Biomedical Science Letters 2018, 24(2): 108~115 https://doi.org/10.15616/bsl.2018.24.2.108 eissn : 2288-7415 Original Article Availability of MADLDI-TOF MS for Identification of Gram Positive Bacilli Isolated from Blood Culture Jin-Un Choi 1,, Sang-Ha Kim 2,, Su-Jeong Hwang 3, Young-Bin Yu 5, Sunghyun Kim 4, and Young-Kwon Kim 5, 1 Department of Laboratory Medicine, Chonnam National University Hospital, Gwangju 61469, Korea 2 Department of Laboratory Medicine, Konyang University Hospital, Daejeon 35365, Korea 3 Department of Dental Hygiene, College of Medical Sciences, Konyang University, Daejeon 35365, Korea 4 Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea 5 Department of Biomedical Laboratory Science, College of Medical Sciences, Konyang University, Daejeon 35365, Korea In the present study, results of the identification of Gram-positive bacilli (GPB) were analyzed by using the MALDI- TOF MS technique to score each 2-year blood culture at a university hospital. In addition, 16S rrna sequence analyses and MALDI-TOF MS results are compared to targeting strains that had been isolated two or more times within the same patient, to evaluate the usefulness of MALDI-TOF MS in GPB identification. According to the cut-off ( 1.7) criteria, there were 410 (57.5%) reliable strains and 303 (42.5%) non-identified strains among the GPB identification results of 713 strains, using a microflex MALDI Biotyper (Bruker Daltonik GmbH, Bremen, Germany). The isolation appeared most often in the following order: Corynebacterium striatum, Bacillus cereus, Bacillus subtilis, Paenibacillus urinalis, and Listeria monocytogenes. Nearly three-fourths, 66 out of 89 (74.2%) of the strains for Corynebacterium striatum; 44 out of 60 (73.3%) strains for Bacillus cereus; and all (25 out of 25, 100%) Listeria monocytogenes strains were identified by their high scores of 2.0 or higher. Most (293 strains out of 303) non-identified strains were strains isolated only once and not significant as infectious bacilli. A total of 43 out of 50 (86.0%) strains matched and were able to be identified based on the 16 rrna sequencing comparison results of strains that were isolated twice or more within the same patient and significant as infection bacilli. Non-matching among 5 out of 7 strains was not identified, even with MALDI-TOF MS. In conclusion, GPB can be identified in blood cultures using MALDI-TOF MS. This can be done accurately with ease, rapidly, and at a low cost. It is also thought to be helpful in GPB diagnosis and treatment. Key Words: Blood culture, Gram positive bacilli, MADLDI-TOF MS, 16S rrna sequencing * Received: March 5, 2018 / Revised: April 25, 2018 / Accepted: May 17, 2018 Jin-Un Choi and Sang-Ha Kim contributed equally to this article. Corresponding author: Sunghyun Kim. Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea. Tel: +82-51-510-0560, Fax: +82-51-510-0568, e-mail: shkim0423@cup.ac.kr Corresponding author: Young-Kwon Kim. Department of Biomedical Laboratory Science, College of Medical Sciences, Konyang University, Daejeon 35365, Korea. Tel: +82-42-660-6371, Fax: +82-42-543-6370, e-mail: ykkim3245@konyang.ac.kr C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. CC 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. - 108 -
서론최근의료기관의임상미생물검사실에서는 Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) 를기반으로한원인균동정시스템의도입이증가하고있는상황이다. 이검사법은임상검체로부터분리배양된단일집락을이온화하여진공관에서검출기에도달하는시간을근거로구성물질의질량을측정하는방법으로동정시간은균주당평균약 6분정도소요된다 (Holland et al., 1996). 동정을위한비용은상품화된진단키트를포함해전통적동정방법 (Seng et al., 2009) 의약 20~30% 정도로미생물검사실에서이용하기에간편하고, 동정에소요되는시간또한매우짧아매우경제적인방법으로알려져있다 (Bizzini et al., 2010; Stevenson et al., 2010). 그람양성막대균 (Gram positive bacilli, GPB) 의대부분은주로환경에서존재하며비병원성으로알려져있었기때문에, 그동안임상미생물검사실에서는이를오염균으로간주하여정확한동정을시행하지않는것이일반적이었다 (Funke et al., 1997). 하지만, GPB 중몇몇병원성세균종은동정을하는것은임상적으로중요하다. 특히, 최근면역저하자나체내삽입기구를처치하는환자가증가하고있으며, 항생제사용또한증가함에따라 GPB에의한기회감염이증가하고있는추세이며, GPB의정확한동정필요성이대두되고있으며, 이를통해감염의정확한원인분석이가능하고, 적절한치료를위해큰도움을줄수있을것으로여겨진다 (Adderso et al., 2008). 일반적으로혈액배양 (Blood culture) 으로부터분리되는 GPB는비용의효용적인측면에서동일환자에서 2회이상분리될경우에만제한적으로원인균동정을시행하지만, 기존에임상미생물검사실에서시행하고있는원인균동정방법은많은시간이소요되고, 항상동일한결과를나타내지않는다 (Barberi et al., 2004). 따라서, 16S rrna 염기서열분석을이용한분자유전학적검사방법을이용하여확인동정을시행하고, 이를표준법 (gold standard method) 으로도입하고있지만, 이검사법은비용과시간이많이소요된다는제한점이있다. MALDI-TOF MS 기반원인균동정시스템을이용한기존의많은연구들은대부분임상미생물검사실에서주로분리되는그람양성알균 (Gram positive cocci, GPC), 그람음성막대균 (Gram negative bacilli, GNB) 을표적으로하고 있으며, 일부는 GPB를대상으로진행되었다 (Carbonnelle et al., 2012; Dubois et al., 2012). 따라서, 본연구에서는국내의한대학병원의혈액배양으로부터분리배양된 GPB를대상으로 MALDI-TOF MS를실시하여혈액배양에서분리된 GPB의동정균주수와미동정균주수를비교해보았고, 동일환자에서 2회이상분리된균주를대상으로 16S rrna 염기서열분석을함께실시하여그결과를비교함으로써, GPB를동정함에있어서 MALDI-TOF MS를기반으로한원인균동정시스템의유용성을평가해보고자하였다. 재료및방법임상검체 2014 년 10월부터 2016 년 9월까지국내의한대학병원에서혈액배양이의뢰된임상검체로부터분리배양된 GPB 총 713주를대상으로연구를실시하였다. 혈액배양혈액배양은산소성배지 (BacT/ALERT FA Plus, FA 와 BACTEC TM Plus Aerobic/F), 무산소성배지 (BacT/ALERT Standard Anaerobic, SN와 BACTEC TM Plus Anerobic/F) 에접종하고각각 BacT/ALERT 3D Blood Culture System (biomérieuxinc., Durham, NC, USA) 과 BD BACTEC TM FX blood culture system (BD, Spark, MD) 에 5일간배양하였다. 양성이나온혈액배양병을 5% Sheep Blood Agar Plate (HANIL KOMED) 에접종하여 5% CO 2, 35 배양기에서 24시간또는 48시간배양하였다. MALDI-TOF MS System을이용한그람양성막대균 (GPB) 의동정 GPB로확인된균주를대상으로 Microflex MALDI Biotyper (Bruker Daltonik GmbH, Bremen, Germany) 장비로검사를수행한후, MALDI Biotyper RTC software (V. 3.1) 를이용해결과를분석하여세균종을동정하였다. 직접도말법은하룻밤배양한신선한집락을멸균 Wooden applicator 를이용해 MSP 96 target polished steel BC microscout Target plate (Bruker Daltonics) 에도말하고, 세균이마른후매트릭스용액 (50% acetonitrile, 2.5% trifluoroacetic acid) 에포화된 (α-cyano-4-hydroxycinnamic acid) 을더한시약 1 μl를가하고, 실온에서완전히건조시킨후 microflex MALDI Biotyper 장비에장착하였다. Bacterial test standard (BTS) 를 - 109 -
