국내임상에서분리된세균의특성화분석에따른병원체자원화, 2014-2016 세브란스병원진단검사의학과김도균, 정석훈, 이경원 * 질병관리본부국립보건연구원병원체자원관리 TF 정향민, 황규잠 * 교신저자 leekcp@yuhs.ac, 02-2228-2240 공동교신저자 kyuhwang@nih.go.kr, 043-719-6870 Characterized Bacteria Resources from Clinical Specimen in Korea, 2014-2016 Department of Laboratory Medicine, College of Medicine, Yonsei University Kim Dokyun, Jeong Seok Hoon, Lee Kyungwon Pathogen Resource TF, Center for Infectious Disease, NIH, CDC Chung Hyangmin, Hwang Kyu Jam Background: The pathogen resources bank for the clinical isolates became an important issue especially after the Nagoya Protocol in 2014. However, the previous system for the bacterium-banking process had limitations including lack of detailed information about the species identification, minimal inhibitory concentration, and mode of resistance. We isolated, characterized, and deposited clinical isolates from the National Culture Collection for Pathogens (NCCP) from 2014 to 2016. Methodology: Survey about the stock and demand of the reference strains was given to the experts of the Korean Society of Clinical Microbiology. Referring to the result of the survey, we collected useful or rare clinical isolates in the tertiary hospitals from 2014 to 2016. The collected isolates were characterized in terms of identification, phenotype and genotype of resistance and toxin, and strain type. Results: We have deposited 416 strains in the bank to characterize bacteria resources. A total of 193 strains were allotted for species identification, 40 strains for antimicrobial susceptibility testing, 14 strains for toxin gene testing, 169 strains for research of epidemiology and mode of resistance. These strains would expand the resources of the National Culture Collection for Pathogen and would also be useful for future medical research and the development of identification devices. 들어가는말 임상미생물검사실및미생물연구소에서는정도관리, 교육, 연구등의목적에사용되는참조균주가중요하다. 특히 2014 년에는 제 10차생물다양성협약에서법적구속력이있는 유전자원접근및이익공유에대한나고야의정서 의발효로인하여병원체자원에대한국가주권이중요한이슈로부각되었다. 또한, 항균제내성은국내 외에서지속적으로증가하고있으며 [1-3], 내성과관련된 www.cdc.go.kr 358
참조균주에대한수요도점점늘어나고있다. 그람양성세균의 methicillin 내성과 vancomycin 내성, 그람음성세균의 extendedspectrum β-lactamase와 carbapenemase에의한 3, 4세대 cephalosporin과 carbapenem 내성등은특히중요하여, 최근 WHO에서도감시체계를확립하고관리를시작하는등전세계적인이슈로자리잡고있다 [4]. 국내에서도병원체에대한자원화필요성을인식하여질병관리본부에서운영중인국가병원체자원은행 (National Culture Collection for Pathogens, NCCP) 은병원체자원네트워크를구축하여국내임상분리병원체를수집, 자원화하고, 관리및분양하여보건의료연구개발의활성화에기여하고있다. 국가병원체자원은행의분양가능한보유자원은 2014년 537종 2,063주대비 2016년 588종 2,428주로수집및자원화건수가 17.7 % 증가하였다. 그러나국외자원은행들의보유균수와는많은차이가있어지속적으로다양한병원체자원의수집및특성분석의확대가요구되고있다. 본내용은임상미생물관련학회전문가들을대상으로참조균주의보유현황과수요를조사한후, 그에맞는임상균주를수집하고, 각균주의특성화분석결과에관한내용이다. 하였다. 그람음성막대균의특성분석에는 16S rrna 또는 rpob gene 염기서열분석을통한정확한균동정, 디스크확산법및한천희석법을통한항생제내성표현형분석, carbapenemase 유전형분석, ESBL 유전형분석, aminoglycoside 수식효소유전형분석, 독소유전자유전형분석, multilocus sequence typing (MLST) 을이용한 strain type 분석등이포함된다. 