도를비교하기위해, PBS 용액 2 ml가채워진각보관용기에녹농균 0.1 ml (2 10 7 /ml) 를접종한후 1, 2, 3세대렌즈와함께보관하였다. 실온에서 24시간동안배양후, 콘택트렌즈를 2 ml PBS로세척하여렌즈에부착되지않은세균들을씻어내었다. 세척된콘택트렌즈를무균적으로시험관에담아 3,000 rpm으로 3분간원심분리시켜콘택트렌즈에부착된세균들을떨어지도록하였다. 이후각각의튜브에서취해진세균부유액을 10 4 배및 10 5 배희석하고 LB 한천배지에접종하여 37 C, 5% CO 2 조건에서 18시간동안배양하였다. 희석된 LB 한천배지에서자란세균집락을 CFU 단위로측정하였으며각각 5회반복수행하여결과를평균하여비교하였다. 기존의다목적용액과연옥수의녹농균에대한항균효과를비교하기위해다목적용액과 1% 및 5% 연옥수각각을전통적인콘택트렌즈보관용기에 2 ml씩채운후 1, 2, 3 세대렌즈를넣고세균부유액 0.1 ml (2 10 7 /ml) 를접종하였다. 실온에서 24시간동안배양한후, 앞서시행한동일한방법으로렌즈에부착된녹농균의수를측정하여비교하였다. 위실험은각각 5회씩반복수행되었다. 통계처리는 SPSS for Windows 18.0 (IBM Corp., Armonk, NY, USA) 통계프로그램을이용하였으며, 녹농균의집락수를 t-test 및 analysis of variance (ANOVA) with the Scheffe test를이용하여비교하였다. 유의도 (p값) 0.05 미만인경우를통계학적으로유의한것으로간주하였다. 결과 배양된균주를각각의렌즈보관용기에접종하여실온에서 24시간동안배양한결과, 기존의콘택트렌즈보관용기 725
- 대한안과학회지 2018 년제 59 권제 8 호 - 에비해 (Fig. 1A), 5% 연옥이포함된보관용기에서 (Fig. 1B) 녹농균의증식이감소하였으며각군의 CFU는 9.62 ± 0.4 및 9.49 ± 0.8로통계적유의성을보였다 (Fig. 2, p=0.03). 일반콘택트렌즈보관용기와 5% 연옥이첨가된보관용기에각세대별콘택트렌즈를보관한후녹농균을배양한결과, 기존의콘택트렌즈보관용기 (Fig. 3A-C) 에서보다 5% 연옥이첨가된보관용기 (Fig. 3D-F) 에서얻은 CFU가모든세대에서낮게측정되었다. 보관용기에따른 CFU는 1세대 콘택트렌즈에서는각각 8.49 ± 0.6 및 8.24 ± 0.8, 2세대콘택트렌즈에서는각각 8.47 ± 0.6 및 8.32 ± 1.4, 3세대콘택트렌즈에서는각각 8.55 ± 0.2 및 8.20 ± 0.4로모든세대에서통계적유의성을보였다 (Fig. 4, 각각 p=0.02, p=0.03 및 A B Figure 1. Representative photographs of bacterial colonies on Luria-Bertani agar plate. The number of Pseudomonas aeruginosa colonies was significantly reduced in 5% contact lens (CL) storage cases containing 5% nephrite (B) compared with traditional CL storage cases (A). Figure 2. Number of Pseudomonas aeruginosa (P. aeruginosa) on contact lens (CL) storage cases. CL storage cases containing 5% nephrite significantly reduced the numbers of P. aeruginosa compared with traditional CL storage cases. Using a unpaired t-test to compare the differences between the two groups. Air optix Oasys Biofinity A B C Traditional CL storage case D E F CL storage case with 5% nephrite FIgure 3. Representative photographs of bacterial colonies on Luria-Bertani agar plate. These pictures (A-F) show the decreased number of Pseudomonas aeruginosa colonies in all generations of silicone hydrogel contact lenses stored at contact lens (CL) storage cases containing 5% nephrite. 726