사용하여장비 Calibration 을실시하였고, m/z 2,000~20,000 범위에서측정된 Mass spectra 동정결과는 MALDI Biotyper RTC software (V. 3.1) 를이용해분석하였다. 장비회사에서제시하는표준기준을기반으로하여 Cut-off score 가 2.0 이상이면균종동정, 1.7 이상이면서 2.0 미만인경우균속동정이가능한것으로판단하였고, 1.7 미만인경우신뢰성이없는것으로간주하였다. 16S rdna 염기서열분석 Genomic DNA 추출 : 혈액배양으로부터분리배양된 GPB의 gdna 추출은 Phosphate buffered saline (PBS) 1 ml 에집락 3~4개를혼탁시킨후 8,000 x G에서 5분간원심분리하였다. 침사층을제외한상층액을제거한후 Lysis solution (DW 1 L, Tris base 1.212 g, EDTA 0.372 g, Triton 1 ml /1 L) 200 μl를첨가하고균을혼탁한후 8,000 x G에서 5분간원심분리하였다. Lysis 과정을 3회반복후, 멸균 DW 200 μl에침사층을혼탁시킨후 100 에서 30분동안가열하여 gdna가유리되도록하였다. 이후 13,000 x G, 4 에서 10분간원심분리하여상층액을취한후사용전까지 -20 에서냉동보관하였고, 염기서열분석을위한 PCR 수행시 gdna의농도를 100 ng에맞추어사용하였다. 16S rdna 염기서열분석을위한 PCR 및염기서열분석 : 16S rdna 염기서열분석을위한 PCR을수행하기위해 Forward primer인 16S-rRNA1 (5'-AGT TTG ATC CTG GCT CAG-3') 와 Reverse primer 인 16S-rRNA2 (5'-GGT TAC CTT GTT ACG ACT T-3') 를이용하였다. Primer 각각 20 pmol 1 μl, dntps 1 μl, 10x Reaction buffer (1x: 10 mm Tris-HCL, 1.5 mm KCL, 0.1% Triton X-100) 5 μl, 2.5 U/μL DyNAzyme TM polymerase 1 μl, 멸균 DW 38.5 μl에추출된 gdna 2.5 μl를넣어총 50 μl로반응액을만들었다. PCR은 TaKaRa PCR Thermal cycler (TP600 Gradient, Roche Molecular System, CA, USA) 를사용하였고, 온도조건을 94 에서 5분 predenaturation 시킨후, 94 에서 1분 denaturation, 49 에서 1분 annealing, 72 에서 1분 extension 을 1주기로 36회실시하여증폭하고 72 에서 10분간 postextension 을실시하였다. PCR 증폭산물은 1.8% agarose gel을사용하여 100 V로 30분간 loading 시켜 UV 투과조명기에서확인하였고, Gene- All PCR DNA Purification kit (Geneall Biotechnology Co. LTD Seoul, Korea) 를사용하여 PCR 산물을정제하였다. 그후 Big dye PCR을시행하였으며 Big dye PCR의경우 Seq primer 를이용하였다. Forward 와 reverse primer 를이용해각각시행하였고, 반응액은 5 pmol seq primer 2 μl, 정제된 PCR 증폭산물 3 μl, Big-dye 0.5 μl, PCR buffer 3 μl, 멸균 DW 4.5 μl를혼합하여총 13 μl가되도록한후반응을진행하였다. 96 에서 1분간 predenaturation 을수행하고, 96 10초, 50 5초, 60 4분으로구성된순환조건을 25회반응시킨후 3 M sodium acetate 를이용한침전과정과 95% EtOH를이용한탈수과정을거쳐반응물질만을정제하였다. 정제된반응물은건조시켜 Hidi 10 μl에녹인후 ABI 3730XL DNA Analyzer (Applied Biosystems) 를이용해염기서열을분석하였다. 분석한염기서열의유사성검색은 NCBI data base인 BLAST 를이용하였다. 분석한자료중 A 99.0% 의유사성을보이는결과를 CLSI의기준을적용하여판독하였다 (Chun et al., 2007; Wayne, 2007). 