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1 대한임상검사학회지 : 38 권제 3 호, , 2006 Metallo-β-lactamase 생성 Pseudomonas aeruginosa의시험관내항균제병합요법에대한연구 분당제생병원진단검사의학과 홍승복 In vitro Antimicrobial Combination Therapy in Metallo-β-lactamase Producing Pseudomonas aeruginosa Seung-Bok Hong Department of Laboratory Medicine, Pundang Jesaeng General Hospital, Seongnam , Korea Metallo-β-lactamase (MBL) can hydrolyze all β-lactams except monobactams and frequently coexists with various antibiotic resistance genes such as aminoglycoside resistance, sulfonamide resistance gene, etc. Therefore, the effective antibiotics against infections by these bacteria are markedly limited or can't even be found. We tried to search in-vitro antimicrobial combinations with synergistic effects for a VIM-2 type MBL producing Pseudomonas aeruginosa, isolated from clinical specimen. On the selection of antibiotic combinations with synergistic effects, we performed a one disk synergy test, modified Pestel's method, in agar without aztreonam (AZT). The bacteriostatic synergistic effects of this tests were scored as S 1 (by susceptibility pattern in agar without antibiotics), S 2 (by the change of susceptibility in agar with or without antibiotics) and S 3 (S 1 + S 2 ) and was classified into weak (1 point), moderate (2 points) and strong (3 points) by S 3 score. Subsequently, we carried out the time-killing curve for the antibiotic combinations with the strong synergistic bacteriostatic effect. One VIM-2 type MBL producing P. aeruginosa confirmed by the PCR showed all resistance against all β-lactams except AZT, aminoglycoside and ciprofloxacin. In the one disk synergy test, this isolate showed a strong bacteriostatic synergistic effect for the antibiotic combination of AZT and piperacillin-tazobactam (PIP-TZP) or AZT and amikacin (AN). On the time-killing curve after six hours of incubation, the colony forming units (CFUs/mL) of this bacteria in the medium broth with both combination antibiotics were decreased to 1/18.7, 1/17.1 of the least CFUs of each single antibiotics. The triple antibiotic combination therapy including AZT, PIP-TZP and AN was shown to be significantly synergistic after 8 hrs of exposure. In a VIM-2 MBL producing P. aeruginosa with susceptibility for AZT, the triple antibiotic combination therapy including AZT, PIP-TZP and AN may be considered as an alternative antibiotics modality against the infection by some MBL type. But the antimicrobial combination therapy for many more MBL producing isolates is essential to know as soon as possible for the selection of effective treatment against the infection by this bacteria. Key words : Metallo-β-lactamase(MBL), Antimicrobial combination therapy, One disk synergy test, Time-killing curve 1) 교신저자 : 홍승복, ( 우 ) 경기도성남시분당구서현동 대진의료재단분당제생병원진단검사의학과 Tel : sbhong8646@hanmail.net 166

I. 서론 확인하고자하였다. Metallo-β-lactamase(MBL) 는그람음성세균이생성하는 β-lactamase 중활성범위가가장넓어서 carbapenem 을포함하여대부분의 β-lactam 항균제를가수분해하므로 MBL 유전자의획득은이들균에의한감염의치료에커다란문제를야기할것으로보고되었다 (Nordamann와 Poirel, 2002).

