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414 Jin-Un Choi, et al. Gram-positive Bacilli Using MALDI-TOF MS ORIGINAL ARTICLE Korean J Clin Lab Sci. 2018;50(4):414-421 https://doi.org/10.15324/kjcls.2018.50.4.414 pissn 1738-3544 eissn 2288-1662 Two-year Quaternary Isolation of Gram-positive Bacilli Using MALDI-TOF MS in Positive Blood Culture of a University Hospital Jin-Un Choi 1, Young-Bin Yu 2, Sang-Ha Kim 3, Seungho Won 4, Young-Kwon Kim 2 1 Department of Laboratory Medicine, Chonnam National University Hospital, Gwangju, Korea 2 Department of Biomedical Laboratory Science, College of Medical Sciences, Konyang University, Daejeon, Korea 3 Department of Laboratory Medicine, Konyang University Hospital, Daejeon, Korea 4 Department of Mechanical Engineering, Konyang University, Nonsan, Korea 일개대학병원의혈액배양에서 MALDI-TOF MS 를이용한 Gram-positive Bacilli 의 2 년간분기별분리율 최진언 1, 유영빈 2, 김상하 3, 원승호 4, 김영권 2 1 전남대학교병원진단검사의학과, 2 건양대학교의과학대학임상병리학과, 3 건양대학교병원진단검사의학과, 4 건양대학교기계공학과 In this study, Gram-positive bacilli (GPB) were identified by MALDI-TOF MS and analyzed according to the quaternary and microbial strains in the blood culture medium over a two year period at a university hospital. The results were as follows. The overall positive rate of blood culture was 9.97%. In 713 isolated GPB, 410 strains (57.5%) were identified using a microflex MALDI Biotyper. The positive rate of GPB among the blood culture positive bacteria was 8.2%, and the quarterly isolation rate was 9.8% in the third quarter of 2015, 8.7% in the second quarter of 2016, 8.1% in the third quarter of 2016, 8.1% in the first quarter of 2015, 7.9% in the first quarter of 2015, 7.9% in the second quarter of 2015, 6.8% in the first quarter of 2016, and 6.7% in the fourth quarter of 2015. The isolates were Corynebacterium striatum 89 (12.4%), Bacillus cereus 60 (8.4%), Bacillus subtilis 30 (4.2%), Paenibacillus urinalis 29 (4.1%), and Listeria monocytogenes 25 (3.5%). The results of 16S rrna sequencing of 43 isolates (86.0%) were consistent with those of the other 50 isolates. Five out of the seven unmatched weeks were not identified by MALDI-TOF MS. Key words: Blood culture, Gram positive bacilli, Isolation rate, MALDI-TOF MS Corresponding author: Young-Kwon Kim Department of Biomedical Laboratory Science, College of Medical Sciences, Konyang University, 158 Gwanjeodong-ro, Seo-gu, Daejeon 35365, Korea Tel: 82-42-600-8431 Fax: 82-42-543-6370 E-mail: ykkim3245@konyang.ac.kr 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. Copyright 2018 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: August 18, 2018 Revised: September 4, 2018 Accepted: September 9, 2018 서론임상미생물검사실에서분리되는그람양성막대균 (Grampositive bacilli, GPB) 의대부분은환경에존재하며비병원성으로알려져있어, 그동안임상미생물검사실에서는오염균으로간주하여정확한동정을시행하지않는것이일반적이었다 [1]. 그러나 GPB 중몇몇병원성균종들은임상적으로중요하며, 최근면역저하환자나체내삽입기구처치를하는환자가증가하고항생제사용도증가함에따라 GPB에의한기회감염이증가하고있다. 따라서 GPB의신속하고정확한동정의필요성이대두되었고, 정확한 GPB의동정은감염의정확한진단과적절한치료에큰도움을주기때문에매우중요하다고볼수있다

