Korean J Clin Lab Sci. 2020;52(2):128-135 https://doi.org/10.15324/kjcls.2020.52.2.128 Korean Society for Clinical Laboratory Science ORIGINAL ARTICLE Molecular Subtyping of Methicillin-Resistant Staphylococcus aureus Isolated from Patients Nasal Cavity Sang-Ha Kim1,2,, Sung-Bae Park3,4,, Heechul Park3,4, Jun Seong Kim3,4, Jungho Kim3, Jiyoung Lee3, Jaewon Lim5, Young Kwon Kim1, Sunghyun Kim3,4 1 Department of Health Sciences, The Graduate School of Konyang University, Daejeon, Korea Department of Laboratory Medicine, Konyang University Hospital, Daejeon, Korea 3 Department of Clinical Laboratory Sciences, College of Health Sciences, Catholic University of Pusan, Busan, Korea 4 Clinical Trial Specialist Program for In Vitro Diagnostics, Brain Busan 21 Plus Program, The Graduate School, Catholic University of Pusan, Busan, Korea 5 Department of Clinical Laboratory Science, College of Medicine and Sciences, Daegu Haany University, Kyungsan, Korea 2 환자의 비강으로부터 분리된 메티실린 내성 황색 포도알균의 분자 아형 분석 김상하1,2,, 박성배3,4,, 박희철3,4, 김준성3,4, 김정호3, 이지영3, 임재원5, 김영권1, 김성현3,4 1 건양대학교 보건복지대학원 보건학과, 2건양대학교병원 진단검사의학과, 3부산가톨릭대학교 보건과학대학 임상병리학과, 4부산가톨릭대학교 일반대학원 BB21+ 사업단, 5대구한의대학교 의과학대학 임상병리학과 ARTICLE INFO ABSTRACT Received April 28, 2020 Revised June 2, 2020 Accepted June 5, 2020 Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that causes infections in different parts of the body and causes skin and soft tissue infections (SSTI). The present study examined the antimicrobial resistance patterns and molecular epidemiological characteristics of MRSA isolated from nasal swabs in clinical patients. of MRSA isolates from clinical patients were analyzed: 24 cases were -II; two cases were type-ii/iva; one case was type-ii/v; one case was type-iva; 11 cases were not-typeable. The type of MRSA isolates from clinical patients were analyzed: 29 cases were type A, and 10 cases were not-typeable, but type B was not found in the present study. In conclusion, -II and mec complex type A were the most dominant MRSA subtypes among the MRSA isolates from a nasal swab of patients, and the results were similar to other studies on hospital-acquired MRSA (HA-MRSA). These results can not only provide basic data for hospital infection management but also be a good guideline for MRSA infections in the Republic of Korea. Key words Infection control Methicillin-resistant Staphylococcus aureus Molecular epidemiology Nasal cavity Copyright 2020 The Korean Society for Clinical Laboratory Science. All rights reserved. 서 론 황색포도알균(Staphylococcus aureus)은 병원감염 및 지 Corresponding author: Sunghyun Kim Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, 57 Oryundae-ro, Geumjeong-gu, Busan 46252, Korea E-mail: shkim0423@cup.ac.kr ORCID: https://orcid.org/0000-0003-2511-6555 The first two authors contributed equally to this work. 역사회 감염을 일으키는 주요 원인균으로, 농양이나 창상감염 등의 피부감염, 패혈증 및 폐렴, 독소쇼크증후군 등 광범위 질환 을 일으키는 것으로 알려져 있다[1, 2]. S. aureus는 자연계에 널리 분포하며, 환경에 대한 저항성이 강하여 생물체에 기생하 pissn 1738-3544 eissn 2288-1662 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.
