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1 J. Exp. Biomed. Sci. 2012, 18(3): 299~306 pissn : Original Article Molecular Characterization and Prevalence of 16S Ribosomal RNA Methylase Producing Bacteria in Amikacin Resistant Gram-negative Bacilli Isolated from Clinical Specimens Kyung-A Shin 1, Seock Yeon Hwang 2 and Seung Bok Hong 3, 1 Department of Laboratory Medicine Bundang Jesaeng Hospital, Sungnam-si, Gyeonggi-do , Korea 2 Department of Biomedical Laboratory Science, Daejeon University, Daejeon , Korea 3 Department of Clinical Laboratory Science, Chungbuk Health & Science University, Cheongwon , Korea Recently, the prevalence of 16S rrna methylase conferring high-level resistance to aminoglycosides has been increasing in Gram-negative bacilli globally. We determined the prevalence and genotype of these methylase-producing bacteria, and characterized the co-resistance to β-lactam antibiotics and quinolone in Gram-negative clinical isolates collected in 2010 at a hospital in Korea. Among 65 amikacin-resistant isolates screened from 864 Gram-negative bacilli (GNB), 16S rrna methylase genes were detected from 49 isolates, including Acinetobacter baumannii (43), Klebsiella pneumoniae (2), Proteus mirabilis (2) and Serratia marcescens (1), Empedobacter brevis (1). All of the 16S rrna methylase genotype was arma and no variant sequences of amplified PCR products for arma were noted. The 16S rrna methylase producing bacteria showed much higher resistance to aminoglycoside for Enterobacteriaceae and glucose non-fermenting (NF)-GNB and to imipenem for glucose NF-GNB, than the non-producing isolates. All of the 16S rrna methylase producing Enterobacteriaceae had the extended-spectrum-β-lactamase. In addition, two K. pneumoniae concurrently produced both plasmid-mediated AmpC β-lactamase and qnrb gene. All of the amikacinresistant A. baumannii (43) co-harbored arma 16S rrna methylase and bla OXA-23 carbapenemase. In conclusion, 16S rrna methylase producing bacteria were very prevalent among GNB in South Korea, and were commonly associated with co-resistance, including carbapenem and quinolone. Key Words: 16S rrna methylase, arma, β-lactams, Quinolone, Aminoglycoside, Gram negative bacilli 서 그람음성간균에의한감염증의치료에는세포벽생성을억제하는 β-lactam계항균제, 단백질합성을억제하는 aminoglycoside계항균제, 그리고 DNA 합성을저해하는 quinolone계항균제가흔히사용된다 (Yao et al., 2007). 이들항균제의사용이증가함에따라세균의내성률도증가하였다. Amikacin과 quinolone에내성인 Klebsiella * Received: August 2, 2012 / Revised: August 13, 2012 Accepted: August 20, 2012 Corresponding author: Seung Bok Hong. Department of Clinical Laboratory Science, Chungbuk Health & Science University, Cheongwon , Korea. Tel: , Fax: sbhong8646@hanmail.net C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. 