- 이상민외 : 콘택트렌즈보관에서연옥의항균효과 - p<0.01). 다목적용액과 1% 및 5% 연옥수에 1, 2, 3세대콘택트렌즈를위치시킨뒤녹농균을접종후배양하여콘택트렌즈에부착된녹농균을관찰한결과연옥이포함된보관용액은기존의다목적용액에보관하였을때와비교하여유의한항균효과를확인할수없었다 (Fig. 5, p>0.05). 고찰 전세계적으로콘택트렌즈의사용이보편화되면서콘택트렌즈와연관된감염각막염발생이증가하게되었고, 16 서양에서는포도상구균이가장흔한원인균으로보고되었지만, 17 국내에서는녹농균이가장흔한원인균으로보고되고있다. 3 콘택트렌즈사용에의한감염각막염이발생하는기전은다음과같다. 콘택트렌즈착용으로인해각막에저산소증과고이산화탄소증이발생하게되고각막상피세포의대사작용과각막지각이감소하여, 미세한손상이발생한다하더라도환자가자각증상을느끼지못하는가운데각막실질에는젖산이지속적으로축적하게되어각막실질부종이발생하게된다. 18 이로인해약해진각막상피에손상이발생하고손상부위로감염원이침투하여감염각막염을유발하게된다. 이중가장중요한단계는저산소증으로인해각막이정상상태를유지하지못하게되는것으로, 이를개선하기위 해현재까지산소투과율과함수율이개선된새로운콘택트렌즈가개발및보급되었다. 19-21 하지만이러한콘택트렌즈재질의발달에도불구하고사용자의관리, 착용습관등으로인해감염각막염은여전히발생하고있어, 콘택트렌즈재질향상만으로는각막염발생을낮추는데한계가있음이보고되었다. 22 특히콘택트렌즈보관용기및세정액의오염이감염각막염을발생시키는중요한유발인자로보고되고있어, 22-24 국외에서는이들관련된제품에서향균효과개선을위한연구들이활발하게이루어지고있는반면, 25-27 국내에서는아직이런연구와제품화에대한노력이부족한실정이다. 연옥은마그네슘, 칼슘, 철분이주성분으로구성되어있어연옥을신체에착용하면당뇨병및고혈압을포함한심혈관계질환에유익할수있음이보고된바있다. 28 특히 Yeom et al 29 은연옥이 interleukin-1-beta, tumor necrosis factor, cyclo-oxygenase-2 같은염증유발의유전자발현을억제함을보고하였으며, Han et al 30 은 12-O-tetradecanoylphorbol-acetate 로유발한염증모델에서연옥이 indomethacin (0.5 mg/20 μl) 과유사한염증억제효과를보였으며, 10 mg 농도에서가장큰효능을나타냄을보고한바있다. 최근 Jung et al 15 은연옥을이용하여콘택트렌즈보관용기및다목적용액에서콘택트렌즈사용자에게흔한가시아메바각막염의발생을낮추는첨가물로서의효과가있음을보고하여연옥이콘택트렌즈와보관용기의첨가물질로서의가능성이있음을제시하였다. 이에따라본연구는녹농균에대한연옥의항균효과를 Figure 4. The number of Pseudomonas aeruginosa (P. aeruginosa) adhering to contact lenses. The number of P. aeruginosa adhering to contact lenses in contact lens storage case containing 5% nephrite was significantly reduced compared with traditional contact lens storage case regardless of the generation of silicone hydrogel contact lenses. Using a unpaired t-test to compare the differences between the two groups. Figure 5. Relationship of the number of Pseudomonas aeruginosa colonies among contact lenses treated with multipurpose solution and 1% and 5% nephrite solutions. Nephrite solutions showed a lot of colonies compared with optifree solution, regardless of the generation of silicone hydrogel contact lenses. Using a unpaired t-test to compare the differences between the two groups. 727
- 대한안과학회지 2018 년제 59 권제 8 호 - 확인해보고자하였으며, 그결과일반적인콘택트렌즈보관용기에서보다 5% 연옥이첨가된콘택트렌즈보관용기에서녹농균의증식이유의하게억제됨이관찰되었다. 또한일반콘택트렌즈보관용기에서보다 5% 연옥이첨가된보관용기에보관하였을때모든세대의콘택트렌즈에서녹농균의부착이감소되는것을확인할수있었다. 그러나상용화된다목적용액과비교시 1% 및 5% 의연옥수에콘택트렌즈를보관하였을때에는녹농균에대한개선된항균효과는확인할수없었다. 하지만다목적용액에연옥을첨가하였을때가시아메바에대한살충효과가기존의다목적용액보다증대됨이보고된바있어, 15 다목적용액에첨가물질로서연옥을포함시킬때녹농균에대한항균효과를증대시킬수있을것으로기대된다. 본연구의제한점으로는 Jung et al 15 이보고한가시아메바각막염에대한연옥의살충효과를확인한보고이외국내외에서연옥의항균작용에대한연구가충분히이루어진바가없어실험결과의비교가어려우며, 콘택트렌즈관리용품의첨가물질로서연옥을사용할경우콘택트렌즈사용자의각막및결막세포에대한안정성이아직평가되지못하였다는것이다. 따라서콘택트렌즈및다목적용액등의관리용품에추가물질로연옥을상용화하기위해서는각막및결막세포에대한연옥의안정성에대한평가가선행되어야할것이다. 결론적으로콘택트렌즈사용자에서발생하는감염각막염, 특히질병의경과가빠르며실명에이르는심각한합병증을초래할수있는녹농균에의한감염각막염을감소시키기위해콘택트렌즈보관용기에첨가물질로서연옥을사용할경우기존의제품들보다녹농균에대한항균효과가증대됨을확인할수있었다. 