2 I. 서론 확인하고자하였다. Metallo-β-lactamase(MBL) 는그람음성세균이생성하는 β-lactamase 중활성범위가가장넓어서 carbapenem 을포함하여대부분의 β-lactam 항균제를가수분해하므로 MBL 유전자의획득은이들균에의한감염의치료에커다란문제를야기할것으로보고되었다 (Nordamann와 Poirel, 2002). 최근에 Pseudomonas aeruginosa(watanabe 등, 1991) 와 Acinetobacter(Yum 등, 2002) 와같은포도당비발효그람음성세균과 Serratia marcescens(osano 등, 1994), Enterobacter cloacae(jeong 등, 2003) 등의장내세균에서 MBL의검출이증가되고있다. 현재까지전세계적으로 5종류의 MBL 유전형이분리되고있는데 (Riccio 등, 2000; Lauretti 등, 1999; Toleman 등, 2002; Castanheira 등, 2004; Lee 등, 2005) 국내에서는 VIM-2형과 IMP-1형이 Pseudomonas species 및 Acinetobacter species에서주로분리되고있으며 (Lee 등, 2003), 2005년세계에서최초로국내에서 SIM-1형 (Lee 등, 2005) 이새로이보고되었다. 현재 MBL 생성균에의한감염의치료제개발에대한연구가진행되고있으나 (Watanabe 등, 1995; Matsumura 등, 1997; Nagano 등, 1999) 임상적으로사용되기위해서는오랜시간이소요될것이다 (Gilpin 등, 1995; Payne, 1993; Walter 등, 1996). 따라서현재사용중인항균제중에서선택해야하며대안으로항균제병합요법이포함될수있으나아직까지 MBL 생성균에서항균제상승효과에대한연구는매우드물다. 항균제의병합은 i) 내성균주의발생억제 ii) 약물농도의감소에의한독성발생의감소 iii) 여러종류의균에의한감염 iv) 항균제상승효과의기대등의이유로이용된다. 항균제상승효과에대한시험은 checkerboard 및 time-killing curve에의한방법이잘알려져있는데, checkerboard 시험은다양한항균제조합에대한시험이가능하나재현성이부족하며, 정균 (bacteriostatic) 효과만볼수있는단점이있다 (Eliopoulos 등, 1996). Time-killing curve는항균제병합에의한살균 (bactericidal) 효과를반영하며 in vivo의결과와가장잘일치되나지루한방법등으로인한시간및노력이많이소요되며병합할수있는항균제의조합도제한될수밖에없다 (Eliopoulos 등, 1996). 이연구의목적은 MBL 생성 P. aeruginosa에서상승효과를보이는항균제조합을 one disk synergy 검사로선별하고선별된항균제조합을 time-killing curve를통하여 1. 대상균주 II. 재료및방법 PCR에의해 VIM-2형 MBL로확인된 P. aeruginosa CBU 01/11/569(Fig. 1) 를대상으로상승효과를보이는항균제조합을선별하였고선별된항균제조합에대한상승효과를 time-killing curve로확인하였다. 2. 항균제감수성검사 (1) 최소억제농도 (minimal inhibitory concentration, MIC) 측정 Imipenem(IMP, Merk/Sharp&Dohme, Rahway, N.J USA), meropenem(mep, Yuhanyanghang Pharmaceutical, Seoul, Korea), aztreonam(azt, BMS, Co, Princeton, N.J. USA), cefepime(fep, Bayer Korea, Seoul, Korea), ceftazidime(caz, Jeiljedang Pharmaceutical, Seoul, Korea), piperacillin(pip, Yuhanyanghang Pharmaceutical, Seoul, Korea), piperacillin-tazobactam(pip-tzp, Yuhanyanghang Pharmaceutical, Seoul, Korea), amikacin(an, Boryeng M 1 2 Fig. 1. Detection of amplified product of blavim-2 gene in 2% agarose gel electrophoresis. Lane M, DNA size marker (100 bp ladder); lane 1, VIM-2 producing P. aeruginosa (801 bp); lane 2, negative control. 167