Korean J Clin Lab Sci. Vol. 52, No. 2, June 2020 129 지않고도장기간생존할수있는세균이다. 건강한사람의피부, 점막, 상기도, 비뇨기, 소화기등에정상상재균및주변환경에도존재하지만, 피부의상처나호흡기를통하여쉽게인체에감염될수도있다. 주요집락형성부위는비강 (nasal cavity) 이며, 비강내집락화된 S. aureus는비말을통하여비강에서부터인체의다른부위로이동및정착하는것으로알려져있다. 특히포도알균속 (genus) 의다양한종 (species) 가운데황색포도알균 ( 황색포도구균, S. aureus) 은세균중가장병독성 (virulence) 이높은것으로알려져다양하고심각한질병을흔히일으키기때문에의학적으로가장중요하다. Staphylococcus 속에속하는 S. aureus는건강인의약 30 60% 가이세균을보유하고있으며, 그중 10 35% 에서비강입구부위 (anterior nares) 에장기간집락화될수있는가능성을지니고있다 [3, 4]. 신체의여러부위에존재하며, 과거에는오염균으로간주되었던혈장응고효소음성포도알균 (coagulase negative Staphylococci, CoNS) 도최근에는면역력이억제되거나저하된환자에서기회감염균으로써다양한감염의원인이됨에따라임상적중요성이부각되고있다. 1960년대초, 영국에서 S. aureus 감염치료에광범위하게사용되던항생제인메티실린에내성을가지는메티실린내성황색포도알균 (methicillin-resistant S. aureus, MRSA) 의출현이알려지면서, 1970 년후반에이르러서는병원내감염을일으키는중요한세균으로인식되기시작하였고, 다제내성 MRSA 분리율의증가는사회적으로심각한보건문제로인식되고있다 [5, 6]. 고전적인 MRSA 는주로병원에서감염되어 β-lactam계이외의항생제에대해서감수성을나타내는경우가많은것으로알려져왔으나, 최근에는 β-lactam 계항생제이외의 ciprofloxacin, clindamycin, gentamycin, trimethoprimsulfamethoxazole 등에대해서다제내성을보이는지역사회관련 MRSA (community-associated MRSA, CA-MRSA) 의발생보고가늘어남에따라이에대한관심또한증가하고있는추세이다 [7]. 2010년발표된소아에서의황색포도알균의비강보균율연구결과에따르면, nasal swab 을시행한소아총 428 명중약 9.3% 에해당하는 40명의비강내에서 MRSA 가분리배양되었다. 이중 2명 (5%) 의소아만이최근 1년이내에입원또는수술력등의의료기관관련감염위험인자를가지고있는의료기관관련 MRSA (hospital-associated MRSA, HR- MRSA) 로확인되었고, 나머지 38 명 (95%) 은 CA-MRSA 로확인되었다 [8]. 따라서본연구에서는추후환자에서분리된 MRSA 분리배 양균주를대상으로분자유전학적인분석을통해 meca 및 mecb 유전자를검출하고, Staphylococcal cassette chromosome (SCC) mec typing 과 typing 을시행하여, MRSA 분리배양균주의아형을분석한후, 관련선행연구를통해의료기관환경에서분리된 MRSA 의분자유전학적특성분석결과와비교분석하여그결과를바탕으로항생제내성세균에관한병원내감염관리및소독체계확립에대한기초자료로제공하고자하였다. 재료및방법 1. 재료 2018년 1월부터 3월까지국내대전지역소재 2차의료기관에서폐렴이나고열및기타폐질환자의 nasal swab 으로부터분리배양된된총 39주의 MRSA 를대상으로 SCCmec과 mec complex 아형의분자유전학적특성을분석하였다. 2. 세균배양및동정환자의비강으로부터채취된모든검체는혈액우무배지 (blood agar plate, BAP) (BNF KOREA, Gimpo, Korea) 에접종하여 37 C 세균배양기에서 18 24시간배양하였다. 계대순수배양을통해단일집락으로분리된세균은 Gram 염색을시행하여 Gram 양성및음성세균을분리하였고, MicroScan WalkAway-96 자동화동정시스템 (Beckman Coulter, West Sacramento, CA, USA) 을이용해세균종을동정하였다. 3. 항생제감수성시험 BAP 배지에서유의한집락을 0.45% 생리식염수에부유시켜 McFarland 탁도가 0.6 0.9 이되게탁도를맞춘후 MicroScan WalkAway-96 자동화동정시스템 (Beckman Coulter) 장비를이용하여항생제감수성시험을시행하였다. 결과의정확도를위해표준균주인 S. aureus ATCC 29213를사용하여주 1회임상검체와동일한방법으로항생제감수성시험을실시하여 Clinical and Laboratory Standards Institute (CLSI, Wayne, PA, USA) 기준허용범위에적절한지확인하였다. 항생제감수성시험시행후감수성과내성여부의판정은 CLSI 가이드라인에서권고하는표준값을적용하여실시하였다. 항생제감수성시험에사용된항생제는 azithromycin, β-lactamase, ciprofloxacin, clindamycin, daptomycin, erythromycin, fosfomycin, fusidic acid, gentamicin, levofloxacin, linezolid, moxifloxacin, mupirocin, ri-