결과분기별혈액배양에서그람양성막대균 (GPB) 의분리빈도 2014 년 10월부터 2016 년 9월까지 2년간혈액배양이의뢰된총 87,241건중 GPB의분리비율은 2014년 4분기 83건 (0.8%), 2015년 1분기 68건 (0.7%), 2015년 2분기 83건 (0.9%), 2015년 3분기 124건 (1.1%), 2015년 4분기 84건 Table 1. Isolation rate of Gram positive bacilli in blood culture (2014-2016) Quarter of a year 4 th quarter of 2014 1 st quarter of 2015 2 nd quarter of 2015 3 rd quarter of 2015 4 th quarter of 2015 1 st quarter of 2016 2 nd quarter of 2016 3 rd quarter of 2016 Total No. (%) of cases 83/ 10168 (0.8%) 68/ 9732 (0.7%) 83/ 9574 (0.9%) 124/ 11255 (1.1%) 84/ 11163 (0.8%) 65/ 11321 (0.6%) 105/ 11937 (0.9%) 101/ 12091 (0.8%) 713/ 87241 (0.8%) 1 st quarter: Jan to Mar, 2 nd quarter: Apr to Jun, 3 rd quarter: Jul to Sep, 4 th quarter: Oct to Dec - 110 -
Table 2. Identification rate of Gram positive bacilli which showed 1.7 to 2.0 of MALDI-TOF MS cut-off scores (2014-2016) Species identification results by MALDI-TOF MS system No. of isolates with the number of positive blood cultures per patient Total No. (%) of patient 1 2 3 4 5 Total No. (%) of species Bacillus subtilis 19 0 0 0 0 19 (15.6) 19 (14.1) Bacillus cereus 14 1 0 0 0 15 (12.3) 16 (11.9) Corynebacterium striatum 4 1 0 2 0 7 (5.7) 14 (10.4) Corynebacterium afermentans 10 0 0 0 0 10 (8.2) 10 (7.4) Bacillus mojavensis 8 0 0 0 0 8 (6.6) 8 (5.9) Bacillus pumilus 8 0 0 0 0 8 (6.6) 8 (5.9) Bacillus sonorensis 7 0 0 0 0 7 (5.7) 7 (5.2) Bacillus licheniformis 6 0 0 0 0 6 (4.9) 6 (4.4) Bacillus vallismortis 5 0 0 0 0 5 (4.1) 5 (3.7) Mycobacterium abscessus 0 0 0 1 0 1 (0.8) 4 (3.0) Bacillus megaterium 4 0 0 0 0 4 (3.3) 4 (3.0) Microbacterium species 3 0 0 0 0 3 (2.5) 3 (2.2) Clostridium perfringens 1 1 0 0 0 2 (1.6) 3 (2.2) Eggerthella lenta 2 0 0 0 0 2 (1.6) 2 (1.5) Brevibacterium iodium 0 1 0 0 0 1 (0.8) 2 (1.5) Actinomyces odontolyticus 2 0 0 0 0 2 (1.6) 2 (1.5) Paenibacillus urinalis 2 0 0 0 0 2 (1.6) 2 (1.5) Solibacillus silvestis 2 0 0 0 0 2 (1.6) 2 (1.5) Bacillus circulans and others a 18 0 0 0 0 18 (14.8) 18 (13.3) Total 115 4 0 3 0 122 (100.0) 135 (100.0) a Bacillus muralis, Bacillus mycoisea, Bacillus niacini, Bacillus thuringiensis, Bacillus weienstephanensis, Clostridium bifermentans, Clostridium carnis, Clostridium innocuum, Corynebacterium singulare, Corynebacterium tuscaniense, Dermabacter hominis, Gordonarubropertincta, Lysinibacillus fusiformis, Microbacterium oxydans, Microbacterium testaceum, Paenibacillus latus, Paenibaillus xylanilyticus (0.8%), 2016 년 1분기 65건 (0.6%), 2016 년 2분기 105건 (0.9%), 2016년 3분기 101건 (0.8%) 이었다. 연구기간동안혈액배양이의뢰된전체건수중혈액배양양성률은 10.0% 였으며, GPB 양성률은 0.8% 였다 (Table 1). MALDI-TOF MS System 을이용한혈액배양으로부터분리된그람양성막대균 (GPB) 의동정 1.7~2.0 의 cut-off score를나타낸 GPB의분리비율 : Cut-off score 1.7~20 을나타낸 GPB 중 Bacillus subtilis 로동정된비율은 19건 (14.1%), Bacillus cereus 는 15건 (11.9%), Corynebacterium striatum 는 14건 (10.4%), Corynebacterium afermentans 는 10건 (7.4%), Bacillus mojavensis 는 8건 (5.9%) 이었다 (Table 2). 