3 Pharmaceutical, Seoul, Korea), ciprofloxacin(cip, Bayer Korea, Seoul, Korea) 등의최소억제농도 (MIC) 를측정하였는데, MIC은 NCCLS 지침 (NCCLS, 2000) 에따라미량액체배지희석법으로다음과같이시행하였다. 혈액한천에서하룻밤동안배양한집락을 McFarland 0.5 관 (10 8 CFU/mL) 에맞춘후다시 10배희석하여 ( CFU/mL) 미리준비한항균제 plate( 각각 100 μl) 에 5 ul씩을접종하여최종농도가 well 당 CFU가되게하였다. 이것을배양기에서 16~18시간배양하였다. 매실험마다정도관리를위하여 P. aeruginosa ATCC 27853를정도관리균주로사용하였다. 최소억제농도는균의성장이육안으로관찰되지않은최소농도로판정하였다. 3. 상승효과를보이는항균제의선별및 time-killing 시험 (1) 상승효과를보이는항균제의선별검사우선 MBL 생성 P. aeruginosa에대한최소억제농도가가장낮은 AZT(MIC; 8 μg/ml) 과여러가지 β-lactam 제그리고 aminoglycoside, CIP, vancomycin(va), tetracycline(te), trimethoprim-sulfamethoxazole(sxt) 의병합효과를디스크확산법으로선별하였다 (one disk synergy test). 상승효과에대한선별검사를위해 AZT의 sub-mic(1/2 MIC, 4 μg/ml) 배지를제조하였다. AZT 4 μg 배지에위에서 McFarland 0.5 관의탁도에맞춘균을접종한후위에서열거한항균제디스크를올려놓고 37 에서 16~18시간배양하였다. 이때동시에항균제를첨가하지않은 MH 배지에상기항균제디스크를올려놓고 AZT 4 μg 배지의억제대와비교하였다. 상승효과의판정은 Pestel 등 (1995) 의방법에따라상승효과를정량화하였다. 우선항균제를넣지않은배지 (S 1) 에서감수성 (S), 중간내성 (I), 저항성 (R) 에따라각각 2, 1, 0 점을부여하였다. 항균제를넣은배지에서억제대의직경이항균제를넣지않은배지의억제대직경과비교하여감수성양상이변할경우 (S2) 점수를차등화하여부여하였다. 즉 R I, I S의경우 2점, R S의경우 3점, S S이나억제대직경이증가한경우는 1점, 그이외에는 0점을부여하였다. 상승효과의최종판정은 S 1 과 S 2 의합 (S3) 으로판정하였는데, 1점의경우 weak, 2점, moderate, 3점, strong으로판정하였다. (2) Time-killing curve One disk synergy 검사에서상승효과가가장큰항균제조합을선택하여 time-killing curve를작성하였다. 우선 AZT과 PIP-TZP의병합에의한상승효과가가장컸기때문에선택하였고, AZT과 AN의병합은 synergy score는 weak 이었지만증가된억제대가 PIP-TZP 다음으로증가되었기때문에선택하였다. Time-killing curve를작성하기위하여 AZT의경우 1/2 MIC( 최종농도 4 μg/ml) 을선택하였고 PIP-TZP도 1/2 MIC(32 μg/ml) 을선택하였다. 그러나 AN의경우 MIC가 >256 μg/ml로 1/2 MIC(128 μg/ml) 는혈중범위에도달할수없는농도 ( 독성혈중농도 ) 였으므로치료적혈중농도를기준으로 16 μg/ml( 치료범위 ; 10~25 μg/ml, 독성범위 : 35 μg/ml, (Moyer와 Pippenger, 1994) 를선택하였다. 균접종농도는 MH 액체배지 7.6 ml에 McFarland 0.5 관 ( CFU/mL) 의균액 0.4 ml를넣어최종균농도가 CFU/mL가되게하였다. 이어서 0시간, 3시간, 6시간 24시간후에균의수를세었다. 균의수는 Eliopoulos 방법 (Eliopoulos 등, 1996) 대로시행하였는데, 각시간마다 0.5 ml를취하여 0.45% 생리식염수 4.5 ml 에섞어 10 1 부터 10 8 까지계단희석하여이중 25 μl를취하여 BAP에접종하였다. 균수의계산은희석배수 (10 n ) 40 균수로계산하였다. III. 결과 1. 상승효과를보이는항균제의선별검사 MBL 생성 P. aeruginosa에대해상승효과를보이는항균제를선별을위해 one disk synergy test를시행하였는데, P. aeruginosa CBU/01/11/569는시험한모든 β -lactam 항균제 (AZT(MIC; 8 μg/ml) 제외 ), aminoglycoside 그리고 CIP에내성을보여 (Table 1) AZT 함유배지를이용하여상승효과를보이는항균제를찾고자하였다. AZT 4 μg 함유배지에서 PIP-TZP는강한상승효과 (strong) 를보였다. AN, CAZ 및 FEP은각각 weak 및 moderate 상승효과를보였다 (Table 2). Carbapenem (IMP, MEM), aminoglycoside 항균제 (gentamicin, tobramycin), cephalosporin(cefazolin, cefuroxime, cefuroxime, cefotaxime), 168