130 Sang-Ha Kim, et al. Molecular Subtyping of MRSA Isolated from Patients fampin, synercid, teicoplanin, tetracycline, trimethoprim-sulfamethoxazole, vancomycin 을포함해총 19 종이다. 4. Genomic DNA 추출 BAP 배지에서배양된세균의집락을 5% Chelex R Resin (Bio-Rad, Hercules, CA, USA)/TBE 용액 500 μl에혼탁시킨후 100 C 에서 10분간끓인후 6,032 g로 2분간원심분리한후상층액을이용하였다. 이후추출한 genomic DNA (gdna) 는 Nanodrop 2,000 (Thermo Fisher Scientific, Waltham, MA, USA) 장비를이용해농도와순도를측정하였다. 5. SCCmec과 아형분석및 meca 유전자검출 SCCmec과 typing 및메티실린내성유전자인 meca 유전자검출을수행하기위해추출한 1 μl gdna ( 약 10 μg/μl) 를주형 DNA 로 10 μl Prime Taq PCR Premix (GeNet Bio, Daejeon, Korea), 각각의 10 pmole/μl primer와 reverse primer를 7 μl 멸균증류수를첨가하여총 20 μl의혼합액을만들어 multiplex PCR 을수행하였다. 본연구에사용한 primer 의염기서열은기존연구자료를바탕으로시행하였다 (Table 1, 2) [9, 10]. 반응이끝난 PCR 산물 3 μl를 1.5% agarose/tbe gel 에분주하여전기영동을통 해그결과를확인하였다 (Figure 1). 결과 1. 환자의비강에서분리배양된 MRSA의항생제감수성시험결과환자의비강에서분리배양된 39주의 MRSA 는 oxacillin 을포함하여 ampicillin, amox/k clav, imipenem, penicillin 에모두내성을나타냈으나, daptomycin, linezolid, synercid, teicoplanin, trimethoprim/sulfamethoxazole, vancomycin 에는모두감수성을나타냈다. Ciprofloxacin, azithromycin, clindamycin, erythromycin, levofloxacin, moxifolxacin 에대해서는내성율이 66.6 74.3% 였으며, fusidic acid와 tetracycline 에대해서는내성율이 43.6 61.5%, 그리고 fosfomycin, mupirocin, rifampin 에는 15.4 30.7% 의내성율을나타냈다. 2. 환자의비강에서분리배양된 MRSA에서의 meca 유전자검출율환자의비강에서분리배양된 39주의 MRSA 임상분리균주를대상으로 meca 유전자검출을위한 PCR 분석을실시한결과총 39주의모든 MRSA 균주 (100%) 에서양성이나오는것을 Table 1. Specific primer sets used in this study for subtyping of & subtypes in methicillin-resistant Staphylococcus aureus isolate Target regions Nucleotide sequences (5-3 ) Amplicon size (bp) Accession No. Reference SCCmec subtypes Methicillin resistance mec complex subtypes Ⅰ Ⅱ Ⅲ Ⅳa Ⅳb Ⅳc Ⅳd Ⅴ meca gene group B 5 -GCT TTA AAG AGT GTC GTT ACA GG-3 5 -GTT CTC TCA TAG TAT GAC GTC C-3 5 -CGT TGA AGA TGA TGA AGC G-3 5 -CGA AAT CAA TGG TTA ATG GAC C-3 5 -CCA TAT TGT GTA CGA TGC G-3 5 -CCT TAG TTG TCG TAA CAG ATC G-3 5 -GCC TTA TTC GAA GAA ACC G-3 5 -CTA CTC TTC TGA AAA GCG TCG-3 5 -TCT GGA ATT ACT TCA GCT GC-3 5 -AAA CAA TAT TGC TCT CCC TC-3 5 -ACA ATA TTT GTA TTA TCG GAG AGC-3 5 -TTG GTA TGA GGT ATT GCT GG-3 5 -CTC AAA ATA CGG ACC CCA ATA CA-3 5 -TGC TCC AGT AAT TGC TAA AG-3 5 -GAA CAT TGT TAC TTA AAT GAG CG-3 5 -TGA AAG TTG TAC CCT TGA CAC C-3 5 -GTG AAG ATA TAC CAA GTG ATT-3 5 -ATG CGC TAT AGA TTG AAA GGA T-3 5 -CCC TTT TTA TAC AAT CTC GTT-3 5 -ATA TCA TCT GCA GAA TGG G-3 5 -TAT TTT TGG GTT TCA CTC GG-3 5 -CTC CAC GTT AAT TCC ATT AAT ACC-3 613 AB033763 Zhang et al, 2005 [10] 398 D86934 280 AB37671 776 AB063172 493 AB063173 200 AB096217 881 AB097677 325 AB12121 147 X52593 146 IS1272 1,305 CA05