각막및결막세포에서연옥의안정성에대한후속연구가이루어진다면, 콘택트렌즈사용자에게서현재보다개선된녹농균에의한감염각막염예방효과를기대해볼수있을것으로사료된다. REFERENCES 1) Buehler PO, Schein OD, Stamler JF, et al. The increased risk of ulcerative keratitis among disposable soft contact lens users. Arch Ophthalmol 1992;110:1555-8. 2) Poggio EC, Glynn RJ, Schein OD, et al. The incidence of ulcerative keratitis among users of daily-wear and extended wear soft contact lenses. N Engl J Med 1989;321:779-83. 3) Hahn YH, Hahn TW, Choi SH, et al. Epidemiology of infectious Keratitis[I] A multi-center study. J Korean Ophthalmol Soc 1998;39:1633-51. 4) Duran JA, Refojo MF, Gipson IK, Kenyon KR. Pseudomonas attachment to new hydrogel contact lenses. Arch Ophthalmol 1987;105:106-9. 5) Sy A, Srinivasan M, Mascarenhas J, et al. Pseudomonas aeruginosa keratitis: outcomes and response to corticosteroid treatment. Invest Ophthalmol Vis Sci 2012;53:267-72. 6) Vazirani J, Wurity S, Ali MH. Multidrug-resistant pseudomonas aeruginosa keratitis: risk factors, clinical characteristics, and outcomes. Ophthalmology 2015;122:2110-4. 7) Kang JS, Koo JY, Shyn KH. Three cases of pseudomonas corneal ulcer in wearer soft contact lens. J Korean Ophthalmol Soc 1985;26:761-7. 8) Jeon SK, Tchah HW. Pseudomonas keratitis due to mishandling of soft contact lenses. J Korean Ophthalmol Soc 1996;37:901-7. 9) Donzis PB, Mondino BJ, Weissman BA. Microbial contamination of contact lens care systems. Am J Ophthalmol 1987;104:325-33. 10) Bailey CS. A review of relative risks associated with four types of contact lenses. Cornea 1990;9 Suppl 1:S559-61; discussion S62-3. 11) Wilson LA, Sawant AD, Simmons RB, Ahearn DG. Microbial contamination of contact lens storage cases and solutions. Am J Ophthalmol 1990;110:193-8. 12) Amos CF, George MD. Clinical and laboratory testing of a silver-impregnated lens case. Cont Lens Anterior Eye 2006;29:247-55. 13) Vermeltfoort PB, Hooymans JM, Busscher HJ, van der Mei HC. Bacterial transmission from lens storage cases to contact lenses-effects of lens care solutions and silver impregnation of cases. J Biomed Mater Res B Appl Biomater 2008;87:237-43. 14) Scheuer C, Zhao F, Erb T, Orsborn G. Multipurpose solutions and rates of biocidal efficacy. Eye Contact Lens 2009;35:88-91. 15) Jung JW, Lee JH, Park SH, et al. Amoebicidal effect of nephrite-containing contact lens storage case. J Korean Ophthalmol Soc 2017;58:509-15. 16) Schein OD, Poggio EC. Ulcerative keratitis in contact lens wearers. Incidence and risk factors. Cornea 1990;9 Suppl 1:555-8; discussion S62-3. 17) Asbell P, Stenson S. Ulcerative keratitis. Survey of 30 years' laboratory experience. Arch Ophthalmol 1982;100:77-80. 