그람양성알균의경우디스크확산법및한천희석법을통한항생제내성표현형분석, meca, vana, vanb 등의주요내성유전자검출, 독소유전자검출, MLST를이용한 strain type 분석등이포함되었다. Clostridium difficile의경우균종동정및독소유전자및조절유전자의검출 (tcdc, tcdd, tcde, binary toxin 생성유전자 ) 등을검출하고 PCR ribotyping을시행하였다. 참조균주보유및수요에대한설문조사에참여한기관은총 77개기관으로, 53개의종합병원, 11개의연구기관, 5개의수탁검사실등이포함되었다. 균종별참조균주보유및필요수조사에서대부분의기관에서흔한세균의 wild type을보유하고있었으나, 균종이다양하지못하며, ESBL, carbapenemase, 독소등 특성분석이된참조균주는대부분보유하지않고있고필요기관의 몸말 저자들은정도관리용및교육용균주의수요및참조균주혹은교육용균주의보유현황과수요를파악하기위해관련학회의전문가들을대상으로설문을시행하였다. 설문조사의결과를토대로, 2014년부터 2016년까지총 3년간종합병원에서분리된세균성임상분리주를대상으로 1차년도에는그람음성막대균특성화및자원화, 2차년도에는그람양성알균특성화및자원화, 3차년도에는무산소성균과기타임상분리주의특성화및자원화를진행하였고, 정도관리균주선별시스템을구축하였다. 참조균주는사용목적에따라, 동정용, 항균제감수성시험용, 내성기전시험용, 독소시험용및감염역학시험용으로구분하였으며, 그용도에따라특성화분석을진행하였다. 연구기간내에개발할참조균주의목표균주수는총 380주이며, 이중 60주의 NCCP 등록을목표로 수가많아특성화분석이된많은균주들이 NCCP에등록이된다면그수요는적지않을것으로판단되었다. 설문조사결과를바탕으로 2014년부터 2016년까지총 416주 (1년차 144주, 2년차 135주, 3년차 137주 ) 를수집하여특성분석을시행하였다. 균주의용도별분류는균종동정용이 193주, 항균제감수성시험용이 40주, 독소유전자시험용이 14주, 역학조사용이 18주, 내성기전조사용이 33주, 내성기전및역학조사용이 118주였다 (Table 1). 균종동정용으로분리된 193주의구성은임상적으로흔히분리되는그람양성세균인 Staphylococcus 균속 12주, Streptococcus 균속 12주, Enterococcus 균속 15주, 및그람음성세균인 Pseudomonas 균속 15주, Acinsetobacter 균속이 19주, 혐기성세균인 Bacteroides 균속 14주와그람양성및음성세균의희귀균주각각 15주및 51주등이포함되었다 (Table 2, Table 3과 Table 4.). 항균제감수성시험용참조균주 40주중 18주는디스크확산법시험에사용할수있는참조균주이며, www.cdc.go.kr 359
Table 1. Number of deposited bacteria according to the classification of the purpose Purpose 2014 2015 2016 Total Species identification 39 79 75 193 Antimicrobial susceptibility testing 22 14 4 40 Toxin gene testing 3 11 0 14 Epidemiology 4 14 0 18 Mode of resistance 18 4 11 33 Epidemiology and mode of resistance 58 13 47 118 Total 144 135 137 416 Table 2. Gram-positive bacteria collected for the species identification Strain No. Name Strain No. Name 15GSB1439 Staphylococcus epidermidis 15GSU830 Enterococcus raffinosus 15GSB1460 Staphylococcus haemolyticus 15GSP143 Enterococcus hirae 15GSB1478 Staphylococcus capitis 15GSB2587 Enterococcus casseliflavus 15GSB1891 Staphylococcus salivarius 14GSB977-2 Kytococcus schroeteri [5] 15GSB1995-2 Staphylococcus warneri 14GS6709854-2 Corynebacterium striatum 15GSP229 Staphylococcus constellatus 14GSB519 Rhothia dentocariosa 15GSP336-2 Staphylococcus lugdunensis 15GSP167 Nocardia nova 15GSU471 Staphylococcus simulans 16GSP140 Nocardia titidiscaviarum 16GSP85 Staphylococcus pasteuri 16GSP409-2 Bergeyella zoohelcum 16GSB2065 Staphylococcus pseudointermedius 16SS91 Globicatella sulfidifaciens 16SS93 Staphylococcus aureus (small colony variant) 16SS92 Lactococcus garvieae 16SS94 Staphylococcus cohnii 16SS99 Weissella viridescens 15GSR203 Streptococcus pyogenes 16SS100 Corynebacterium diphtheriae 15GSR173 Streptococcus agalactiae 16SS101 Listeria monocytogenees 15GSP363 Streptococcus dysgalactiae 