Korean J Clin Lab Sci. Vol. 52, No. 2, June 2020 131 Table 2. Multiplex PCR conditions for SCCmec & subtyping Pre-denaturation Targets meca Denaturation Annealing 1 cycle 94 C/5 min Extension 40 cycle 94 C/30 sec I II III IVa IVb IVc IVd V A B Final extension 1 cycle 50 C/30 sec 55 C/30 sec 72 C/30 sec 72 C/30 sec 50 C/30 sec 55 C/30 sec 72 C/1 min 72 C/30 sec 54 C/30 sec 72 C/1 min 72 C/10 min Figure 1. Multiplex PCR assay to identify SCCmec subtypes. Multiplex PCR assay identifies SCCmec subtypes I, II, III, IVa, IVb, IVc, IVd, V. LM, ladder marker; 1 to 23, MRSA isolates; NC, Negative control. 확인하였다. SCCmec과 아형은 ampicillin과 amox/k clav에 모두 내성을 나타냈다. 3. 환자의 비강에서 분리 배양된 MRSA의 SCCmec과 mec -II형에 속하는 총 24주의 MRSA 중 22주 complex 아형 분자 특성 (91.7%)가 ampicillin, amox/k clav, imipenem, oxacillin, 환자의 비강으로부터 분리 배양된 MRSA 39주의 SCCmec penicillin, azithromycin, clindamycin, ciprofloxacin, 아형 분석 결과 -II가 24건(61.5%), SCCmec erythromycin, levofloxacin, moxifloxacin 등 11개 항생 type-ii/iva가 2건(5.1%), -II/V가 1건(2.6%), 제에 대해 내성을 나타냈으며, 19주(79.2%)는 앞에서 언급한 -IVa가 1건(2.6%), SCCmec not-typeable이 11개 항생제와 더불어 tetracycline에 대해서도 추가적인 내성 11건(28.2%)으로 분석되었다. 환자로부터 분리 배양된 MRSA 을 나타냈고, 17주(70.8%)는 앞에서 언급한 12개 항생제와 더 39주의 아형 분석 결과, 가 29건 불어 fusidic acid에 대해서도 추가적인 내성을 나타냈다. (74.4%), not-typeable이 10건(25.6%)이었으며, group B는 -II형에 속하는 총 24주의 MRSA 중 16주 없는 것으로 분석되었다(Table 3). (66.7%)가 gentamycin에 대한 내성을 나타냈으며, 11주 (45.8%)가 fosfomycin에 대한 내성을 나타냈고, rifampin과 4. MRSA의 SCCmec과 아형 분자 특성에 mupirocin에 대해서는 각각 4주(16.7%)가 내성을 나타냈다. 따른 항생제 내성 양상 -II/IVa형에 속하는 것으로 보이는 총 2주의 환자의 비강에서 분리 배양된 MRSA 39주에 속하는 모든 MRSA의 항생제 감수성 시험 결과는 ampicillin, amox/k
132 Sang-Ha Kim, et al. Molecular Subtyping of MRSA Isolated from Patients Table 3. Comparison of SCCmec & subtypes of MRSA isolates from nasal swab of clinical patients Targets Results No. of samples, N (%) Total meca gene Positive 39 (100.0) 39 (100.0) Negative 0 (0.0) subtypes Group A 29 (74.4) Group B 0 (0.0) Not-typeable 10 (25.6) SCCmec subtypes I 0 (0.0) II 24 (61.5) III 0 (0.0) IVa 1 (2.6) IVb 0 (0.0) IVc 0 (0.0) IVd 0 (0.0) V 0 (0.0) II/IVa 2 (5.1) II/V 1 (2.6) Not-typeable 11 (28.2) clav, imipenem, oxacillin, penicillin, azithromycin, clindamycin, ciprofloxacin, erythromycin, levofloxacin, moxifloxacin 등 11개의항생제에대한공통내성을나타냈으며, 1주 (50%) 는앞에서언급한 11개항생제와더불어 gentamycin 에대한추가적인내성을나타냈다. -II/V 형에속하는것으로보이는 1주의 MRSA 의항생제감수성시험결과는 ampicillin, amox/k clav, imipenem, oxacillin, penicillin, azithromycin, clindamycin, ciprofloxacin, erythromycin, levofloxacin, moxifloxacin, tetracycline, fusidic acid, gentamycin, fosfomycin, rifampin 등 15개의항생제에대한내성을나타냈다. -IVa 형에속하는 1주의 MRSA 의항생제감수성시험결과는 ampicillin, amox/k clav, imipenem, oxacillin, penicillin, azithromycin, clindamycin, ciprofloxacin, erythromycin, levofloxacin, moxifloxacin, gentamycin 등 12개의항생제에대한내성을나타냈다. 