Korean J Clin Lab Sci. Vol. 50, No. 4, December 2018 415 [2]. 일반적으로혈액배양에서분리되는 GPB들은비용의효율적인측면에서동일환자에서 2회이상분리되는경우에만감염의의미를가지고동정을시행하지만, 임상미생물검사실에서시행하고있는전통적인동정방법으로는동정시간이많이걸리고항상정확한결과를얻기가어렵다 [3]. 특히패혈증의경우는미생물에감염되어일어나는전신성반응으로사망률또한높은질환이며경제적부담을증가시킨다 [4]. 중환자실환자의경우패혈증으로인한중증패혈증으로 20 50% 의사망률을나타내며, 패혈성쇼크환자는다른질환에비해사망률이각각 30 87% 에이를정도로심각한질환으로많은경제적부담을증가시키므로패혈증의원인균을신속하게진단하고치료의방향을신속하게제시해줌으로써환자의예후에큰영향을미칠수있다 [5]. 따라서임상미생물검사실에서는저렴한비용으로좀더신속하게원인균을동정해내고자노력하고있다. 그러나일반적으로행해지고있는생화학적동정방법으로는최소 2일최대 4일정도의시간이소요된다 [6]. 최근개발된 microflex MALDI Biotyper (Bruker Daltonik GmbH, Bremen, Germany) 는 MALDI-TOF MS 기법을이용하여얻은세균의단백질정보를이미구축된각균종에대한정보와비교분석하여균종을동정하는원리를이용한다. Seng 등 [6] 의연구에의하면 MALDI-TOF MS 동정시간은균주당평균 6분정도소요되며, 동정을위해산출되는비용은상품화키트를포함한전통적동정방법의 22 32% 로임상미생물검사실에서이용하기에간편하고동정에소요되는것으로보고하였다 [7, 8]. 혈액배양시료에서직접세균동정과항생제감수성시험실시하는방법은정확하고결과를보고하는데까지소요되는시간을크게감소시켜효율적인치료에유용하게활용될수있다 [9]. 대부분의 MALDI-TOF MS 장비를이용한연구에서는임상미생물검사실에서주로동정이되는그람양성알균, 그람음성막대균및매우소수의 GPB을포함하여동정하고있다 [10, 11]. 그러나국내에서는 MALDI-TOF MS를이용한혈액배양에서 GPB분리동정률에대한자료가부족하여, 본연구에서는일개대학병원의혈액배양에서분리된전체세균을대상으로 MALDI-TOF MS를이용하여 GPB의균종별, 분기별분리비율을조사하여혈액배양에서전통적방법으로동정되지못하거나오염균으로간과할수있는오류를최소화하기위한기초자료를알아보고자하였다. 재료및방법 2014년 10월부터 2016년 9월까지 2년간일개대학병원임 상미생물검사실로혈액배양이의뢰된 87,241건중에서 GPB 로분리된 713균주를대상으로하였으며, 분기별혈액배양양성률중 GPB의분리율을분석하였다. 1. 혈액배양혈액배양은산소성배지인 (BacT/ALERT FA Plus, FA) 와 BACTEC TM Plus Anerobic/F), 무산소성배지인 (BacT/ALERT Standard Anaerobic, SN) 와 (BACTEC TM Plus Anerobic/F) 에각각 5 ml 씩접종하였으며, 각각 BacT/ALERT 3D blood culture system (biomérieux Inc., Durham, NC, USA) 과 BD BACTEC TM FX blood culture system (BD, Spark, MD) 에서 5 일간배양하였다. 혈액배양양성배양액을 5% sheep blood agar plate (Hanil Komed) 에접종하여 5% CO 2, 35 C 배양기에서 24시간에서 48시간동안배양하였다. 분리된균종들은집락성상과그람염색결과를이용하여 GPB로일차분류하였다. 2. MALDI-TOF MS를이용한동정전통적혈액배양에서양성인배양액을혈액우무배지에배양한후집락성상과그람염색결과 GPB로확인된균주를대상으로 microflex MALDI Biotyper (Bruker Daltonik GmbH, Bremen, Germany) 로검사하고 MALDI Biotyper RTC software (V. 3.1) 을이용하여직접도말법으로균종을동정하였다. 직접도말법은하룻밤배양한신선한집락을멸균 Wooden applicator 를이용해 MSP 96 target polished steel BC microscout Target plate (Bruker Daltonics) 에도말하고, 세균이마른후매트릭스용액 (50% acetonitrile, 2.5% trifluoroacetic acid) 에포화된 ( -cyano-4-hydroxycinnamic acid) 을더한시약 1 L를가하고, 실온에서완전히건조시킨후 microflex MALDI Biotyper 장비에장착하였다. Bacterial test standard (BTS) 를사용하여장비 Calibration 을실시하였고, m/z 2,000 20,000 범위에서측정된 Mass spectra 동정결과는 MALDI Bio- typer RTC software (V. 3.1) 를이용해분석하였다. 동정은기기회사의기준을이용하여판정하였는데, 일반적으로동정치 (cut-off score) 가 2.0 이상이면균종동정이, 1.7 이상이면서 2.0 미만인경우균속동정이가능한것으로판단하였고 1.7 미만인경우신뢰성이없는것으로조작적정의를하였다. 결과 1. 혈액배양분기별분리양성률분석 1 2014년 10월부터 2016년 9월까지 2년간혈액배양에서의