4 Table 1. The minimal inhibitory concentration (MIC) of VIM-2 MBL producing P. aeruginosa Antibiotics MIC (μg/ml) Imipenem 256 Meropenem 64 Aztreonam 8 Cefepime 32 Ceftazidime 32 Piperacillin 128 Piperacillin-tazobactam 64/8 Amikacin >256 Ciprofloxacin 128 penicillin(penicillin G, ampicillin, oxacillin), SXT, VA, TE, clindamycin, erythromycin, chloramphenicol 등의항균제는모두상승효과가관찰되지않았다. 위의결과를바탕으로두가지항균제조합을선택하여 time-killing curve로상승효과를확인하고자하였다. 우선강한상승효과를보인 AZT과 PIP-TZP의조합을선택하였고, 두번째로미약한상승효과 (weak) 를보였지만의미있는억제대의증가 ( mm) 를보인 AZT과 AN 조합을선택하였다. 2. Time-killing 실험 AZT 와 PIP-TZP 의병합은항균제노출 6 시간후에 AZT( CFU/mL) 또는 PIP-TZP( CFU/mL) 의단독항균제투여보다균수가 1/18.7로감소하였다 ( CFU/mL). 그리고 AZT와 AN의병합에서도항균제노출 6시간후에 AZT 또는 AN ( CFU/mL) 의단독항균제에투여보다균수가 1/17.1로감소하였다 ( CFU/mL). 결국위두조합은의미있는상승효과가나타나지않아위세항균제의병합을시험하였다. 위세항균제를병합하였을때항균제노출 8시간에 AZT( CFU/mL), PIP-TZP ( CFU/mL) 및 AN ( CFU/mL) 의단독투여에비하여균수가 1/183.3로감소하여 ( CFU/mL) 의미있는상승효과를보였다 (Figs. 2, 3, 4). IV. 고찰 MBL을생성하는균에의한감염을치료하기위한항균제의선택은매우어렵다 (Payne, 1993). 현재 MBL에의해가수분해되지않은항균제의개발및임상시험이진행중에있지만 (Matsumura 등, 1997; Nagano 등, 1999) 이항균제를사용하려면오랜시간이필요할것이다. 따라서기존에사용되고있는항균제중에서선택하여야하며항균제의병합요법이하나의대안이될수있을것이다. 호중구감소증환자나면역이결핍된환자에서다제내성인 P. aeruginosa에의한감염에병합요법이종종이용되고있는데 (Dejongh 등, 1986), 이중 β-lactam 항균제와 aminoglycoside의병합에의한상승효과가잘알려져있다 (Dejongh 등, 1986; Miller 등, 1986). 그러나아직까지 MBL를생성하는 P. aeruginosa에서병합요법의효과에대한보고는극히드물다. 본연구에서는 MBL을생 Table 2. Screening for the antibiotics with the synergistic bacteriostatic effect in combination in aztreonam in VIM-2 MBL producing P. aeruginosa Agar with 0.5 MIC of aztreonam Antibiotic disc PIP-TZP CAZ FEP AN Inhibition zone 19.0 mm 16.0 mm 15.5 mm 13.5 mm Increased inhibition zone* 8.5 mm 5.5 mm 5.0 mm 7.5 mm Change of Susceptibility R -> S R -> I R -> I R -> R Synergic score, S S S 3 (S 1+S 2) * Increased inhibition zone (mm) in agar with 0.5 MIC of aztreonam than control (without antibiotics) Synergic score were defined as Pestel's method (Pestel et al. 1995) (see text for material and methods) Abbreviation; PIP-TZP, piperacillin-tazobactams; CAZ, ceftazidime; FEP, cefepime; AN, amikacin. 169

5 Fig. 2. Time-killing curves of a VIM-2 MBL producing P. aeruginosa after the exposure of AZT (4 μg/ml), TZP (32/4 μ g/ml), or the combination of both. MIC: AZT; 8 μg/ml, TZP; 64/8 μg/ml. Abbreviation: A+T, aztreonam plus piperacillin-tazobactam. Fig. 4. Time-killing curves of a VIM-2 MBL producing P. aeruginosa after the exposure of AZT (4 μg/ml), TZP (32/4 μ g/ml), AN (16 μg/ml), or the combination of the three. Abbreviation: A+T+A, aztreonam plus piperacillin-tazobactam plus amikacin. Fig. 3. Time-Killing curves of a VIM-2 MBL producing P. aeruginosa after the exposure of AZT (4 μg/ml), AN (16 μg/ ml), or the combination of both. MIC : AZT; 8 μg/ml, AN; >256 μg/ml Abbreviation: A+A; aztreonam plus amikacin. 