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1 Korean J Clin Microbiol Vol. 12, No. 3, September, 2009 Metallo-β-lactamase Producing Gram-negative Bacilli Dongeun Yong Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea Among gram-negative bacteria, rate of antibiotic resistance has been increasing. As a result, carbapenem is now considered as a last resort of therapeutic regimens for gram-negative bacterial infections. The choice of antibiotics has been impeded by the spread of organisms producing metallo-β-lactamases (MBL), which can confer resistance to nearly all β-lactams. MBLs have extremely diverse structures and are carried by various organisms including human pathogens. This review will focus on the classification and current status of MBL reported in Korea. (Korean J Clin Microbiol 2009;12: ) Key Words: Metallo-β-lactamase, Gram-negative bacilli 서 론 분 류 효소중에는활성을나타내기위하여다른효소혹은금속이온등의보조인자를필요로하는효소가있다. 이들중금속이온은효소, 기질의복합체혹은효소단백질구조안정화에관여한다 [1,2]. 이러한 metalloenzyme에는 alcohol dehydrogenase (Zn 2+ ), phosphotransferase (Mg 2+ ) 및 cytochrome oxidase (Cu 2+ ) 등이있다. 최근항균제내성연구분야에서큰관심을끌고있는 carbapenem 분해 β-lactamase에는 Ambler class A 및 class D 에속하는 serine β-lactamase 와 Ambler class B, 즉 metallo-β-lactamase (MBL) 가있다. Class A serine carbapenemase는 clavulanic acid에의해그활성이저해되며그종류로는 NMC, IMI, SME, KPC 및 GES가있다 [3-8]. 이들은 carbapenem 고도내성과연관성이높지는않은것으로알려져있다 [9]. Class D serine carbapenemase는주로 Acinetobacter baumannii에서생성되는 OXA형효소중일부가해당된다. 그러나 carbapenem 분해능은높지않으며 clavulanic acid에의해서부분억제된다 [10]. 본종설에서다룰 MBL은효소활성에 Zn 2+ 가필요한 carbapenemase이다. Received 8 June, 2009, Revised 19 August, 2009 Accepted 25 August, 2009 Correspondence: Dongeun Yong, Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, 250, Seongsan-ro, Seodaemun-gu, Seoul , Korea. (Tel) , (Fax) , ( ) deyong@yuhs.ac 1997년 Rasmussen과 Bush[11] 는 MBL을 imipenem 가수분해능, EDTA 억제정도, serine β-lactamase 억제제에의한억제정도에따라서 3개 subgroup (B1, B2 및 B3) 로나누었다 (Table 1). 한편 Walsh 등 [12] 은염색체에존재하여자연내성으로판단되는 MBL과 integron과같은이동성유전단위에존재하여서다른세균에서유래된것으로판단되는 MBL로분류하였다. 1. 염색체성 MBL 일부환경분리세균이염색체에 MBL 유전자를가지고있는이유는아직밝혀지지않았다. 과거부터 β-lactam 항균제에노출되어왔거나혹은 MBL이우리가모르는대사과정의일부분에기여하는것이아닌지추측되고있다 [12]. 이들세균은 B. anthracis, S. maltophilia, P. aeruginosa 등을제외하고는감염을일으키는일이드물다. 각 subgroup별균종별염색체성 MBL의종류는 Table 1에상술하였다. 2. 획득성 MBL 현재까지 MBL 획득에관여하는것으로알려진기전은 plasmid[14], integron[15-17], transposon[18], insertion sequence common region[19] 이있다. 많은 MBL 유전자가 kb 크기의 plasmid에존재하는것으로보고되었다. 그러나미국에서분리된 VIM-7 유전자는 24 kb 크기의접합성 plasmid에존재함이보고되기도하였다 [20]. 이러한 plasmid 등은동시에많은내성유전자를운반할수있으므로다양한항균제가 MBL의선택압력으로작용할수있음을염려하게한다. IMP, VIM 및 GIM 유전자는대부분 class 1 integron 안에 103