론 pneumoniae가 1997년에각각 8% 이었으나 2003년에는 13%, 19% 로증가하였으며 2009년에 15%, 33% 까지증가하였다 (Lee et al., 2006; Lee et al., 2011). 한편 Acinetobacter 균종에서는 1997년에 amikacin과 quinolone에대한내성이각각 50%, 2003년에 56%, 58% 그리고 2009년에는 66%, 67% 로급격히증가하였다 (Lee et al., 2006; Lee et al., 2011). 한편가장흔한 aminoglycoside의내성기전은수식효소에의한것이었으나최근 30S 리보좀을구성하는 16S rrna의 methylation에의해 amikacin 및 gentamicin을포함한모든 4,6-substituted deoxystreptamine 항균제에고도내성을나타나게하는내성기전이증가하고있으며, 이들내성기전은플라스미드를통해쉽게다른균으로전달될수있다 (Galimand et al., 2003). 2003년프랑스에서처음으로 K. pneumoniae에서 arma형 16S rrna methylase

2 가보고되면서 (Galimand et al., 2003), 최근까지장내세균뿐만아니라 Pseudomonas spp. 와 Acinetobacter spp. 를포함한다양한그람음성간균에서 rmta (Yokoyama et al., 2003), rmtb (Doi et al., 2004), rmtc (Wachino et al., 2006), rmtd (Doi et al., 2007a), rmte (Davis et al., 2010) 및 npma (Wachino et al., 2007) 가전세계적으로보고되었다. Bogaerts 등 (Bogaerts et al., 2007) 은 2000년에서 2005년까지 Belgium에서분리된 16S rrna methylase를생성하는균중모두가 extended-spectrum-β-lactamase (ESBL) 을생성함을보고하였으며, 국내에서 Kim 등 (Kim et al., 2008) 은 16S rrna methylase 생성 A. baumannii가 bla OXA-23 을동시에생성하고 aminoglycoside와 carbapenem에동시내성을보이는균에의한감염의창궐을보고하였다. 한편 2010 년 Lee 등은 arma 양성장내세균의 91% 에서 plasmidmediated quinolone resistance (PMQR) 유전자인 qnrb와연관됨을보고하였다 (Lee et al., 2010). 이와같이 16S rrna methylase 생성균주는 aminoglycoside에고도내성을보일뿐아니라다양한 β-lactam 항균제와 quinolone 등에도내성을보일수있다. 따라서이들균에의한감염의치료를더욱어렵게할수있으나이에대한국내연구는드물다. 본연구의목적은국내한지역의 2차병원에서분리되는그람음성간균중에서 16S rrna methylase를생성하는균주의빈도및유전형을조사하고, β-lactam 및 quionolone 내성유전자의동시생성등을조사하여이들항균제내성기전을밝히고자하였다. 재료및방법시험균주의수집및균종동정 2010년 4월부터 8월까지 5개월동안경기도지역한병원에서분리된그람음성간균 864주중에서 amikacin에내성인 65주를대상으로하였으며동일한환자에서반복분리된균은제외하였다. 균의동정은기본적인생화학적검사와자동동정기기인 MicroScan WalkAway 96 plus system (Siemens Healthcare Diagnostics Inc, West Sacramento, CA, USA) 을이용하였다. 항균제감수성검사항생제감수성검사는 Clinical and Laboratory Standards Institute (CLSI, 2012) 한천희석법으로시험하였다. 시험항균제는 amikacin (Boryung Pharmaceutical Co., Seoul, Korea), ceftazidime (CJ Jeiljedang Co., Seoul, Korea), ciprofloxacin (Bayer Korea Co., Seoul, Korea), gentamicin (Dongshin Pharmaceutical Co., Seoul, Korea), cefoxitin 및 imipenem (Merk & Co., Inc., Elkton, MD, USA), tobramycin (Daewoong Pharmaceutical Co., Seoul, Korea), arbekacin (Joongwae Pharmaceutical Co., Seoul, Korea) 이었다. 최소억제농도 (MIC) 는균의증식이관찰되지않는최소항균농도로정의하였으며, 매실험마다정도관리를위해 Escherichia coli ATCC 및 P. aeruginosa ATCC 를동시에시험하였다. β-lactamase 선별검사 Extended-spectrum β-lactamase 생성시험 : Double disk synergy (DDS) 시험으로 ESBL 생성을확인하였다. DDS 시험은 amoxacillin-clavulanic 디스크 (BBL, Microbiology Systems, Cockeysville, MD., USA, 20/10 μg) 를가운데두고 3세대 cephalosporin (cefotaxime, ceftazidime, aztreonam: BBL, 30 μg) 과 cefepime 디스크 (BBL, 30 μg) 를 20 mm 간격으로위치하게한후 16~18시간배양후상승효과가존재하면 ESBL로판단하였다 (Jarlier et al., 1988). DDS에서 ESBL 생성균주로의심되면 CLSI ESBL phenotypic confirmatory test (CLSI, 2012) 로 ESBL 생성유무를다시확인하였다. Plasmid-mediated AmpC β-lactamse 생성시험 : Plasmidmediated AmpC β-lactamase (PMABL) 검사는 Song 등의방법으로 (Song et al., 2007) 시행하였으며방법을간략히기술하면다음과같다. 