2.0 이상의 cut-off score를나타낸그람양성막대균 (GPB) 의분리비율 : Cut-off score를 1.7 이상나타내며, GPB로동정된총 410건중 275건 (67.1%) 은 2.0 이상의높은 cut-off score로 GPB의종동정이가능하였다. Cut-off score 2.0 이상을나타낸 GPB 중 C. striatum 은 66건 (27.3%), B. cereus 는 44건 (16.0%), Paenibacillus urinalis 은 27건 (9.8%), Listeria monocytogenes 는 25건 (9.1%), Clostridium perfringens 는 15건 (5.5%) 이었다 (Table 3). MALDI-TOF MS 분석 cut-off score에따른그람양성막대균 (GPB) 의분리비율 : C. striatum 과 B. cereus 는각각 2년동안분리된총 89건중 66건 (74.2%), 60건중 44건 (73.3%) 이 cut-off score 2.0 이상의높은수치로동정가능하였고, L. monocytogenes 와 C. perfringens 는각각 25건중 25건 (100%), 18건중 15건 (83.3%) 이 cut-off score 2.0 이상의 - 111 -
Table 3. Identification rate of Gram positive which showed above 2.0 of MALDI-TOF MS cut-off scores (2014-2016) Species identification results by MALDI-TOF MS system No. of isolates with the number of positive blood cultures per patient Total No. (%) of patient 1 2 3 4 5 6 7 8 Total No. (%) of species Corynebacterium striatum 17 9 1 3 2 0 1 1 34 (16.7) 75 (27.3) Bacillus cereus 35 3 1 0 0 0 0 0 39 (19.1) 44 (16.0) Paenibacillus urinalis 25 1 0 0 0 0 0 0 26 (12.7) 27 (9.8) Listeria monocytogenes 3 7 0 2 0 0 0 0 12 (5.9) 25 (9.1) Clostridium perfringens 9 3 0 0 0 0 0 0 12 (5.9) 15 (5.5) Bacillus subtilis 11 0 0 0 0 0 0 0 11 (5.4) 11 (4.0) Bacillus licheniformis 10 0 0 0 0 0 0 0 10 (4.9) 10 (3.6) Clostridium tertium 6 0 0 0 0 0 0 0 6 (2.9) 6 (2.2) Microbacterium species 1 0 0 1 0 0 0 0 2 (1.0) 5 (1.8) Actinomyces oris 4 0 0 0 0 0 0 0 4 (2.0) 5 (1.8) Bacillus infantis 4 0 0 0 0 0 0 0 4 (2.0) 4 (1.5) Bacillus pumilus 4 0 0 0 0 0 0 0 4 (2.0) 4 (1.5) Bacillus megaterium 3 0 0 0 0 0 0 0 3 (1.5) 3 (1.1) Bacillus sonorensis 3 0 0 0 0 0 0 0 3 (1.5) 3 (1.1) Clostridium carnis 0 0 1 0 0 0 0 0 1 (0.5) 3 (1.1) Eggerthella lenta 3 0 0 0 0 0 0 0 3 (1.5) 3 (1.1) Bacillus mojavensis 2 0 0 0 0 0 0 0 2 (1.0) 2 (0.7) Bacillus mycoisea 2 0 0 0 0 0 0 0 2 (1.0) 2 (0.7) Corynebacterium minutissimun 2 0 0 0 0 0 0 0 2 (1.0) 2 (0.7) Corynebacterium urealyticum 0 1 0 0 0 0 0 0 1 (0.5) 2 (0.7) Lactobacillus paracasei 2 0 0 0 0 0 0 0 2 (1.0) 2 (0.7) Microbacterium aurum 2 0 0 0 0 0 0 0 2 (1.0) 2 (0.7) Mycobacterium abscessus 0 1 0 0 0 0 0 0 1 (0.5) 2 (0.7) Paenibacillus illinoisensis 2 0 0 0 0 0 0 0 2 (1.0) 2 (0.7) Actinomyces odontolyticus a 16 0 0 0 0 0 0 0 16 (7.8) 16 (5.8) and others Total 166 25 3 6 2 0 1 1 204 (100.0) 275 (100.0) a Arthrobacteroxydans, Bacillus atrophaeus, Bacillus flexus, Bacillus thermoamylovorans, Bacillus thuringiensis, Corynebacterium amycolatum, Corynebacterium falsenii, Corynebacterium pseudodiphthriticum, Exigubacterium aurantiacum, Lactobaillus salivarius, Lysinibacillus fusiformis, Paenibacillus barengoltzii, Paenibacillus rhizosphaerae, Paenibacillus odorifer, Paenibacillus pauli 높은수치로동정가능하였다 (Table 4). 