2 104 Korean J Clin Microbiol 2009;12(3): Table 1. Classification of MBLs[1,12,13] Subgroup Organism Enzyme B1 (chromosomal) Bacillus cereus BCII-5/B/6, BCII-569/H Bacillus anthracis Bla2 Chryseobacterium meningosepticum BlaB, BlaB2-8 Chryseobacterium gleum CGB-1 Myroides odoratus TUS-1 Myroides odoratimimus MUS-1 Flavobacterium johnsoniae JOHN-1 Bacteroides fragilis CfiA/CcrA B1 (transferable) Pseudomonas spp. IMP, VIM, SPM-1, GIM-1 Acinetobacter spp. IMP, VIM, SIM-1 Escherichia coli VIM, NDM-1 Klebsiella pneumoniae IMP, VIM, NDM-1 Citrobacter freundii IMP, VIM, KHM-1 Enterobacter aerogenes VIM Enterobacter cloacae IMP, VIM Serratia marcescens IMP, VIM Proteus mirabilis VIM Providencia stuartii VIM Shigella flexneri IMP Achromobacter xylosoxidans IMP, VIM Alcaligenes spp. IMP B2 (chromosomal) Aeromonas hydrophilia CphA Aeromonas veronii ImiS, AsbM1 Serratia fonticola SFH-1 B3 (chromosomal) Caulobacter crescentus Mb11B, CAU-1 Janthinobacterium lividium THIN-B Legionella gormanii FEZ-1 Chryseobacterium meningosepticum GOB-1-7 Stenotrophomonas maltophilia L1a, L1-BlaS, L1c, L1d, L1e B3 (transferable) Pseudomonas aeruginosa AIM-1 gene cassette로존재한다. 2003년이태리에서분리된 IMP-13 유전자양성 P. aeruginosa에서이내성유전자를포함한 class 1 integron이 Tn5051형 transposon에존재하며, 폴란드 P. aeruginosa에서도같은형의 transposon에 IMP-13과 VIM-2 유전자가존재함이보고되었다 [18]. 이는 integron뿐아니라 transposon이 MBL 유전자전파에관여함을보인것이라하겠다. SPM-1은브라질 P. aeruginosa에서발견되었는데이유전자의바로곁에 ISCR4가존재하여유전자전파에관여하는것으로추정되었다 [19]. 1) IMP 형 MBL (Table 2): 1988년최초의전이성 MBL인 IMP-1이일본 P. aeruginosa에서보고되었다 [14]. 그유전자는접합성 plasmid와 class 1 integron에위치하였다. 3년후다른병원에서분리된 S. marcescens에서도 IMP-1 유전자가발견되었다 [21]. 2년후다른병원에서분리된 S. marcescens의 class 3 integron 안에서 IMP-1 유전자가보고되었다 [22]. 이는향후 carbapenem 선택압력이있는조건에서 MBL 생성세균이증가할수있음을시사하는보고이다. 유럽에서는 1997년이태리에서 IMP-2가 [23], 1998년포르투갈에서 IMP-5가보고되었다 [24]. 아시아에서는 1994년홍콩에서 IMP-4를생성하는 Acinetobacter를보고하였고 [25] 국내에서는 2000년분리 Acinetobacter spp. 및 P. aeruginosa에서 IMP-1을보고하였다 [26]. 2009년 5월 25일까지세계적으로 26종류의 IMP형 MBL 이보고되었다. gov/genbank/index.htm/[online]]. 2) VIM형 MBL (Table 3): 세계적으로매우흔한 MBL이다. VIM-1은 1997년이태리에서분리된 P. aeruginosa에서발견되었다 [16]. 같은병원에서분리된 P. putida, Achromobacter xylosoxidans에서도연이어서 VIM-1이보고되었다 [28,29]. VIM-2 는 1997년프랑스에서분리된 P. aeruginosa에서처음발견되었다 [30]. 이는 45 kb의비접합성 plasmid에위치하였고 integron에 gene cassette로존재하였다. 1995년국내에서분리된 5주의 P. aeruginosa에서 VIM-2가규명되었다 [31]. 2009년 5월 25일까지세계적으로 22종류의 VIM형 MBL이보고되었다 ( gov/genbank/index.htm[online]). 3) SPM-1, GIM-1, KHM-1, NDM-1: SPM-1은 1997년브라질 P. aeruginosa에서발견되었고 ISCR4와연관되어있을뿐 transposon 및 integron과연관되었다는증거가없다 [32]. SPM-1 은활성부위 HFHLD 뒤에 24개의아미노산이삽입되어있는