3-aminophenylboronic acid (Sigma- Aldrich Inc, Steinhelm, Germany) 400 μg을 cefoxitin disk에첨가하여억제대가 5 mm 이상증가한경우양성으로판단하였다. Carbapenemase 생성시험 : imipenem 내성원인을조사하기위하여 CLSI guideline (2012) 에따라 modified Hodge test를시행하여 carbapenemase를선별하였으며, 양성인균주는 imipenem-edta double disk synergy (EDTA-DDS) 법으로 metallo-β-lactamase (MBL) 생성을확인하였다 (Lee et al., 2003). 한편 modified Hodge test에서양성이고 EDTA-DDS 시험에음성인 A. baumannii에서는 OXA-type carbapenemase 유전자검출을시도하였다 (Table 1)

3 Table 1. Primers used for detection of 16S rrna methylase, plasmid mediated quinolone resistance and OXA-type carbapenemase genes Name Nucleotide sequence (5' 3') Product size (bp) References. arma F a arma R rmta F a rmta R rmtb F a rmtb R rmtc F a rmtc R rmtd F a rmtd R qnra F b qnra R qnrb F b qnrb R qnrs F b qnrs R qepa F b qepa R OXA23F c OXA23R OXA24F c OXA24R AGG TTG TTT CCA TTT CTG AG TCT CTT CCA TTC CCT TCT CC CTA GCG TCC ATC CTT TCC TC TTT GCT TCC ATG CCC TTG CC CCC AAA CAG ACC GTA GAG GC CTC AAA CTC GGC GGG CAA GC CGA AGA AGT AAC AGC CAA AG ATC CCA ACA TCT CTC CCA CT ATG AGC GAA CTG AAG GAA AAA CTG C GCT CCA AAA GCG GCA GCA CCT TA AGA GGA TTT CTC ACG CCA GG TGC CAG GCA CAG ATC TTG AC GGA ATT GAA ATT CGC CAC TG TTT GCC GCC CGC CAG TCG AA GCA AGT TCA TTG AAC AGG GT TCT AAA CCG TCG AGT TCG GCG CCG ACA GGC CCA CGA CGA GGA TGC TCG GCG GCG TGT TGC TGG AGT TCT GAT CGG ATT GGA GAA CCA GA ATT TCT GAC CGC ATT TCC AT GGT TAG TTG GCC CCC TTA AA AGT TGA GCG AAA AGG GGA TT a Primers for 16S rrna methylase genes b Primers for plasmid mediated quinolone resistance genes c Primers for OXA-type carbapenemase genes 590 Lee et al., Lee et al., Lee et al., Lee et al., Lee et al., Cattoir et al., Cattoir et al., Cattoir et al., Lee et al., Woodford et al., Woodford et al., S rrna methylase 유전자검출 Amikacin 내성 65주에대하여 16S rrna methylase 유전자 (arma, rmta, rmtb, rmtc 및 rmtd) 에대한 PCR을시행하였다 (Table 1). PCR 반응액은 polymerase 1 U가들어있는 premix (Bioneer, Daejeon, Korea) 에 DNA 추출액 2 μl 와 20 pmol의 primer를각각 1 μl를넣고, 최종용량이 20 μl가되게증류수를넣고시행하였다. PCR 증폭은 Gene Amp PCR system 9600 (Perkin-Elmer Cetus Corp., Norwalk, CT, USA) 을사용하였고, 94 에서 15분 predenaturation 후 94 30초, 55 30초, 72 1분씩 35회증폭반응시키고 72 에서 5분간연장반응시켰다. rmtc 및 rmtd은 annealing 온도만각각 55 및 62 로사용하였고, 기타조건은위와동일하게시행하였다. PCR 증폭산물은 1% 의아가로스겔을이용하여, 100 V에서 30분간전기영 동하였다. 증폭된유전자를 ABI 3700 (Applied Biosystem, Foster city, CA, USA) 로염기서열을분석하였으며염기서열은 GenBank 염기 database (wwww.ncbi.nlm.nih.gov/blast/) 와비교하였다 Plasmid-mediated quinolone 내성유전자검출플라스미드에의해전달되는 quinolone 내성유전자인 qnra, qnrb, qnrs 및 qepa를 PCR로검출하였다 (Cattoir et al., 2007). 사용한시발체는 Table 1과같으며, PCR 반응액은 polymerase 1 U가들어있는 premix (Bioneer) 에 DNA 추출액 2 μl와 20 pmol의 primer를각각 1 μl 넣고, 최종용량이 20 μl가되게증류수를넣고반응시켰다. PCR 증폭은 Gene Amp PCR system 9600 (Perkin-Elmer Cetus) 을사용하였고, 94 에서 15분 predenaturation 후 94 30초, 55 30초, 72 1분씩 35회증폭반응시키고 72 에서