416 Jin-Un Choi, et al. Gram-positive Bacilli Using MALDI-TOF MS 뢰건수총 87,241건중전체양성은 8,699건으로 9.97% 를나타내었고, 그중 Gram positive cocci는 4,198건 (48.3%), Gram negative bacilli 3,245건 (37.3%), GPB 713건 (8.2%), Candida species는 513건 (5.9%), Gram negative cocci 30건 (0.3%) 순으로분리되었다. 2014년 4분기의뢰건수총 10,168 건중전체양성률은 1,028건으로 10.1% 를나타내었고, 그중 Gram positive cocci는 562건 (54.7%), Gram negative bacilli 343건 (33.4%), GPB 83건 (8.1%), Candida species 38건 (3.7%), Gram negative cocci 2건 (0.2%) 를나타냈으며, 2015 년 1분기는의뢰건수총 9,732건중전체양성률은 861건으로 8.8% 를나타내었고, 그중 Gram positive cocci 457건 (55.2%), Gram negative bacilli 266건 (30.9%), GPB 68건 (7.9%), Candida species 46건 (5.3%), Gram negative cocci 6 건 (0.7)% 를나타냈으며, 2015년 2분기총 9,574건중양성은 998건 (10.4%) 을나타내었고, 그중 Gram positive cocci 517 건 (51.8%), Gram negative bacilli 339건 (34.1%), GPB 83건 (7.9%), Candida species 52건 (5.2%), Gram negative cocci 7 건 (0.7%) 를나타냈다. 2015년 3분기총의뢰건수 11,255건중양성은 1,263건으로 1.2% 를나타냈으며, 그중 Gram positive cocci 572건 (45.3%), Gram negative bacilli 481건 (38.1%), GPB 124건 (9.8%), Candida species 81건 (6.4%), Gram negative cocci 5건 (0.4%) 를나타냈다. 2015년 4분기총 11,163건중양성은 1139건 (10.2%) 이었으며, 그중 Gram positive cocci 508건 (44.6%), Gram negative bacilli 412건 (36.2%), Candida species 131건 (11.5%), GPB 84건 (6.7%), Gram negative cocci 4건 (004%) 의순으로나타났다. 2016년 1분기는총 11,321건중양성은 950건 (8.4%) 이었으며, 그중 Gram positive cocci 415건 (43.7%), Gram negative bacilli 409건 (43.1%), GPB 65건 (6.8%), Candida species 57건 (6.6%), Gram negative cocci 4건 (0.4%) 을나타냈다. 2016년 2분기총의뢰건수 11,937건중양성은 1212건 (10.2%) 이었으며, 그중 Gram positive cocci 537건 (43.3%), Gram negative bacilli 507건 (41.8%), GPB 105건 (8.1%), Candida species 62 건 (5.1%), Gram negative cocci 1건 (0.01%) 로나타났으며, 2016년 3분기총의뢰건수 12,091건중양성은 1,248주 (10.3%) 이었으며, 그중 Gram positive cocci 612건 (49.0%), Gram negative bacilli 488건 (39.1%), GPB 101건 (8.1%), Candida species 46건 (3.7%), Gram negative cocci 1건 (0.01%) 순으로분리되었다. 총혈액배양양성률은 10.0% 였으며양성세균중 GPB양성률은 8.2% 였다 (Table 1, Figure 1). 2. MALDI-TOF 를이용한그람양성막대균의분리율 총 713건의 GPB를 microflex MALDI Biotyper를이용해동정하여기기회사의기준에따라균속동정이가능한스코어 Figure 1. Quarterly in year positive blood cultures at one university hospital (2014 2016). Table 1. Quarterly in year positive blood cultures at one university hospital (2014 2016) No. (%) of results obtained from blood culture Quarter in year Gram positive bacilli Gram positive cocci Gram negative bacilli Gram negative cocci Candida species Positivity (%) 2014-4Q* 83 (8.1) 562 (54.7) 343 (33.4) 2 (0.2) 38 (3.7) 1028/10168 (10.1) 2015-1Q 68 (7.9) 475 (55.2) 266 (30.9) 6 (0.7) 46 (5.3) 861/9732 (8.8) 2015-2Q 83 (7.9) 517 (51.8) 339 (34.1) 7 (0.7) 52 (5.2) 998/9574 (10.4) 2015-3Q 124 (9.8) 572 (45.3) 481 (38.1) 5 (0.4) 81 (6.4) 1263/11255 (11.2) 2015-4Q 84 (6.7) 508 (44.6) 412 (36.2) 4 (0.4) 131 (11.5) 1139/11163 (10.2) 2016-1Q 65 (6.8) 415 (43.7) 409 (43.1) 4 (0.4) 57 (6.6) 950/11321 (8.4) 2016-2Q 105 (8.7) 537 (43.3) 507 (41.8) 1 (0.01) 62 (5.1) 1212/11937 (10.2) 2016-3Q 101 (8.1) 612 (49.0) 488 (39.1) 1 (0.01) 46 (3.7) 1248/12091 (10.3) Total 713 (8.2) 4198 (48.3) 3245 (37.3) 30 (0.3) 513 (5.9) 8699/87241 (100/9.97) Abbreviations: No, number; Q, quarter. 1st quarter: Jan to Mar, 2nd quarter: Apr to Jun, 3rd quarter: Jul to Sep, 4th quarter: Oct to Dec.