16SS102 Nocardia farcinica 15GSP1-2 Streptococcus sanginosus 16SS103 Nocardia nova 15GST109 Streptococcus anginosus 16SS104 Trueperella bernardiae 15GSP114 Streptococcus dentisani 16SS105 Tsukamurella inchonensis 15GSP334 15GSB1124 16SS95 16SS96 16SS97 16SS98 Streptococcus mitis Streptococcus infantarius Streptococcus gallolyticus Streptococcus gallolyticus subsp. pasteurianus Streptococcus pyogenes (nonhemolytic variant) Streptococcus salivarius subsp. thermophilus www.cdc.go.kr 360
Table 3. Gram-negative bacteria collected for the species identification Strain No. Name Strain No. Name 14P430-1 Pseudomonas mosselii GS15B302 Agrobacterium tumefaciens 14U220-2 Pseudomonas putida GS15G75 Cupriavidus basilensis 14P311 Pseudomonas stutzeri GS15T131 Ralstonia mannitolilytica 14R783 Pseudomonas plecoglossicida GS14R463 Kerstersia gyiorum 14U1436 Pseudomonas fulva GS14P62 Pasteurella multocida 14R868 Pseudomonas fulva (fluorecens) GS15P222 Pasteurella canis GS15p149 Pseudomonas oryzihabitans 15 Elizabethkingia anophelis 16 Pseudomonas otitidis [6] 15-A Elizabethkingia anophelis GS14U220-2 Pseudomonas plecoglossicida 15-B Elizabethkingia meningoseptica GS14P429 Pseudomonas stutzeri 15-C Elizabethkingia meningoseptica 16GSC295 Pseudomonas nitroreducens 15-D Elizabethkingia miricola 16GSP350 Pseudomonas hunanensis 15-E Elizabethkingia miricola 16GSP93 Pseudomonas guariconensis 15-F Elizabethkingia anophilus 16GSP223 Pseudomonas oryzihabitans GS15P301 Ochrobactrum tritici 16GSP90 Pseudomonas rhodesiae GS15P2 Nassilia varians KS201314 Acinetobacter nosocomialis GS14P66 Bordetella petrii [7] KM201301 Acinetobacter pittii GS14G59 Neisseria lactamica EUIM2012S13 Acinetobacter soli GS15R617 Neisseria cinerea PS2012F15 Acinetobacter oleivorans 16SS84 Campylobacter hyointestinalis [8] KB2012S13 Acinetobacter bereziniae 15GSB1609 Ochrobactrum anthropi IH201312 Acinetobacter gyllenbergii 15GSB1420 Burkholderia multivorans DJM201307 Acinetobacter haemolyticus 15GSU1075 Myroides phaeus JB2013N07 Acinetobacter junii 15GST131 Ralstonia mannitolilytica KSN2013N10 Acinetobacter radioresistens 16GSP261 Ralstonia pickettii GS2013B1272 Acinetobacter ursingii (red brown colony) 16GSG50-3 Roseomonas sp. KSN2013N16 Acinetobacter ursingii 15GSB1553 Roseomonas massiliae/mucosa EUMC201415 Acinetobacter calcoaceticus 15GSR172 Bordetella bronchiseptica IH201408 Acinetobacter genomosp. 