4 Table 2. Distribution of 16S rrna methylase and plasmid mediated quinolone (PMQ) resistance genes in amikacin resistant gram-negative bacilli Bacterial species No. isolated No. of AMK resistance 16S rrna methylase genes arma RmtA, B, C, D No. of AMK resistance PMQ resistance genes qnra, S qnrb qepa E. coli K. pneumoniae K. oxytoca 15 0 NT NT 0 NT NT NT E. cloacae E. earogenes 21 0 NT NT 0 NT NT NT C. freundii 21 0 NT NT 0 NT NT NT P. mirabilis S. marcescens P. aeruginosa A. baumannii E. brevis Total Abbreviations: AMK, amikacin; PMQ, plasmid mediated quinolone; NT, not tested 5분간연장반응시켰다. qepa PCR은 annealing 온도만각각 61 로사용하였고, 기타조건은위와동일하게시행하였다. OXA-carbapenemase 유전자검출 Modified Hodge test에서양성이고, EDTA-DDS 시험에음성인균주는 OXA-type carbapenemase 생성을시사하므로, bla OXA-23, bla OXA-24 를 PCR로검출하였다. 사용한시발체와반응은 Woodford의방법대로시행하였다 (Woodford et al., 2006). 결과 16S rrna methylase 양성률및유전형 2010년 4월부터 5개월동안경기도지역에서분리된그람음성간균 864주중에서 65주 (7.52%) 가 amikacin에내성이었으며이중 49주 (5.67%) 가 16S rrna methylase 를생성하였다. 16S rrna methylase 양성인균종은 A. baumannii 43주 (43/82), K. pneumoniae 2주 (2/6), Proteus mirabilis 2주 (2/2), Serratia marcescens 1주 (1/1) 및 Empedobacter brevis 1주 (1/1) 였다 (Table 2). 유전형으로는 16S rrna methylase 양성 49주모두 arma이었다. Plasmid mediated quinolone 내성유전자는 E. coli 1주와 K. pneumoniae 5주에서 qnrb가검출되었으며 arma 양성 K. pneumoniae 2주에서 arma와 qnrb 유전자가동시에검 출되었다 (Table 2). 16S rrna methylase 생성균주의항균제내성양상 Amikacin 내성장내세균 14주는 imipenem을제외하고시험한항균제에모두내성을보였다. 이들중 16S rrna methylase (arma) 양성균주 (5주) 는 aberkacin, amikacin, gentamicin 및 tobramycin의 MIC 50 이모두 256 μg/ml 이상으로 16S rrna methylase 음성균주에비하여고도내성을보였다. 또한 16S rrna methylase 양성장내세균은모두 ESBL을생성하고있었으며, 이들중 K. pneumoniae 2주는 ESBL과 ampc β-lactamase를동시에생성하였다 (Table 3 & 4). Amikacin 내성포도당비발효세균 51주도 imipenem을제외한시험한모든항균제에내성을보였으며 imipenem 내성률은 16S rrna methylase 생성균주및미생성균주에서각각 95% 와 71% 이었다. 한편 A. baumannii 44주는모두 16S rrna methylase (arma) 양성이었으며, 이들은 16S rrna 음성포도당비발효세균에비하여 arberkacin 과 amikacin에대한 MIC 50 이의미있게증가되어있었으나 gentamcin과 tobramycin의 MIC 50 은유사하였다. 16S rrna methylase 양성 A. baumannii는 imipenem MIC 50 이음성주에비하여증가되어있었다 (Table 3). 이들균은모두 modified Hodge 검사에서양성, EDTA-DDS에서음성을보여 Class D carbapenemase를시사하였으며 Class D OXA-23 β-lactamse 생성균이었다 (Table 4)