Korean J Clin Lab Sci. Vol. 50, No. 4, December 2018 417 1.7 이상균주는 410주 57.5% 였다. 동정은되었으나스코어 1.7 아래로신뢰할수없는결과를보였던균주는 213주 29.9% 였고 no peaks found로동정이안되는균주는 90주 12.6% 였다. 스코어 1.7 이상에서동정된균주별분리는 Corynebacterium striatum 89건 (12.4%), Bacillus cereus 60건 (8.4%), Bacillus subtilis 30건 (4.2%), Paenibacillus urinalis 29건 (4.1%), Listeria monocytogenes 25건 (3.5%), Clostridium perfringens 18건 (2.5%), Bacillus licheniformis 16건 (2.2%), Bacillus pumilus 12건 (1.7%) 이었으며, Bacillus mojavensis, Corynebacterium afermentans, Bacillus sonorensis 각각 10건 (1.4%) 이었고, Microbacterium spp. 8건 (1.1%), Bacillus megaterium 7건 (1.0%) 순으로나타났다 (Table 2). 3. 16S-rRNA sequencing 분석과 MALDI-TOF 동정결과염기서열분석을위한 PCR을수행하기위해 Forward primer 인 16S-rRNA1 (5'-AGT TTG ATC CTG GCT CAG-3') 와 Reverse primer인 16S-rRNA2 (5'-GGT TAC CTT GTT ACG ACT T-3') 를이용하였다. Primer 각각 20 pmol 1 L, dntps 1 L, 10x Reaction buffer (1x: 10 mm Tris-HCL, 1.5 mm KCL, 0.1% Triton X-100) 5 L, 2.5 U/ L DyNAzymeTM polymerase 1 L, 멸균 DW 38.5 L에추출된 gdna 2.5 L를넣어총 50 L로반응액을만들었다. PCR은 TaKaRa PCR Table 2. Identification of Gram positive bacilli by MALDI-TOF from blood culture (2014 2016) ID result by MALDI-TOF No. of isolates with the number of positive blood cultures per patient Total No. (%) of patient Species a 1 2 3 4 5 6 8 11 Total No. (%) of species Corynebacterium striatum 17 9 2 3 1 2 1 1 36 (5.9) 89 (12.4) Bacillus cereus 47 5 1 0 0 0 0 0 53 (8.6) 60 (8.4) Bacillus subtilis 30 0 0 0 0 0 0 0 30 (4.9) 30 (4.2) Paenibacillus urinalis 27 1 0 0 0 0 0 0 28 (4.6) 29 (4.1) Listeria monocytogenes 3 7 0 2 0 0 0 0 12 (2.0) 25 (3.5) Clostridium perfringens 8 5 0 0 0 0 0 0 13 (2.1) 18 (2.5) Bacillus licheniformis 16 0 0 0 0 0 0 0 16 (2.6) 16 (2.2) Bacillus pumilus 12 0 0 0 0 0 0 0 12 (2.0) 12 (1.7) Bacillus mojavensis 10 0 0 0 0 0 0 0 10 (1.6) 10 (1.4) Corynebacterium afermentans 10 0 0 0 0 0 0 0 10 (1.6) 10 (1.4) Bacillus sonorensis 10 0 0 0 0 0 0 0 10 (1.6) 10 (1.4) Microbacterium species 3 0 0 0 1 0 0 0 4 (0.7) 8 (1.1) Bacillus megaterium 7 0 0 0 0 0 0 0 7 (1.1) 7 (1.0) Mycobacterium abscessus 0 0 0 0 0 1 0 0 1 (0.2) 6 (0.8) Clostridium tertium 6 0 0 0 0 0 0 0 6 (1.0) 6 (0.8) Actinomyces oris 5 0 0 0 0 0 0 0 5 (0.8) 5 (0.7) Bacillus vallismortis 5 0 0 0 0 0 0 0 5 (0.8) 5 (0.7) Eggerthella lenta 5 0 0 0 0 0 0 0 5 (0.8) 5 (0.7) Bacillus infantis 4 0 0 0 0 0 0 0 4 (0.7) 4 (0.6) Clostridium carnis 0 0 0 1 0 0 0 0 1 (0.2) 4 (0.6) Actinomyces odontolyticus 3 0 0 0 0 0 0 0 3 (0.5) 3 (0.4) Bacillus mycoisea 3 0 0 0 0 0 0 0 3 (0.5) 3 (0.4) Bacillus thuringiensis 2 0 0 0 0 0 0 0 2 (0.3) 2 (0.3) Brevibacterium iodium 0 1 0 0 0 0 0 0 1 (0.2) 2 (0.3) Corynebacterium minutissimun 2 0 0 0 0 0 0 0 2 (0.3) 2 (0.3) Corynebacterium urealyticum 0 1 0 0 0 0 0 0 1 (0.2) 2 (0.3) Lactibacillus paracasei b 10 0 0 0 0 0 0 0 10 (1.6) 10 (1.4) Arthrobacter oxydans c 27 0 0 0 0 0 0 0 27 (4.4) 27 (3.8) Not reliable identification 205 4 0 0 0 0 0 0 209 (34.0) 213 (29.9) No peaks found 88 1 0 0 0 0 0 0 89 (14.5) 90 (12.6) Total 565 34 3 6 2 3 1 1 615 (100.0) 713 (100.0) a ID result cut-off criteria: score 1.7. b Microbacterium aurum, Lysinibacillus fusiformis, Solibacillus silvestis, Paenibacillus illinoisensis. c Bacillus atrophaeus, Bacillus circulans, Bacillus flexus, Bacillus muralis, Bacillus niacini, Bacillus thermoamylovorans, Bacillus weienstephanensis, Clostridium bifermentans, Clostridium innocuum, Corynebacterium amycolatum, Corynebacterium falsenii, Corynebacterium pseudodiphthriticum, Corynebacterium singulare, Corynebacterium tuscaniense, Dermabacter hominis, Exigubacterium aurantiacum, Gordona rubropertincta, Lactobaillus salivarius, Microbacterium oxydans, Microbacterium testaceum, Paenibacillus barengoltzii, Paenibacillus latus, Paenibacillus rhizosphaerae, Paenibacillus odorifer, Paenibacillus pauli, Paenibaillus xylanilyticus.

418 Jin-Un Choi, et al. Gram-positive Bacilli Using MALDI-TOF MS Thermal cycler (TP600 Gradient, Roche Molecular System, CA, USA) 를사용하였고, 온도조건을 94 C에서 5분 predenaturation시킨후, 94 C 에서 1분 dena- turation, 49 C에서 1분 annealing, 72 C 에서 1분 extension을 1주기로 36회실시하여증폭하고 72 C 에서 10분간 postextension 을실시하였다. microflex MALDI Biotyper의동정결과와생화학적동정결과와일치하지않는총 50주중 43주는 16S-rRNA sequencing 분석결과와일치하였으나 7주는일치하지않았다. 일치된균주들은 Corynebacterium striatum 19주, Clostridium perfringens 5주, Listeria monocytogenes 9주, Bacillus iodinum 1주, Clostridium canis 1주, Corynebacterium urealyticum 1주, Mycobacterium abscessus 1주, Paenibacillus urinalis 1주이었다. 1.7 이상의스코어에서동정되었던균주중 Bacillus cereus는 Lysinbacillus spp. 로 Microbacterium spp. 