3 Dongeun Yong : Metallo-β-lactamase Producing Gram-negative Bacilli 105 Table 2. IMP-type MBL[12,27] MBL Species Nation IMP-1 P. aeruginosa Japan, Korea, Brazil P. putida, P. fluorescens Japan, Singapore P. stutzeri Japan Acinetobacter spp. Japan, Korea, UK IMP-2 Acinetobacter spp. Japan, Italy P. aeruginosa Japan IMP-3 Shigella flexneri Japan IMP-4 A. baumannii Hong Kong C. freundii China, Australia P. aeruginosa China, Australia IMP-5 A. baumannii Portugal P. aeruginosa Korea IMP-6 A. baumannii Brazil S. marcescens Japan P. aeruginosa Korea IMP-7 P. aeruginosa Malaysia, Canada IMP-8 E. cloacae, K. pneumoniae Taiwan IMP-9 P. aeruginosa China IMP-10 P. aeruginosa, A. xylosoxidans Japan IMP-11 P. aeruginosa, A. baumannii Japan IMP-12 P. putida Italy IMP-13 P. aeruginosa Italy IMP-14, 15 P. aeruginosa Thailand IMP-16 P. aeruginosa Brazil IMP-17, Assigned Assigned -23, -24 IMP-18 P. aeruginosa USA IMP-19 Aeromonas punctata France IMP-20, -21 P. aeruginosa Japan IMP-22 P. fluorescens Italy IMP-25 P. aeruginosa China IMP-26 P. aeruginosa Korea 것이 IMP형및 VIM형 MBL과상이하다. GIM-1은 2002년독일에서분리된 P. aeruginosa에서규명되었다 [15]. IMP형 MBL과약 43% 의상동성을보이고 45 kb의 plasmid와 class 1 integron에들어있었다. KHM-1은 1997년일본 C. freundii에서발견되었다 [13]. 200 kb의접합성 plasmid에존재하였고 IMP-1과 59% 상동성을보였다. Integron 등유전자전이구조들과연관된증거는찾을수없었다. NDM-1은스웨덴거주인도환자의요검체에서분리된 K. pneumoniae에서규명되었고접합에의하여내성이전이되었다. Integron과연관되었다는증거는찾을수없었다. 우리나라에서의보고 1995년국내일개대학병원에서분리된 P. aeruginosa에서 VIM-2 유전자가보고되었고 1997년분리된 imipenem 내성 P. aeruginosa 중 9% 가 VIM-2를생성하였다. 이들 VIM-2 유전자는 class 1 integron에존재하였고일부균주에서는접합에의하 Table 3. VIM-type MBL[12,27] MBL Species Nation VIM-1 P. aeruginosa, Italy P. putida A. xylosoxidans Italy E. coli Greece, France K. pneumoniae Italy VIM-2 A. baumannii, Korea Acinetobacter spp. A. xylosoxidans Japan P. aeruginosa Korea, Japan, Italy, Greece, France, Spain, Portugal, Poland, Croatia, USA, Argentina, Chile, Venezuela P. putida Korea, Japan P. fluorescens Chile P. stutzeri Taiwan C. freundii Taiwan E. cloacae, Korea S. marcescens VIM-3 P. aeruginosa Taiwan VIM-4 P. aeruginosa Greece, Poland, Sweden E. cloacae, Italy K. pneumoniae