혈액배양으로부터분리된그람양성막대균 (GPB) 동정을위한 16S rdna 염기서열분석과 MALDI-TOF MS system 의결과비교세균의 16S rdna 염기서열분석과 MALDI-TOF MS 분석을동시에실시한총 50건중 43건 (86.0%) 은두분석에서결과가일치하였으며, 7건 (14.0%) 은결과가일치하 지않았다. 두분석에서일치한결과를나타낸경우는 C. striatum 19건, L. monocytogenes 9건, C. perfringens 5건, B. iodinum 1건, Clostridium canis 1건, Corynebacterium urealyticum 1건, Mycobacterium abscessus 1건, P. urinalis 1건이었다. 두분석에서불일치를나타낸 7건중 MALDI-TOF MS 분석에서 cut-off score 1.7 이상을나타냈던경우는총 2건으로 B. cereus 는 Lysinbacillus spp. 로, Microbacterium spp. 는 Mycobacterium paraoxydans 로각각불일치한결과를나타 - 112 -
Table 4. Distribution of Gram positive bacilli species from blood culture according to the cut-off scores of MALDI-TOF MS (2014-2016) Species identification results by MALDI-TOF MS system Score Score 2.0 ~ 1.7 2.0 Total No. of species Corynebacterium striatum 14 75 89 Bacillus cereus 16 44 60 Bacillus subtilis 19 11 30 Paenibacillus urinalis 2 27 29 Listeria monocytogenes 0 25 25 Clostridium perfringens 3 15 18 Bacillus licheniformis 6 10 16 Bacillus pumilus 8 4 12 Other species 67 64 131 Not reliable identification - - 213 No peaks found - - 90 Total 135 275 713 Table 5. Comparison of the results of MALDI-TOF MS and 16S rdna sequence analysis with double positive cases in blood culture of the same patient Species identification results by MALDI-TOF MS System 2.0 Score 1.7 Score 2.0 No. of isolates Score < 1.7 Concordant result Discordant result Corynebacterium striatum 17 2 19 0 Fianl ID result by 16S-rRNA sequencing Bacillus cereus 5 1 5 1 Lysinbacillus sp. Clostridium perfringens 4 1 5 0 Listeria monocytogenes 9 9 0 Bacillus iodinum 1 1 0 Clostridium carnis 1 1 0 Corynebacterium urealyticum 1 1 0 Microbacterium species 1 0 1 Mycobacterium abscessus 1 1 0 Paenibacillus urinalis 1 1 0 Mycobacterium paraoxydans Not reliable identification 4 0 4 Cellulomonas hominis Corynebacterium amycolatum Catabacter hongkongenesis Bacillus sp. No peaks found 0 1 Bacillus velezensis Total 39 6 4 43 7 냈다. 두분석에서불일치한결과를나타낸나머지 5 주는 MALDI-TOF MS 분석에서 not reliable identification 혹은 no peaks found 의결과를나타냈고, 16S rdna 분석을실 시한결과각각은 Cellulomonas hominis, Corynebacterium - 113 -
amycdatum, Catabacter hongkongenesis, Bacillus spp., Bacillus velezensis 로동정되었다 (Table 5). 고찰기존의연구들을통해임상적으로의의가있는 GPC, GNB, 진균의대부분은 MALDI-TOF MS 분석으로종과속의수준까지간편하고신속하게동정이가능함이현재까지확인되었다 (Bernard et al., 2012; Levesque et al., 2015). 하지만, 상대적으로오염균으로여겨지던 GPB에대한기존의연구와임상적유용성평가결과는결여되어있어본연구를통해혈액배양에서분리되는 GPB의동정이 MALDI-TOF MS 분석을통해유용성이있는지분석해보았다. 따라서, 국내의한대학병원에서 2년간시행된총 87,241건의혈액배양중 GPB가분리된 713건 (0.8%) 을대상으로연구를실시하였다. 장비회사에서제공하는기준에따라균과속의동정이가능한 cut-off score 1.7 이상을나타낸경우는총 410건 (57.5%) 이었다. 