는 Mycobacterium paraoxydans로각각 16S-rRNA sequencing 결과와불일치하였고, 5주는 MALDI-TOF MS에서도 not reliable identification 혹은 no peaks found 를나타내동정해내지못했던균주들에대한 16S-rRNA sequencing 분석결과 Cellulomonas hominis, Corynebacterium amycdatum, Catabacter hongkongenesis, Bacillus spp., Bacillus velezensis로동정되었다 (Table 3). 고찰혈액배양양성은세균성균혈증을나타낼수있는것으로생 각할수있지만일부는오염에의한혈액배양양성이존재할수있기때문에모두가진정한감염을나타내지않는다. 혈액배양에서분리되는많은 GPB의대부분은정상적인피부정상상재균의일부로혈액배양을오염시키거나정맥내카테터에집락화될수있다 [2]. 혈액배양에서오염의주된원인균으로는 Propionibacterium acnes, Corynebacterium spp. 및 Bacillus spp. 가대표적이다. 그러나노령인구의증가와면역기능저하환자의증가, 장기이식의발전과항암제, 방사선치료등으로과거에비병원성으로생각하였던균종에의해서도기회감염및중증의균혈증이생길수있음이보고되었으며 [12], 실제로이러한세균들은심내막염, 인공심장판막, 관절감염, 호중구감소증등과같은특정임상조건의맥락에서병원성일수있음을기억해야한다 [13]. 국내의한연구에서도혈액배양오염균의가능성이높은피부상재균으로분류되는 Coagulase-negative Staphylococcus (CoNS), Bacillus spp., Corynebacterium spp., Enterococcus spp., Micrococcus spp., Propionibacterium spp. 이혈액배양에서동정되면임상적인의미의해석없이모두분석에포함하여연구하였다는 GPB의분리동정에대한제한점에대해보고한바있다 [14]. 이러한제한점이있음에도불구하고임상검사실에서는확실한오염이라고판단되지않는한일단양성세균의분리결과를보고하고, 그결과에대해서는임상의의판단에맡기는경우가많다. 국내외많은연구에서는 GPB을포함한혈액배양에서분리되는미생물들에대해서균혈증과관련된높은이환율과사망률때문에신속한평가와적절한경험적항생 Table 3. Comparison of identification results between MALDI-TOF and 16S-rRNA sequencing of two or more isolates from one patient ID result by Species No. of concordant and discordant by MALDI-TOF Concordant result Discordant result Fianl ID result by 16S-rRNA sequencing Corynebacterium striatum 19 0 Bacillus cereus 5 1 Lysinbacillus sp. Clostridium perfringens 5 0 Listeria monocytogenes 9 0 Bacillus iodinum 1 0 Clostridium carnis 1 0 Corynebacterium urealyticum 1 0 Microbacterium species 0 1 Mycobacterium paraoxydans Mycobacterium abscessus 1 0 Paenibacillus urinalis 1 0 Not reliable identification 0 4 Cellulomonas hominis Corynebacterium amycolatum Catabacter hongkongenesis Bacillus sp. No peaks found 0 1 Bacillus velezensis Total 43 7 Abbreviation: No, number.

Korean J Clin Lab Sci. Vol. 50, No. 4, December 2018 419 제치료가매우중요하다고강조하고있다 [15]. 혈액배양에서세균검출을위한분자진단기술이보다쉽게이용가능하고구현하기가용이해질때까지임상의는경험적치료결정을내리는데필요한예비자료로그람염색법을계속의존해야한다 [16]. 실제적으로 MALDI-TOF MS 기술이도입되기전까지는혈액배양에서뿐만아니라임상검체에서도 GPB의분리율이낮을뿐만아니라분리되는종 (species) 의수도극히제한되는경우가많았다 [17]. 하지만상대적으로오염균으로간주되어지던 GPB에대한국내연구는많은부족함을느껴본연구를통해 GPB의분리율과분리되는종의다양성에대해알아보았다. 본연구에서는일개대학병원에서 2년간의뢰된총 87,241 건중양성은 8,699건으로전체양성율은 9.97% 였으며, 양성세균중 Gram positive cocci는 4,198건 (48.3%), Gram negative bacilli 3,245건 (37.3%), GPB 713건 (8.2%), Candida species는 513건 (5.9%), Gram negative cocci 30건 (0.3%) 순으로분리되었다. 