17BJ 15GSP76 Massilia timonae GS15B2974 Acinetobacter grimontii 16GSR139 Elizabethkingia anophelis MJNS4 Acinetobacter ursingii (IMP producing) 16GSC131 Comamonas sp. 15GSB893 Acinetobacter soli 16GSR711 Ewingella americana 16GSB984 Acinetobacter guillouiae 15GSP235 Pasteurella dagmatis 16GSU1570 Acinetobacter schindleri 16SS85 Capnocytophaga ochracea 16GSG24 Acinetobacter radioresistens 16SS87 Dysgonomonas capnocytophagoides 14P329-3 Neisseria cinerea 16SS88 Eikenella corrodens 14P433-1 Comamonas testosteroni 16SS89 Gordonia bronchialis 14P78-1 Shewanella haloitis 16SS90 Kerstersia sp. 14P99 Ochrobactrum galinifaecis 16SS106 Mycoplasma homonis 14P382-1 Moraxella lacunata 16GSR297 Sphingomonas echinoides 14R463-2 Kerstersia gyiorum 16GSC293 Moraxella osloensis 14U913 Stenotrophomonas maltophillia 16GSU538 Massilia timonae GS15P199-1 Rahnella aquatillis 16GSR933 Shewanella spp. GS15P402 Eikenella corrodens www.cdc.go.kr 361
Table 4. Anaerobic bacteria collected for the species identification Strain No. Name Strain No. Name 2 Clostridium perfringens 16SS83 Lactobacillus rhamnosus 4 Ruminococcus gnavus 16SS84 Lactobacillus casei 15GSP201 Actinomyces neuii 1 Bacteroides fragilis 5 Actinomyces europaeus 3 Bacteroides thetaiotaomicron 6 Actinomyces odontolyticus 4 Bacteroides ovatus 15GSB2549 Actinomyces odontolyticus 5 Bacteroides uniformis 7 Eggerthella lenta 6 Bacteroides vulgatus 8 Lactobacillus casei 14-B Bacteroides faecis [9] 9 Lactobacillus gasseri 14-C Bacteroides intestinalis 9-A Bifidobacterium pseudocatenulatum 14-A Bacteroides salyersiae 10 Propionibacterium acnes GS14B3015 Bacteroides massiliensis 10-A Finegoldia magna GS14C211 Bacteroides cellulosilyticus 12 Parvimonas micra 16SS70 Bacteroides cellulosilyticus 12-A Anaerococcus vaginalis 16SS71 Bacteroides faecis 14 Peptostreptococcus anaerobium 16SS72 Bacteroides intestinalis 16 Peptoniphilus indolicus 16SS73 Bacteroides saalyersiae 17 Peptoniphilus rhinitidis 7 Parabacteroides distasonis 14GSB22-2 Flavonifractor plautii 8 Prevotella intermedia 15GSB2059 Clostridium subterminale 9 Fusobacterium nucleatum 16GSC61 Atopobium parvulum 10 Fusobacterium mortiferum 16SS77 Porphyromonas pogonae 11 Fusobacterium varium 16SS78 Alloscardovia omnicolens 15 Leptotrichia trevisanii 16SS79 Clostridium innocuum (vanb positive) 3 Dialister pneumosintes 16SS80 Propionibacterium avidum 16SS69 Alistipes onderdonkii 16SS81 Propionimicrobium lymphophilum 16SS74 Butyricimonas spp. 16SS82 Slackia exigua 16SS75 Odoribacter splanchnicus 균주별로디스크확산법시험 50s회를반복하여참조범위를설정하였다 ( 평균값 ± 2SD) (Table 5). 독소유전자용참조균주는 bft-1, bft-2 그리고 bft-3를독소유전자를가지고있는 Bacteroides fragilis와 sea, sec, seg, seh, sei, tst 등의독소유전자를가진 Staphylococcus aureus를포함하였다. 역학조사용참조균주는 Clostridium difficile 중임상적으로중요한 ribotype인 159, 106, 078, 027, 084, 085, 010를포함하는 14균주와다제내성과연관된 sequence type (ST) 1249, 454, 701, 739의 Acinetobacter baumannii 를 포함하였다. 내성기전시험용참조균주는그람양성세균에서 vancomycin 내성과관련된 vana 유전자와그람음성세균에서중요한내성과연관된 ESBL 유전자와 carbapenemase 유전자을가진 33주를분석하여기탁하였다. 그리고내성기전및역학조사용참조균주로 118주를확보하였는데균종별로는 A. baumannii 59주, P. aeruginosa 32주, E. coli 10주, K. pneumoniae 4주및 E. faecium이 13주였다. A. baumannii 일부는 PCR mapping으로 OXA-23 유전자를지닌 transposon을규명하였고, colistin 내성 www.cdc.go.kr 362