동정은가능하였으나, cut-off score가 1.7 이하로신뢰할수없는결과를보였던경우는총 213건 (29.9%) 이었으며, 분석결과가 No peaks found 로동정조차힘들었던경우는 90건 (12.6%) 에달했다. Cut-off score 1.7 이상을나타낸경우 C. striatum 으로동정된경우는 89건 (12.4%), B. cereus 는 60건 (8.4%), B. subtilis 30건 (4.2%), P. urinalis 는 29건 (4.1%), L. monocytogenes 는 25건 (3.5%) 이었다. Corynebacterium spp., Bacillus spp., Listeria spp. 은임상적으로도의의가많은감염균으로서높은비율을보임을알수있었다 (Renom et al., 2012; Severo et al., 2014). 또한분리비율은적지만 Mycobacterium spp. 과 Clostridium spp. 은 cut-off score 1.7 이상의신뢰할수있는결과로동정이가능함을알수있었다. 물론 not reliable identification 혹은 no peaks found의결과를나타낸경우도 42.5% 로높은비율을차지하였으나, 거의대부분의경우 1회분리균은오염균으로간주되기때문에감염균으로의의의가없는것으로사료된다. Cut-off score 2.0 이상으로동정된균종과 cut-off score 1.7~2.0 으로동정된균종을비교해보면 cut-off score 1.7 이상으로동정된총 410건중 275건 (67.1%) 은 2.0 이상의높은 cut-off score 로동정이가능했다. Cut-off score 2.0 이상인경우는 C. striatum 이 66건 (27.3%), B. cereus 가 44건 (16.0%), P. urinalis 가 27건 (9.8%), L. monocytogenes 가 25건 (9.1%), C. perfringens 가 15건 (5.5%) 이었고, cut-off score 1.7~ 2.0로동정된균종은 B. subtilis 가 19건 (14.1%), B. cereus 가 15건 (11.9%), C. striatum 가 14건 (10.4%), C. afermentans 가 10건 (7.4%), B. mojavensis 가 8건 (5.9%) 이었다. C. striatum 과 B. cereus 는각각 2년동안분리된총 89건중 66건 (74.2%), 60건중 44건 (73.3%) 을 cut-off score 2.0 이상의높은신뢰도를가지고동정이가능했다. 또한, 1회만분리되어도의의가있는 L. monocytogenes 는 25건모두가 cut-off score 2.0 이상을나타냈다. GPB이지만동일환자에서 2회이상분리된경우감염원으로간주되기때문에이러한조건에해당하는총 50건은 16S rdna 염기서열분석을통해재확인절차를진행하였으며, 최종결과를비교해보았다. 총 50건중 43건 (86.0%) 은 MALDI-TOF MS와 16S rdna 염기서열분석결과가일치하였고, 나머지 7건 (14.0%) 은불일치하는결과를나타냈다. 본연구의결과를토대로지난 2년간의혈액배양에서분리된 GPB의절반이상이 MALDI-TOF MS 분석시 cutoff score 1.7 이상의신뢰할수있을만한수준으로균종동정이가능했고, 그중 1회만분리되어도임상적의의가있는균종, 동일환자에서 2회이상분리되어오염균이아닌감염균으로간주되는균종은세균 16S rdna 염기서열분석결과와 86.0% 의높은일치율을나타냈다. 추가적으로, 세균의 16S rdna 염기서열분석과 MALDI-TOF MS 분석을동시에실시한총 50건중 39건 (78.0%) 은 cutoff score 2.0 이상, 6건 (12.0%) 은 cut-off score 1.7 이상의높은신뢰도로균종동정이가능했으며, 총 2건 (4.0%) 에서만 MALDI-TOF MS와세균의 16S rdna 염기서열분석결과가일치하지않았다. 이러한결과들을기반으로하여, 기존에오염균으로생각되어거의동정되지않았던 GPB 균종을 MALDI-TOF MS System 을이용해 1차동정을실시하고, 임상적의의가있는균종에대해서는 16S rdna 염기서열분석을추가적으로적용하여결과를활용한다면, 향후감염원으로서의의가있는 GPB 동정에매우유용할것으로사료된다. 하지만, 환자로부터흔히분리되지않고, 비병원성세균이많이포함되어있는 GPB의특성으로인해현재 MALDI-TOF MS를이용해균종동정을하기위한데이터베이스가부족하고, 협막이존재하는일부 GPB의구조적특성상단백질분리의어려움이있기때문에동정이힘든경우가많이확인되었다. 추후 MALDI-TOF MS를이용해 GPB 균종을동정할수있는데이터베이스를더욱확충하고, 협막보유세균을위해추가보완된단백질 - 114 -