VIM-5 P. aeruginosa, Turkey K. pneumoniae VIM-6 P. putida Singapore VIM-7 P. aeruginosa USA VIM-8 P. aeruginosa Chile VIM-9, 10 P. aeruginosa UK VIM-11 P. aeruginosa Italy, Argentina VIM-12 K. pneumoniae Greece VIM-13 P. aeruginosa Spain, Korea VIM-14 P. aeruginosa Italy, Spain VIM-15, -16 P. aeruginosa Germany VIM-17 P. aeruginosa Greece VIM-18 P. aeruginosa India VIM-19, Assigned Assigned 20, 21, 22 여내성을전달하였으나플라즈미드에존재하는지에대하여는확실하지않다 [31]. 1998년부터 1999년사이에국내일개대학병원에서분리된 28주의 carbapenem 내성 Acinetobacter spp. 중 VIM-2 와 IMP-1 이검출되었다 [33]. Oh 등은 imipenem 혹은 ceftazidime 비감수성 P. aeruginosa 와 A. baumannii 중 VIM-2 혹은 IMP-1 을검출하여보고하였다 [34]. 전국 KONSAR 참여 28개병원에서 2003년수집된 imipenem 비감수성 Pseudomonas spp. 중 11.1% 가 bla VIM-2 양성이었다 [26]. 또한수집된 imipenem 비감수성 Acinetobacter spp. 중 15.1% 가 bla VIM-2 와 bla IMP-1 양성이었다 [35]. 대구에서분리된 P. aeruginosa 2주에서 IMP-5, 5주에서 IMP-6, Acinetobacter spp. 1주에서 VIM-13 이보고되었는데이는국내에서분리되는 MBL이다양해지고있다는증거이다 [36]. 2005년분리된 A. baumannii에서는 SIM-1과 VIM-2가동시에생성됨이보고되기도하였다

4 106 Korean J Clin Microbiol 2009;12(3): [unpublished data]. Pseudomonas spp. 와 Acinetobacter spp. 외의세균에서도 MBL 생성이보고되었다. Imipenem 내성 S. marcescens 1주에서 class 1 integron에존재하는 VIM-2 유전자가보고되었고 [37] 그외에도국내에서분리된 K. pneumoniae, E. cloacae, Achromobacter xylosoxidans[38,39] 등에서 MBL 유전자가발견되었다. 임상적중요성 MBL을구성적으로생성하는 Janthinobacterium lividium, Chromobacter vibrioides 및 Bradyrhizobium japonicum 등은병독성이낮다. 따라서이들균종의 MBL은임상적인중요성이낮다고판단된다. 그러나 S. maltophilia, B. fragilis, Aeromonas spp., C. meningosepticum 및 C. indologenes 같은비교적흔하게분리되는기회감염균에서생성되는 MBL은임상적으로중요하다고판단된다 [1]. S. maltophilia는면역저하환자에서균혈증, 심내막염, 피부및연조직감염의원인균이며 L1 (MBL) 과 L2 (serine β-lactamase) 를생성하여항균제에내성을나타낸다 [40]. B. fragilis는임상적으로중요한혐기성세균감염의가장흔한원인균이다. Insertion sequence 에의한활성화로 CfiA/CcrA를생성하며 carbapenem을비롯한대부분의 β-lactam 제제에내성을나타낸다 [41]. CfiA/CcrA는일본에서 % 로보고되었고 [42] 프랑스에서 2.4%[43], 우리나라에서는 B. fragilis 임상분리주의 4% 가 CfiA/CcrA 유전자를보유하였고그중 27% 가 insertion sequence에의한활성화로 CfiA/CcrA 를생성하였다. Aeromonas는위장관감염이외에도창상감염, 패혈증등을일으킨다. A. hydrophila, A. salmonicida, A. veronii 및 A. jandaei에서 MBL인 CphA/Imi를생성하는것으로보고되었다 [44]. 특이한것은이들 MBL 생성 Aeromonas가통상감수성시험에서 carbapenem에내성을보이지않는다는것이다. 그러나접종균수를증가시키면내성을보이므로 Aeromonas 감염시 carbapenem을권장하지않는것이좋다는의견도있다 [45]. C. meningosepticum은포도당비발효그람음성간균인데신생아뇌수막염등의기회감염균이며, MBL 중 BlaB와 GOB 를생성한다 [46,47]. C. indologenes도포도당비발효그람음성간균이나임상적인중요성은비교적낮으며 MBL인 IND를생성한다 [48]. MBL 중임상적으로염려되는것은 IMP와 VIM 등이동성유전자에의한효소들이다. 1990년대에 P. aeruginosa, Acinetobacter spp. 