형으로만분류된총 3주의 MRSA 는모두 ampicillin, amox/k clav, imipenem, oxacillin, penicillin 등 5가지항생제에공통으로내성을나타냈으며, 1 주 (33.3%) 는 tetracycline, gentamycin, mupirocin 등 3가지항생제에대한추가적인내성을나타냈으며, 1주 (33.3%) 는 azithromycin, clindamycin, ciprofloxacin, erythromycin, levofloxacin, moxifloxacin, tetracycline, fosfomycin, rifampin, mupirocin 등 10가지항생제에대한추가적인내성을나타냈다. SCCmec과 아형분석에서 not-typeable로분류된 MRSA 8주모두 ampicillin, amox/k clav, imipenem, oxacillin, penicillin 등 5가지항생제에공통으로내성을나타냈으며, 그중 1주 (12.5%) 는 tetracycline 과 gentamycin 등 2가지항생제에대해추가적인내성을나타냈으며, 1주 (12.5%) 는 azithromycin, clindamycin, erythromycin 를포함해 3가지항생제에추가적인내성을나타냈고, 1 주 (12.5%) 는 azithromycin, clindamycin, ciprofloxacin, erythromycin, levofloxacin, moxifloxacin, tetracycline, gentamycin, mupirocin 을포함해 9가지항생제에추가적인내성을나타냈다 (Table 4). 고찰 MRSA 의감염에의해야기되는피부와연조직감염은집단생활을하는사람들에게서많이발생되고있으며인플루엔자나폐렴과함께의료기관으로의입원의주요한감염원인이되고있다 [11]. 일반적으로당뇨및투석환자, 장기의료ㆍ복지시설등에서발생하는 HA-MRSA 의 SCCmec 아형은주로 SCCmec type-i, -II, -III 아형으로다약제에대한내성 (multi-drug resistance) 을가지고있는것으로알려져있다 [12]. 과거국내에서실시된연구결과에따르면, 일반입원실과중환자실에서분리된 MRSA 중약 25% 가 CA-MRSA 로조사되었으며, 항생제감수성시험결과 Oxacillin 약제에대해모두 (100%) 내성을보였고, 그외 erythromycin, ciprofloxacin, trime-
Korean J Clin Lab Sci. Vol. 52, No. 2, June 2020 133 Table 4. Comparison of phenotypic antibiotic resistance patterns of MRSA isolates according to SCCmec & subtypes Patients nasal cavity Strains Phenotypic antimicrobial resistance profile Clinical diagnosis SCCmec & mec complex subtypes HA-MRSA-35 AM, AUG, IMP, OX, P Diabetes mellitus -II, HA-MRSA-1 AM, AUG, IMP, OX, P, GM General weakness HA-MRSA-15 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF Pneumonia HA-MRSA-36 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, MUP Dyspnea HA-MRSA-12 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, GM Bacterial pneumonia HA-MRSA-25 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, GM Fever HA-MRSA-31 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA Pneumonia HA-MRSA-7 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, FOS Pneumonia HA-MRSA-26 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, FOS Hyperglycemia HA-MRSA-29 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, FOS Acute respiratory failure HA-MRSA-17 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, FOS Chronic obstructive airway disease HA-MRSA-22 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, FOS