성하는 P. aeruginosa에서 one disk synergy 검사를이용하여상승효과를보이는항균제를찾아보았다. One disk synergy test는 Pestel 등 (Pestel 등, 1995) 의방법을변형하여사용하였는데, 항균제를섞은배지와섞지않은배지에서억제대직경의차이로상승효과를정량적으로측정할수있는방법으로상승효과를보이는항균제를찾는데유용한방법이다. 본연구에서는 P. aeruginosa CBU 01/11/569가 AZT에감수성을보여이항균제와병합에의해상승효과를보이는항균제조합을찾았다. AZT와 PIP-TZP의병합에서억제대의증가가가장컸으며이어서 AZT과 AN의병합에서도의미있는억제대증가가관찰되어 (Table 2) 이들항균제조합으로 time-killing curve 를작성하였다. 그람음성간균에서 time-killing 시험의기준은아직까지정해져있지않는데, 균의응집및 biofilm의형성에 의해균집락내부에도달되는항균제농도가낮게되며 24시간후에흔히재증식을보일수있다 (Cappelletty와 Rybak 등, 1996). 따라서항균제노출후 6~8시간내에상승효과유무를판독하자는보고가있으며 (Glew와 Pavuk, 1984), 실제로 β-lactam 항균제및대부분의항균제는 8시간이내에재투여가되므로위제안이의미있을것으로생각된다. Time-killing 시험에서항균제농도는 MIC와혈중농도를고려해야하는데, 일반적으로 1/4 MIC 또는 1/2 MIC 그리고혈중도달농도를고려하여정한다 (Elliopoulos 등, 1996). 본실험에서는기본적으로 1/2 MIC를선택하였으며 AN은 1/4 MIC(>64 μg/ml) 이독성범위 (toxic level>35 μg/ml, Zaske, 1980) 이었으므로독성범위이하의치료농도 (therapeutic range; 10~25 μ g/ml) 를선택하여항균제노출 6시간또는 8시간에판독하였다. 실험결과 AZT와 PIP-TZP의병합에의해서각각의항균제보다억제효과는컸지만의미있는상승효과 (100배이상의균수감소 ) 는관찰되지않았다 (Fig. 2). 그러나일부균에서 aminoglycoside에고도내성일지라도 β -lactam제와의병합은의미있는상승효과를나타낼수있다고알려져있어자주병합요법에이용되고있다 (Miller 등, 1986). P. aeruginosa CBU 01/11/569는 AN에대한 MIC가 256 μg/ ml 이상을보였으나 AZT와 one disk synergy 검사에서억제대가의미있게증가되었다. 그리고 time-killing curve에서는 AN과 AZT(β-lactams) 의병합은 AZT과 PIP-TZP의병합효과와유사하였다 (Fig. 3). 따라서이세항균제를병합하여시험하게되었는데, 이시험에서는각각의항균제조합보다항균제노출후 8 시간에의미있는상승효과 (1/183로감소 ) 를보여 (Fig. 4) 어떤 MBL 생성균주에서 aminoglycoside를포함하는삼 170

6 중요법은유용할것으로사료되었다. 결론적으로 MBL 생성균은종종 MBL 유전자와동시에 aminoglycoside 내성유전자를포함하고있기때문에 aminoglycoside에고도내성을보이지만 MBL 생성균에서병합요법에 aminoglycoside( 특히, AN) 를포함하는것은효과적일수있을것이다. 뿐만아니라통상적으로 MBL 생성균은 AZT을가수분해하지못하여감수성일수있으므로 AN과 AZT의이중병합은유용할것이다. 그러나이들균에서변이에의한 AmpC β-lactamse를갖고있으면 AZT에내성을보일수있으므로 (Korfmann 등, 1991; Stapleton 등, 1995) AZT의효과는감소할수있으며, 이런경우 AZT, PIP-TZP, 그리고 AN의삼중병합도고려해볼수있을것이다. 앞으로보다많은수및다양한유전형의 MBL 생성균을대상으로병합요법에대한시험이필요할것으로사료된다. 참고문헌 1. Castanheira M, Toleman MA, Jones RN, Schimidt FJ, Walsh TR. Molecular characterization of a betalactamase gene, blagim-1, encoding a new subclass of metallo-β-lactamases. Antimicrob Agents Chemother 48: , Cappelletty DM and Rybak MJ. Comparison of methodologies for synergism testing of drug combination against resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 40: Dejongh CA, Joshi JH and Thompson BW. A double β-lactam combination versus an aminoglycoside- containing regimen as empiric antibiotic therapy for febrile granulocytopenic cancer patients. Am J Med Supp 5C: , Eliopoulos GM, Moellering Jr RC. Antimicrobial combination. In Lorian V(eds). Antibiotics in laboratory medicine. 