이는 Ahn 등 [12] 이보고한, 2002년부터 2006년까지혈액배양양성율은 18.5% 로본연구에서의 9.97% 보다 2배가량높은비율의양성율을나타냈으며, Gram positive cocci의분리율은 35.1% 로본연구에서 48.3% 보다낮게나타났고, Gram negative bacilli 는 23.9% 로본연구에서의 37.3% 로본연구보다낮은양상을나타내었으며, GPB의분리율은 5.2% 를나타내본연구에서의 8.2% 보다는낮은분리율을나타냈다. GPB의 5.2% 중 Bacillus spp. 0.8%, Erysiphelothrix spp. 0.2%, 기타 GPB의분리율은 12.8% 로보고하였으며, Candida species는 2.8%, Gram negative cocci 0.0% 로본연구에서의 0.3% 와약간의차이가있음을보고하였다. 국내다른연구에서 1998년부터 2010년까지 13년간의보고에서는 [14] 전체양성률에대한보고는없었으나 Gram positive cocci의분리율은 50.1% 로본연구에서 48.3%, Gram negative bacilli 는 38.4% 로본연구에서의 37.3% 로본연구에서와비슷한양상을나타내었으며, GPB는무산소성막대균을포함하여 7.7% 를나타내본연구에서의 8.2% 보다는약간낮은분리율을나타냈다. Yeasts 는 2.8% 로본연구에서 5.9% 보다낮게나타났다. 결론적으로 2년간총혈액배양양성률은평균 9.97% 였으며 microflex MALDI Biotyper 를이용하여총 713 건의 GPB을기기제조회사의기준에따라균속동정이가능한스코어 1.7 이상균주는 410주 57.5% 이었다. 동정은되었으나스코어 1.7 아래로신뢰할수없는결과를보였던균주는 213주로 29.9% 였으며 no peaks found로동정조차힘들었던균주는 90주 12.6% 이었다. 혈액배양양성세균중 GPB의양성률은평균 8.2% 였다. 분기별분리율은 2015년 3분기는 9.8 %, 2016년 2분기는 8.7 %, 2016년 3분기는 8.1 %, 2014년 4분기는 8.1%, 2015년 1분기는 7.9 %, 2015년 2분기는 7.9 %, 2016년 1분기는 6.8 %, 2015년 4분기는 6.7 % 순으로분리되었으며, 분기별분리률의차이는유의성이없는것으로나타났다. 분리균종별로는 Corynebacterium striatum 89주 12.4%, Bacillus cereus 60주 8.4%, Bacillus subtilis 30주 4.2%, Paenibacillus urinalis 29주 4.1%, Listeria monocytogenes 25주 3.5% 순으로분리되었다. Listeria spp. 등과같은임상적으로도의의가많은감염균의동정은다른연구에서와비슷한비율을나타내었다 [18-20]. 또한분리건수는적지만 Mycobacterium spp., 각종 Corynebacterium spp., Bacillus spp., Clostridium spp. 은 1.7 이상에서신뢰할수있을만한스코어로동정할수있었다. 물론동정해내지못하거나신뢰할수없는결과도 42.5% 로절반정도를차지하였다. 여러개의혈액배양병에서 1개에서만양성으로분리된것은거의대부분이오염균으로간주되어감염균으로의의의가없는것이었다. 그러나 1개만분리되었어도감염의의의가있는 Listeria monocytogenes는 25주가분리되었다. 최근우리나라에공급되기시작한 MALDI-TOF MS 기술은임상미생물검사실에서세균과진균을동정함에있어효과적인방법으로인식되고있고, 사용또한늘어나고있다. 많은논문에서임상적으로의의가있는그람양성알균과그람음성막대균, 진균의대부분이 MALDI-TOF MS를통해속수준까지간편하고신속하게동정가능하다는것을보고했다 [21]. 본연구에서는 2년간의혈액배양에서분리된 GPB의절반이상을 1.7 이상의스코어에서신뢰할수있을만한종 (species) 수준까지다양한균종의동정이가능하다는것을확인하였다. 우리의경험에의하면임상미생물검사실에서분리된 GPB을 MALDI-TOF MS 기술을사용하여 1차동정해보고의의가있는균주에대해서는이를확인하는전통적방법이나 16S-rRNA sequencing 방법을적용하여판독한다면향후감염균으로서의의가있는 GPB의동정에매우유용하리라판단된다. 제한점으로는흔히분리되지않고비병원성오염세균이많이포함될수있는 GPB의특성으로인하여데이터베이스가부족하고, 협막이있는일부 GPB의구조적특성상단백질분리의어려움이있어동정해내지못하는제한점이있다. 앞으로데이터베이스를더확충하고, 단백질분리가어려운 GPB의경우다른분리방법을추가하여동정한다면정확한동정에더많은도움이되리라생각된다.