Pneumonia HA-MRSA-2 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, GM Pneumonia HA-MRSA-5 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, GM Acute respiratory failure HA-MRSA-30 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, GM Spontaneous bacterial Peritonitis HA-MRSA-9 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, GM, FOS Acute traumatic subarachnoid hemorrhage HA-MRSA-14 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, GM, FOS Pneumonia HA-MRSA-34 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, GM, FOS Fever HA-MRSA-20 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, Bronchitis GM, FOS, RIF HA-MRSA-24 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, GM, FOS, MUP Malignant neoplasm of stomach HA-MRSA-4 AM, AUG, IMP, OX, P, AZI, CD, CP,E, LVX, MXF, TE, FA, Acute pneumonia GM, RIF, MUP HA-MRSA-10 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, Pneumonia GM, RIF, MUP HA-MRSA-3 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, Malignant neoplasm of -II GM, FOS, RIF main bronchus HA-MRSA-11 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, GM Pneumonia HA-MRSA-16 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, GM Lung mass -II/ HA-MRSA-32 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF Panperitonitis IVa mec, complex HA-MRSA-27 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FA, GM, FOS, RIF Bronchitis -II/V, HA-MRSA-21 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, GM Acute cholecystitis -IVa, HA-MRSA-13 AM, AUG, IMP, OX, P, TE, GM, MUP Fever SCCmec HA-MRSA-23 AM, AUG, IMP, OX, P Cerebral infarction not-typeable, HA-MRSA-39 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, FOS, Dyspnea RIF, MUP HA-MRSA-6 AM, AUG, IMP, OX, P Alcoholic liver cirrhosis SCCmec & mec HA-MRSA-8 AM, AUG, IMP, OX, P General medical examination complex HA-MRSA-18 AM, AUG, IMP, OX, P Anaphylatic reaction not-typeable HA-MRSA-19 AM, AUG, IMP, OX, P Acute Pneumonia HA-MRSA-28 AM, AUG, IMP, OX, P, AZI, CD, E Hyperplasia of prostate HA-MRSA-33 AM, AUG, IMP, OX, P Bronchopneumonia HA-MRSA-37 AM, AUG, IMP, OX, P, TE, GM Acute bronchiolitis HA-MRSA-38 AM, AUG, IMP, OX, P, AZI, CD, CP, E, LVX, MXF, TE, GM, MUP Intestinal obstruction Abbreviations: HA-MRSA, Hospital acquired-mrsa; AM, Ampicillin; AUG, Amox/K clav; IMP, Imipenem; OX, Oxacillin; P, Penicillin; GM, Gentamycin; AZI, Azithromycin; CD, Clindamycin; CP, Ciprofloxacin; E, Erythromycin; LVX, Levofloxacin; MXF, Moxifloxacin; MUP, Mupirocin; TE, Tetracycline; FA, Fusidic acid; FOS, Fosfomycin; RIF, Rifampin.