18) Bruce AS, Brennan NA. Corneal pathophysiology with contact lens wear. Surv Ophthalmol 1990;35:25-58. 19) Teichroeb JH, Forrest JA, Ngai V, et al. Imaging protein deposits on contact lens materials. Optom Vis Sci 2008;85:1151-64. 20) Giraldez MJ, Serra C, Lira M, et al. Soft contact lens surface profile by atomic force microscopy. Optom Vis Sci 2010;87:E475-81. 21) Shayani Rad M, Khameneh B, Sabeti Z, et al. Antibacterial activity of silver nanoparticle-loaded soft contact lens materials: the effect of monomer composition. Curr Eye Res 2016;41:1286-93. 22) Stapleton F, Dart JK, Seal DV, Matheson M. Epidemiology of Pseudomonas aeruginosa keratitis in contact lens wearers. Epidemiol Infect 1995;114:395-402. 23) Donzis PB, Mondino BJ, Weissman BA, Bruckner DA. Microbial contamination of contact lens care systems. Am J Ophthalmol 1987;104:325-33. 24) Bowden FW 3rd, Cohen EJ, Arentsen JJ, Laibson PR. Pattern of lens care practices and lens product contamination in contact lens associated microbial keratitis. CLAO J 1989;15:49-54. 25) Lee GH, Lee JE, Park MK, Yu HS. Adhesion of Acanthamoeba on silicone hydrogel contact lenses. Cornea 2016;35:663-8. 26) Imayasu M, Shimizu H, Shimada S, et al. Effects of multipurpose contact-lens care solutions on adhesion of Pseudomonas aeruginosa to corneal epithelial cells. Eye Contact Lens 2009;35:98-104. 27) Dantam J, Zhu H, Stapleton F. Biocidal efficacy of silver-impreg- 728
- 이상민외 : 콘택트렌즈보관에서연옥의항균효과 - nated contact lens storage cases in vitro. Invest Ophthalmol Vis Sci 2011;52:51-7. 28) Lee JY, Seo BI, Lee IH, Park BJ. A mineralogical geochemical and oriental medical study on nephrite well waters. J Science Education Kyungpook National University 2001;25:43-52. 29) Yeom MJ, Choi BH, Han DO, et al. In vitro inhibition of pro-inflammatory mediator mrna expression by nephrite in lipopolysaccharide-induced mouse macrophage cells. Korean J Orient Physiol Pathol 2004;18:1622-7. 30) Han DO, Choi BH, Lee HJ, et al. In vivo studies on anti-inflammatory activity of nephrite. Korean J Orient Physiol Pathol 2005;19:977-81. = 국문초록 = 연옥이첨가된콘택트렌즈보관용기의항녹농균효과 목적 : 연옥이첨가된콘택트렌즈보관용기의항녹농균효과를일반콘택트렌즈보관용기와비교하였다. 대상과방법 : 5% 연옥이첨가된콘택트렌즈보관용기와일반콘택트렌즈보관용기를이용하여, 각각의보관용기에따른녹농균의증식정도와 1-3 세대실리콘하이드로겔콘택트렌즈에대한부착정도를비교하였다. 또한 1% 및 5% 연옥수와상용다목적관리용액에서콘택트렌즈에대한녹농균의부착정도를비교하였다. 결과 : 일반콘택트렌즈보관용기에비해 5% 연옥이첨가된콘택트렌즈보관용기에서녹농균의증식이유의하게감소되었으며 (p=0.03), 콘택트렌즈에부착된녹농균의수도모든세대에서유의하게감소하였다 ( 각각 p=0.02, p=0.03 및 p<0.01). 1% 및 5% 연옥수는다목적관리용액과비교시녹농균부착정도에서유의한차이를보이지않았다. 결론 : 연옥은콘택트렌즈보관용기에첨가시녹농균에의한감염각막염을감소시킬수있는차세대물질로서가능성이있을것으로사료된다. < 대한안과학회지 2018;59(8):724-729> 729