Table 5. Reference strains for the antimicrobial susceptibility testing Strain No. Name Characteristic Stain No. Name Characteristic ISM201312* Acinetobacter baumannii Susceptible to all antibiotics tested 03-10-P113 Bacteroides fragilis Resistant to carbapenem CS201310* Acinetobacter baumannii Resistant to carbapenem 12 Neisseria gonorrhoeae Susceptible to all antibiotics tested GS2011R111* Acinetobacter baumannii Resistant to carbapenem and colistin 13 Neisseria gonorrhoeae Penicillinase production PSB200807* Pseudomonas aeruginosa Susceptible to all antibiotics tested 14 Neisseria gonorrhoeae High level resistant to tetracycline BDC200843* Pseudomonas aeruginosa Resistant to carbapenem 15 Neisseria gonorrhoeae Non-susceptible to cefixime and cefpodoxime AS201312* Escherichia coli Susceptible to all antibiotics tested 15-A Neisseria gonorrhoeae Resistant to Cefixime GS2013U443* Escherichia coli Resistant to carbapenem (KPC-2) 16 Neisseria meningitidis Susceptible to all antibiotics tested KSS201302* Escherichia coli Resistant to carbapenem (OXA-232) 17 Campylobacter jejuni Susceptible to all antibiotics tested KS201308* Klebsiella pneumoniae Susceptible to all antibiotics tested 18 Porphyromonas sp. Susceptible to all antibiotics tested MP014* Klebsiella pneumoniae Resistant to carbapenem (KPC-2) 11 Finegoldia magna Resistant to moxacillin KSS201301* Klebsiella pneumoniae Resistant to carbapenem (OXA-232) 11-A Finegoldia magna Resistant to clindamycin YN1312* Staphylococcus aureus Susceptible to all antibiotics tested 13 Anaerococcus vaginalis Resistant to clindamycin EUIM1304* Staphylococcus aureus Susceptible to methicillin 13-A Peptostreptococcus anaerobius Resistant to penicillin SB1311* Staphylococcus aureus Resistant to methicillin GS15R637 Streptococcus pneumoniae Susceptible to penicillin SS1311* Enterococcus faecalis Susceptible to all antibiotics tested SS15169 Streptococcus pneumoniae Resistant to penicillin GS1305* Enterococcus faecium High level resistant to aminoglycoside INHA1404 Enterococcus faecium High level resistant to Gentamicin KM1409* Enterococcus faecium Resistant to vancomycin 15USP3 Pseudomonas aeruginosa Susceptible to all antibiotics tested WK1306* Enterococcus faecium Susceptible to all antibiotics tested 15EUIME1 Escherichia coli Susceptible to all antibiotics tested 2012-5-B6158 Bacteroides fragilis Susceptible to all antibiotics tested 15KMS15 Staphylococcus aureus Susceptible to all antibiotics tested 2010-3-P152 Bacteroides fragilis Resistant to carbapenem 15IH-EFM5 Enterococcus faecium Susceptible to all antibiotics tested * These 18 strains are reference strains for antimicrobial susceptibility testing with reference interval of zone diameter. (mean ± 2 SD). www.cdc.go.kr 363