134 Sang-Ha Kim, et al. Molecular Subtyping of MRSA Isolated from Patients thoprim/sulfamethoxazole, rifampin 약제에대해각각 83.6%, 53.4%, 10.9%, 4.1% 내성을나타내는것으로조사되었다 [13]. SCCmec과 아형분석결과, 환자의비강에서분리배양된 MRSA 39주중 22주가 group A이면서 -II 아형으로분류되어가장많은분포를나타냈고, 다음으로 와 SCCmec 아형모두유형이밝혀지지않은 not-typeable 이 8주로많은분포를나타낸반면, 이면서 SCCmec type-ii/v와 -IVa 가각각 1주로가장적은분포를나타냈다. 이결과는 HA-MRSA 의 SCCmec 아형중 s-ii 형이가장우세하다는과거의연구결과와유사한양상을나타내었다 [9, 12]. 본결과에서나타난 nottypeable 8 주의경우기존에알려지지않은다른아형의존재임을확인하기위해 SCCmec 부위의 DNA sequencing 분석을통한추가적인연구가필요할것으로사료된다. 본연구에서 MRSA 로분리배양된총 39주에대한항생제감수성시험결과, 환자의비강에서분리배양된 MRSA 총 39주모두 ampicillin, amox/k clav, imipenem, oxacillin, penicillin 등 5가지항생제에공통으로내성을나타냈다. 본연구를통해환자의비강에서분리된 MRSA 아형중가장많은비중을차지하는 -II아형에속하는총 24주 (61.5%) 의 MRSA 중 22주 (91.7%) 가 ampicillin, amox/k clav, imipenem, oxacillin, penicillin, azithromycin, clindamycin, ciprofloxacin, erythromycin, levofloxacin, moxifloxacin 등 11개항생제에대해내성을나타냈으며, 12가지항생제에대한내성을나타낸 MRSA가 19주 (79.2%), 13가지항생제에대한내성을나타낸 MRSA 는 17주 (70.8%) 에달했다. 반면에, 환자의비강에서분리된총 8주 (20.5%) 의 not-typeable MRSA 균주중 5주 (62.5%) 는 ampicillin, amox/k clav, imipenem, oxacillin, penicillin 등 5가지항생제에대해서만내성을나타내어, 환자의비강에서주로분리배양되는 -II 아형이지니는다약제에대한내성특성과분명한차이가있는것으로나타났다 [3]. 현재까지밝혀진 은 type I에서 VI까지크게분류되며내성유전자를포함하는 plasmid 나 transposon 등에따라 subtype 이매우다양한것으로나타났다. 본연구에서는환자의전체를대상으로 MRSA 의보균율을조사한것이아니기때문에, 환자로부터분리배양된 MRSA 특성을대변하기에는통계학적유의성이낮을수있기때문에분자아형에따른항생제내성패턴의특성을정확하게비교분석하기에는제한점 이있을수있다. 하지만, 본연구결과와의료기관획득MRSA 에관한다른연구결과와유사한결과를나타내었으며 [14], 이는본연구가병원내에서언제든 MRSA 집단감염으로인한피부와연조직감염환자가발생할가능성이있으며, 이로인해집단감염초래할수있다는중요성을지니고있다. 따라서병원내 MRSA 감염환자발생시이에따른후속조치를실시함에앞서병원내에서 MRSA 의전파를예방할수있는사전대책을더욱강구하고적극적으로감염의예방을홍보하는것이매우중요할것으로판단된다. 또한, 면역력이저하및억제된환자나기저질환을가지고있는고령자등에서새롭게출현하는항생제내성균의발생위험이높고, 균의국내유입및확산이감염관리의새로운문제로대두되고있기때문에이러한내성균의출현을최대한억제하고, 조기에발견하여적절한예방지침을시행함으로써감염과확산을예방하는것이중요할것으로사료된다. 요약본연구에서는환자의비강으로부터분리배양된총 39건의 MRSA 분리배양균주를이용해 meca 유전자검출, SCCmec typing 과 typing 을분석해보고자하였다. 임상환자의비강으로부터분리배양된 MRSA 총 39주중 -II 가 24건, type-ii/iva 가 2건, type-ii/v가 1 건, type-iva 가 1건, not-typeable 이 11건으로분석되었으며, type A가 29 건, not-typeable이 10건이었으며, type B는없는것으로분석되었다. 결론적으로, 환자의비강으로부터분리된 MRSA 분리배양균주중 SCCmec type-ii 와 type A 아형이가장많이분포하고있었으며, 이결과는의료기관획득 MRSA 에관한다른연구결과와유사한결과를나타냈다. 이후환자의비강으로부터분리배양된 not-typeable 아형의 MRSA 균주를대상으로국내에서발견되는새로운 MRSA 아형규명에관한추가연구가필요할뿐만아니라, MRSA 분리배양균주의아형을분석함으로써그분자적특성을분석한결과를바탕으로병원감염관리를위한기초자료를제공할수것으로사료된다. Acknowledgements: This paper was supported by Brain Busan 21 Plus project. Conflict of interest: None Author s information (Position): Kim SH 1,2, M.T.; Park SB 3,4, Park H 3,4, Kim JS 3,4, Graduate student; Lee J 3,
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