420 Jin-Un Choi, et al. Gram-positive Bacilli Using MALDI-TOF MS 요약 본연구에서는일개대학병원에서 2년간분리된혈액배양양성배지에서 MALDI-TOF MS system을이용하여 Grampositive bacilli를동정한결과를균종별, 분기별로분석하였다. Corynebacterium striatum은총 89균주중 66균주 (74.2%), Bacillus cereus는 60균주중 44 균주 (73.3%), Listeria monocytogenes는 25균주중 25균주 (100%) 로 2.0 이상의높은스코어에서동정되었다. 미동정된균주는 303균주중 293균주는혈액배양에서 1회분리균주로감염균으로서의의의가없는오염균주로간주되었다. 감염균으로서의의가있는동일환자 2회이상분리균주대상 16S-rRNA sequencing 비교결과총 50균주중 43균주가일치해 86.0% 동정이가능하였다. 일치하지않은 7균주중 5균주는 MALDI-TOF MS로도동정이되지않았다. 결론적으로혈액배양에서 Gram-positive bacilli 가동정되는경우, 일차적으로 MALDI-TOF MS를이용하여동정해보고이를활용한다면어렵고비용이많이들던 Gram-positive bacilli 동정이저비용으로더욱간편하고정확해지며, Grampositive bacilli 에의한감염진단에도도움이될것으로판단된다. Acknowledgements: None Conflict of interest: None Author s information (Position): Choi JU 1, M.T.; Yu YB 2, Professor; Kim SH 3, M.T.; Won S 4, Professor; Kim YK 2, Professor. REFERENCES 1. Funke G, von Graevenitz A, Clarridge J3, Berneard KA. Clinical microbiology of coryneform bacteria. Clin Microbiol Rev. 1997;10:125-159. 2. Adderso EE, Boudreaux JW, Hayden RT. Infections caused by coryneform bacteria in pediatric oncology patients. Pediatr Infect Dis J. 2008;27:136-141. https://doi.org/10.1097/inf.0b013 e31814fab12. 3. Barberi SC, Almuzara M, Join-Lambert O, Ramirez MS, Famiglietti A, Vay C. Comparison of the Bruker MALDI-TOF mass spectrometry system and conventional phenotypic methods for identification of Gram-positive rods. PLoS One. 2014: 9:e106303. https://doi.org/10.1371/journal.pone.0106303. 4. Watson RS, Carcillo JA, Linde-Zwirble WT, Clermont G, Lidicker J, Angus DC. The pidemiology of severe sepsis in children in the united stated. Am J Respir Crit Care Med. 2003; 167:695-701. https://doi.org/10.1164/rccm.200207-682oc. 5. Holland RD, Wilkes JG, Rafii F, Sutherland JB, Persons CC, Voorhees KJ, et al. Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 1996;10:1227-1237. https:// doi.org/10.1002/(sici)10970231(19960731)10:10<1227::aid- RCM659>3.0.CO;2-6. 6. Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, et al. Ongoing evolution in bacteriologyl routine identification of bacteria by matrix-assisted laser desorption ionization itme-offlight mass spectrometry. Clin Infect Dis. 2009; 49:543-551. https://doi.org/10.1086/600885. 7. Bizzini A, Durussel C, Bille J, Greub G, Prod hom G. Performance of matrix-assisted laser desorption ionizationtime of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory. J Clin Microbiol. 2010;48: 1549-1554. https://doi.org/10.1128/ JCM.01794-09. 8. Stevenson LG, Drake SK, Shea YR, Zelazny AM, Murray PR. Evaluation of matrix-assisted laser desorption ionization-time of Flight mass spectrometry for identification of clinically important yeast species. J Clin Microbiol. 2010;48:3482-3486. https://doi.org/10.1128/jcm.00687-09. 9. Park KG, Yu YB, Yook KD, Kim SH, Kim SH, Kim YG. An evaluation of the rapid antimicro bial susceptibility test by VITEK MS and VITEK 2 Systems in blood culture. Korean J Clin Lab Sci. 2017;49:279-284. https://doi.org/10.15324/kjcls. 10. Carbonnelle E, Grohs P, Jacquier H, Day N, Tenza S, Dewailly A, et al. Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification. J Microbiol Methods. 2012;89:133-136. https://di.org/10.1016/j.mimet.2012.03.003. 11. Dubois D, Grare M, Prere MF, Segonds C, Marty N, Oswald E. Performances of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for rapid identification of bacteria in routine clinical microbiology. J Clin Microbiol. 2012;50:2568-2576. https://doi.org/10.1128/ JCM. 00343-12. 12. Ahn GY, Jang SJ, Lee SH, Jeong OY, Bidur Prasad Chaulagain BP, Moon DS, et al. Trends of the species and antimicrobial susceptibility of microorganisms isolated from blood cultures of patients. Korean J Clin Microbiol. 2006;9:42-50. 13. Navas M, Pincus DH, Wilkey K, LaSalvia M, Wilson D, Procop GW, et al. Identification of aerobic Gram-positive bacilli by use of Vitek MS. J Clin Microbiol. 2014;52:1274-1277. https://doi.org/10.1128/jcm.03483-13. 14. Kim NH, Hwang JH, Song KH, Choe PG, Park WB, Kim ES, et al. Changes in antimicrobial susceptibility of blood isolates in a university hospital in South Korea, 1998-2010. Infect Chemother. 2012;44:275-281. https://doi.org/10.3947/ic.2012. 44.4.275. 15. Woo PC, Lau SK, Teng JL, Tse H, Yuen KY. Then and now: use of 16S rdna gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect. 2008;14:908 934. https://doi.org/ 10.1111/j.1469-0691.2008.02070.x. 16. Bruins MJ, Bloembergen P, Ruijs GJ, Wolfhagen MJ. Identification and susceptibility testing of enterobacteriaceae and pseudomonas aeruginosa by direct inoculation from pos-

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