에서처음으로보고된이후다른포도당비발효그람음성막대균과 Enterobacteriaceae에서도발견되었다. 이들에의한원내감염집단발생이보고되고있어서전파방지및감염관리를위한노력이계속요구된다. 우리나라에서분리된 VIM-2 생성 P. aeruginosa에서 imipenem MIC 범위는 8 128μg/mL이어서감수성은없었으나중간이 6.7% 였다 [31,35]. VIM-2 혹은 IMP-1 생성 Acinetobacter spp. 에서 imipenem MIC 범위는각각 4 32μg/mL 및 8 32 μg/ml로 P. aeruginosa보다낮았고 VIM-2 생성 Acinetobacter spp. 의 6.3% 는 imipenem 감수성이었다. Enterobacteriaceae 균종은 MBL을생성하더라도 carbapenem MIC가 Pseudomonas spp. 혹은 Acinetobacter spp. 만큼높지않다 [12]. S. marcescens는 32μg/mL 혹은 64μg/mL 이상의 imipenem MIC를보였으나 E. coli, K. pneumoniae, Shigella flexneri, E. cloacae 및 Citrobacter spp. 들에서 MIC는감수성기준보다낮은경우가보고되었다 [27]. 이는통상검사시 MBL 생성균을선별하 Table 4. Methods for MBL detection[12,27] Methods Materials Advantages Disadvantage Phenotype Double disk synergy CAZ or IPM Easy Need standardization, vague to read plus MPA or SMA IPM + EDTA/SMA Easy Need standardization, vague to read Disk potentiation IMP vs. IMP+EDTA Easy Need standardization, false negative by EDTA Microdilution IPM, EDTA, 1,10-phenanthroline Easy Labor intensive, false negative to IPM susceptible MBL producer E test IPM, EDTA Easy False negative to IPM susceptible MBL Carbapenem hydrolysis Spectrophotometer Applicable to all MBL Labor intensive, vague to read detection Standard methods Genotype PCR Primer, thermocycler Easy, MBL type specific Primer design May not applicable to New MBL type DNA probe Probe, hybridizer Group specific probe Cloning, sequencing Nucleotide sequencer Applicable to new MBL type Labor intensive

5 Dongeun Yong : Metallo-β-lactamase Producing Gram-negative Bacilli 107 지못할수도있다는사실을시사한다. 검사실의검출 2009년 CLSI에서 carbapenemase KPC 생성세균검출을위하여 modified Hodge test를시행할것을권장하였다 [49]. 그러나이방법으로다른 MBL 생성도검출할수있는지에대하여는언급하지않았다. 현재 MBL 생성균의검출방법은여러가지가제안되었다. 크게표현형과유전자형을검출하는방법으로나눌수있다 [12]. 임상미생물검사실에서시행하기에가장간편한방법은 double disk synergy 법이다 (Table 4). Imipenem 대신 ceftazidime 디스크를사용하도록권하는보고도있으나 [22] AmpC 다량생성균인경우위음성결과를보일수있음을주의해야한다. Double disk synergy 시험에사용하는 MBL 저해제중 EDTA 는 MBL 비생성균주의증식도억제하는경우가있어서위양성의우려가있다 [50]. Mercaptopropionic acid가권장되기도하였으나 [22] 악취와독성이문제되므로 sodium mercaptoacetic acid가사용되기도한다. EDTA 의비특이적균독성을줄이기위하여 EDTA 750μg과 sodium mercaptoacetic acid 2 mg 을함께점적한디스크를사용하기도하였다 [51]. Imipenem- Hodge 시험으로 serine carbapenemase도선별할수있으므로 MBL 확인시험전에시행하는것이권장되기도한다. E test MBL strip은사용하기는쉬우나비용이많이소요된다 [52]. PCR을이용한방법은민감도는높으나새로운 MBL은검출할수없고일반임상미생물검사실에서시행하기에는비용과노력이많이소요되는단점이있다 [53]. 치료 MBL 생성균감염증치료에대한연구는매우제한되어있다. 따라서적절한약제선택에대한정설이없는상태이다. 