A. baumannii 는 pmrcab 유전자및 LPS 유전자의염기서열을 분석하였다. P. aeruginosa 일부는전유전체염기서열분석 (whole 관련된병원체균주를지속적으로확보하고자원화할수있는 기반을마련하는것이필요할것이다. genome sequencing, WGS) 으로 carbapenemase 유전자를지닌 genomic island (PAGI) 를규명하였다. K. pneumoniae 일부는 WGS로 KPC 유전자를지닌 plasmid를규명하였고, Enterococci 일부는 vana operon과 plasmid 구조를규명하였다 (Data not shown). 세균의항균제내성은계속확산되고있으며, 특히그람음성세균에서는내성기전의확산이 clone 에의한경우가많다. OXA-23 을생성하는 A. baumannii CC92, metallo-β-lactamase 생성 Pseudomonas aeruginosa ST235, CTX-M-15 ESBL 생성 E. coli ST131 등이대표적인예이다. 그리고이들유행내성 clone 은계속바뀌는경향이있어그특성을분석하여자원은행에자원화하는노력이필요하다. 본연구는특성이분석된균주를 NCCP에공급하여 NCCP의자원을확대하고수요에맞는균주를갖추는것을목표로진행하였다. 균종동정용으로분석된 193주는임상적으로중요한균종이거나, 분리가드문균종이포함되었으며, 앞으로 matrix-associated laser desorption/ionization-time of flight mass spectrometry(maldi-tof MS) 의동정용 database 구축에도유용할것으로생각된다. 항균제감수성시험용참조균주역시임상미생물검사실에서항균제감수성내부정도관리용으로그유용성이클것으로예상되며, 독소유전자시험용, 역학조사용, 내성기전조사용으로분석된균주들역시각각의시험에서양성참조균주로사용될수있을것으로여겨진다. 맺음말 임상미생물검사실및교육기관에서참조균주에대한수요는많이증가하였지만, 특성이분석된세균자원은행은부족한상황이다. 본연구를통하여기탁된총 416주는 NCCP 병원체자원의확대에일조하였다고여겨진다. 뿐만아니라지속적으로 참고문헌 1. Hong SG, Yong D, Lee K, Kim EC, Lee WK, Jeong SH, et al. Antimicrobial resistance of clinically important bacteria isolated from hospitals located in representative provinces of Korea. Korean J Clin Microbiol 2003;6:29-36. 2. Lee K, Lee MA, Lee CH, Lee J, Roh KH, Kim S, et al. Increase of ceftazidime- and fluoroquinolone-resistant Klebsiella pneumoniae and imipenem-resistant Acinetobacter spp. in Korea: analysis of KONSAR study data from 2005 and 2007. Yonsei Med J 2010;51:901-11. 3. Shibayama K, Lee H, Kim S. Comparison of antibiotic resistance rate of medically important microorganisms between Japan and Korea. Ann Clin Microbiol 2015;18:111-8. 4. World Health Organization; Global Antimicrobial Resistance Surveillance System.(http://www.who.int/antimicrobial-resistance/ global-action-plan/surveillance/glass/en/) 5. Ha J, Kim KH, Kim JO, Hong J, Jeong SH and Lee K. Bacteraemia caused by Kytococcus schroeteri in a pneumonia patient. J Med Microb Diagn 2015;4:4. 6. Kim D, Hong SK, Seo YH, Kim MS, Kim HS, Yong D, et al. Two non-otic cases of POM-1 metallo-β-lactamase-producing Pseudomonas otitidis infection: Necrotizing fasciitis and panperitonitis.j Glob Antimicrob Resist. 2016;7:157-158. 7. Kwon SS, Kim JO, Kim KH, Jeong SH and Lee K. Persistent Bordetella petrii infection related to bone fractures. Ann Lab Med. 2016;36:70-72. 8. Kim DK, Hong SK, Kim M, Ahn JY, Yong D and Lee K. Campylobacter hyointestinalis isolated from a human stool specimen. Ann Lab Med. 2015;35:657-659. 9. Lee Y, Kim HS, Yong D, Jeong SH, Lee K and Chong Y. Bacteroides faecis and Bacteroides intestinalis recovered from clinical specimens of human intestinal origin. Yonsei Med J. 2015;56:292-4. 이글은 2014~2016년질병관리본부용역과제인 특성화세균자원은행 결과보고서를일부요약정리한내용입니다. 늘어나고있는항균제내성에대비하기위해서이와관련된 참조균주역시필요성이점점증가하고있어, 앞으로내성기전과 www.cdc.go.kr 364