4th ed. p Williams & Willkins, Baltimore, Gilpin ML, Fulston M, Payne D, Cramp R and Hood I. Isolation and structure determination of twe novel phenazines from a Streptomyces with inhibitory activity against metallo-enzymes, including metallo-β -lactamases. J Antibiot (Tokyo) 48: , Glew RH and Pavuk RA. Early synergistic interaction between amikacin and six β-lactam antibiotics against multiply resistant members of the famili Enterobacteriaceae. Antimicrob Agents Chemother 26: , Jeong SH, Lee K, Chong Y, Yum JH, Lee SH, Choi HJ, Kim YM, Park KH, Han BH, Lee SW and Jeong TS. Characterization of a new integron containing VIM-2, a metallo-β-lactmase gene cassettes, in a clinical isolate of Enterobacter cloacae. J Antimicrob Chemother 51: , Korfmann G, Sanders CC and Moland ES. Altered phenotypes associated with ampd mutation in Enterobacter cloacae. Antimicrob Agents Chemother 35: , Lauretti L, Ricco MI, Mazzriol A, Cornaglia G, Amicosante G, Fontana R and Rossolini M. Cloning and characterization of blavim, a new integron-borne metallo-β-lactamase gene form a Pseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother 43: , Lee K, Lee WG, Uh Y, Ha GY, Cho J and Chong Y. VIM-and IMP-type metallo-β-lactamase producing Pseudomonas spp. and Acinetobacer spp. in Korean Hospitals. Emerg Infect Dis 9: , Lee K, Yum JH, Young D, Lee HM, Kim HD, Docquier JD, Rossolini GM, Chong Y. Novel acquired metallo-β-lactamase gene, bla (SIM-1), in a class 1 integron from Acinetobacter baumannii clinical isolates from Korea. Antimicrob Agents Chemother 49: , Matsumura N, Minami S and Mitsuhashi S. Antibacterial activity of T-5575, a novel 2-carboxypenem, and its stability to β-lactamase. J Antimicrob Chemother 39:31-34, Miller MH, El-Sokkary MA, Feinstin SA and Lowy FD. Penicillin-induced effects on streoptomycin uptake and early bactericidal activity differ in viridans group and enterococcal streptococci. Antimicrob Agents Chemother 30: , Moyer TP and Pippenger CE. Therapeutic drug 171

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12_HM hwp The Korean Journal of Microbiology (2010) Vol. 46, No. 3, pp. 303-307 Copyright c 2010, The Microbiological Society of Korea 단보 국내에서분리된 Acinetobacter baumannii 의 Integron 과연관된다제내성분석 김성환 최지혜 박은진 서인원 손승렬