동물에서 VIM-2 생성 P. aeruginosa 폐렴을고농도의 aztreonam 으로치료하여균수를감소시켰다는보고가있다 [54]. 또한일부 carbapenem 제제도효과가있었으나실제임상에서이와같은제제를사용할수있을지에대하여는추가연구가필요하다. VIM-1 생성 K. pneumoniae에대한 imipenem 생체내항균력이호중구가감소된생쥐의대퇴감염모델에서보고된바있다 [55]. 시험균주는 imipenem MIC가 2, 4, 32μg/mL인 VIM-1 생성균주와, VIM-1 음성이면서 imipenem MIC가 0.125μg/mL 인균주이었다. Imipenem 치료후세균수의감소는 VIM-1 음성세균이가장현저하였고 VIM-1 을생성하는균중 imipenem 에감수성인세균은중등도의감소를보였다. 이는 imipenem 투여가 imipenem에대한 MIC가낮은순서대로유효할것임을 시사하는결과이다 년캐나다에서 MBL 생성 P. aeruginosa 감염에대한치료성적이보고되었다 [56]. 균주들은 VIM-2 생성 53주, IMP-7 생성 1주및미상 2주이었다. 전체환자 56명중 29% 가사망하였는데사망의중요인자는부적절한항균제치료, 면역억제및균혈증이었다. Piperacillin-tazobactam에내성인균주는없었으나이약제로치료한사망환자가흔하였다. Aztreonam의효과는다른항균제와유사하였고감수성을보인항균제를병합하는경우좋은생존율을보였다. MBL 억제제와의병용가능성도현재는낮다 [12]. 현재사용가능한 MBL 억제제가없고또한이약제의개발이요원한실정이다. 이는 MBL은활성부위구조가다양하여서모든 MBL 에공통적으로작용하는억제제를구하기가어렵고사람에도 MBL과유사한활성부위를가지는효소가많기때문이다. Aminoglycoside와의병용투여효과에대한연구도 MBL 생성균주가이들약제에동시내성을보이는경향때문에제한되어있다. 현재선택가능한치료제로는 polymyxin 계열의약제가있는데, polymyxin이알려진것처럼독성이강하지않았다는보고가있어다행이다 [57]. 참고문헌 1. Rossolini GM and Docquier JD. Class B β-lactamases. In: Bonomo RA and Tomasky ME, eds. Enzyme-mediated Resistance to Antibiotics: Mechanisms, Dissemination, and Prospects for Inhibition. Washington, DC.; ASM Press, 2007: Kim YS. Biochemistry. 1st ed. Seoul; Shinkwang, 1987;7: Nordmann P, Mariotte S, Naas T, Labia R, Nicolas MH. Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli. Antimicrob Agents Chemother 1993;37: Poirel L, Weldhagen GF, Naas T, De Champs C, Dove MG, Nordmann P. GES-2, a class A beta-lactamase from Pseudomonas aeruginosa with increased hydrolysis of imipenem. Antimicrob Agents Chemother 2001;45: Queenan AM, Torres-Viera C, Gold HS, Carmeli Y, Eliopoulos GM, Moellering RC Jr, et al. SME-type carbapenem-hydrolyzing class A beta-lactamases from geographically diverse Serratia marcescens strains. Antimicrob Agents Chemother 2000;44: Rasmussen BA, Bush K, Keeney D, Yang Y, Hare R, O'Gara C, et al. Characterization of IMI-1 beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae. Antimicrob Agents Chemother 1996;40: Yang YJ, Wu PJ, Livermore DM. Biochemical characterization of a beta-lactamase that hydrolyzes penems and carbapenems from two Serratia marcescens isolates. Antimicrob Agents Chemother 1990;34: Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward CD, et al. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother 2001;45: Nordmann P and Poirel L. Emerging carbapenemases in Gram-

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