분리방법이사용된다면, GPB의신속정확한동정에많은도움이될수있을것으로사료된다. ACKNOWLEDGEMENTS None. CONFLICT OF INTEREST The authors have no conflicts of interest to disclose. REFERENCES Adderso EE, Boudreaux JW, Hayden RT. Infections caused by coryneform bacteria in pediatric oncology patients. Pediatr Infect Dis J. 2008. 27: 136-141. Barberi C, Almuzara M, Join-Lambert O, Ramirez MS, Famiglietti A. Comparison of the Bruker MALDI-TOF Mass Spectrometry System and Conventional Phenotypic Methods for Identification of Gram-Positive Rods. PLoS ONE. 2014. 9: e106303. Bernard K. The genus corynebacterium and other medically relevant coryneform-like bacteria. J Clin Mircobiol. 2012. 50: 3152-3158. Bizzini A, Durussel C, Bille J, Greub G, Prod'hom G. Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory. J Clin Microbiol. 2010. 48: 1549-1554. Carbonnelle E, Grohs P, Jacquier H, Day N, Tenza S, Dewailly A, Vissouarn O, Rottman M, Herrmann JL, Podglajen I, Raskine L. Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification. J Microbiol Methods. 2012. 89: 133-136. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, et al. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol. 2007. 57: 2259-2261. 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. Funke G, von Graevenitz A, Clarridge JE 3rd, Berneard KA. Clinical microbiology of coryneform bacteria. Clin Microbiol Rev. 1997. 10: 125-129. 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 desor- ption/ionization with time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 1996. 10: 1227-1237. Levesque S, Dufresne PJ, Soualhine H, Doming M-C, Bekal S, Lefebvre B, et al. A side by side compariton of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technolo gy for Microorganism Identification in a Public Health Reference Laboratory. PLoS ONE. 2015. 10: e0144878. Renom F, Gomila M, Garau M, Gallegos MDC, Guerrero D, Lalucat J, Soriano JB. Respira tory infection by Corynebacterium striatum: epidemiological and clinical determinants. New Micorbes New Infect. 2014. 2: 106-114. Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, et al. Ongoing evolution in bacteriologyl routine identifi- cation of bacteria by matrix-assisted laser desorption ionization itme-offlight mass spectrometry. Clin Infect Dis. 2009. 49: 543-551. Severo CB, Guazzelli LS, Barra MB, Hochhegger B, Severo LC. Multiple pulmonary nodules caused by Corynebacterium striatum in an immunocompetent patient. Rev Inst Med Trop Sao Paulo. 2014. 56: 89-91. 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. Wayne PA. Clinical and Laboratory Standards Institute. Interpre- tive criteria for identify cation of bacteria and fungi by DNA target sequencing; approved guideline MM-18A. Clinical and Laboratory Standards Institute. 2007. https://doi.org/10.15616/bsl.2018.24.2.108 Cite this article as: Choi JU, Kim SH, Hwang SJ, Yu YB, Kim S, Kim YK. Availability of MADLDI-TOF MS for Identification of Gram Positive Bacilli Isolated from Blood Culture. Biomedical Science Letters. 2018. 24: 108-115. - 115 -