5 16S rrna methylase 유전자및 quinolone 내성유전자염기서열분석유전자의변이형을선별하기위해서 16S rrna methylase 양성 49주에대해 arma 유전자와 pasmid-mediated quinolone 내성 qnrb 유전자 2주에대해염기서열을분석 하였으나모든균주에서변이형은관찰되지않았다. 고찰 864주의그람음성간균중 49주 (5.7%) 가 16S rrna methylase를생성하였으며이중에는 K. pneumoniae (2), P. Table 3. Comparison of the MICs (μg/ml) for antimicrobial agents between 16S rrna methylase- positive and -negative gram negative bacilli Enterobacteriaceae (14) a 16S rrna methylase-positive (49) 16S rrna methylase-negative (16) MIC range MIC 50 MIC 90 %R MIC range MIC 50 MIC 90 %R Amikacin >256 >256 > ~> > Arbekacin 256~>256 >256 > ~> > Tobramycin 256~>256 >256 > ~> > Gentamicin 256~>256 >256 > ~> > Ciprofloxacin 64~> > ~> > Ceftazidime 64~> > ~> Cefoxitin 32~> > ~> Imipenem 0.25~ ~ Glucose nonfermenting GNR (51) b Amikacin >256 >256 > ~> Arbekacin 256~>256 >256 > ~ > Tobramycin 256~>256 >256 > ~>256 >256 > Gentamicin 256~>256 >256 > ~256 >256 > Ciprofloxacin 64~> > ~ Ceftazidime 64~> ~ Imipenem 1~ ~ Abbreviations: GNR, Gram negative bacilli; MIC, minimal inhibitory concentration; R, resistance a composed with 5 positive isolates and 9 negative isolates for 16S rrna methylase. b composed with 44 positive isolates and 7 negative isolates for 16S rrna methylase, which included 44 Acinetobacter baumannii, 7 Pseudomonas aeruginosa and 1 Empedobacter brevis Table 4. Determinants of antimicrobial resistance in 49 Gram-negative bacilli with 16S rrna methylase Carbapenemase Isolates (No) 16S M a DDS test Hodge c bla OXA-23 Susceptibility qnrb b IMP FOX CIP K. pneumoniae (2) arma b ESBL + ampc Negative - + S/R R R P. mirabilis (2) arma ESBL Negative - - R/S R R S. marcescens (1) arma ESBL Negative - - S R R A. baumannii (43) arma Negative Positive + - R R R E. brevis (1) arma Negative Negative - - R R R Abbreviations: DDS, double disk synergy test; ESBL, extended spectrum-β-lactamase; IMP, imipenem; FOX, cefoxitin; CIP, ciprofloxacin; NT, not tested; S, susceptibility; R, resistance a 6S rrna methylase gene b no variant sequences of amplified PCR products for arma and qnrb were noted. c modified Hodge test

6 mirabilis (2), S. marcescens (1), A. baumannii (43), E. brevis (1) 등이포함되었는데, E. brevis에서 16S rrna methylase 의보고는전세계적으로처음이다. 2000년부터 2005년사이에벨기에서수집된 15,386주의장내세균에서총 19주 (0.12%) 가 16S rrna methylase를생성하였으며 (Bogaerts et al., 2007), 2004년일본의 169개병원에서수집한 87,626 주의그람음성간균에서총 26주 (0.03%) 의 16S rrna methylase 생성주가분리되었다 (Yamane et al., 2007). 한편 년국내의두병원에서수집된 2,722주의그람음성간균에서 123주 (4.5%) 의 16S rrna methylase 생성주가분리되어 (Lee et al., 2010) 이들내성기전이점차증가하며다양한균으로확산됨을시사하였다. 16S rrna methylase 유전형은 49주가모두 arma로이전의국내의보고와유사하였다 (Lee et al., 2010). 흥미로운것은지리적으로가까운일본에서는 rmta가주로분리되었으며 (Yamane et al., 2007), 유럽에서는 arma가흔히분리되었는데 (Bogaerts et al., 2007) 일본및유럽에서분리되는균주와의 clonality에대한추가연구가필요할것이다. 16S rrna methylase 생성균주는다양한항균제에내성을보일수있는데, 특히장내세균에서 ESBL의생성을흔히동반한다. 유럽에서분리되는 arma 유전자양성균주모두에서 CTX-M형 ESBL이생성됨이보고되었으며 (Bogaerts et al., 2007), 내에서도 19주의 arma 양성장내세균 14주 (73.7%) 가 ESBL 생성균주이었다 (Lee et al., 2010). 그러나 CTX-M은 1주로유럽과대만의경우와상이하였다 (Yan et al., 2004). 본연구에서도 5주의 arma 양성장내세균은모두 ESBL을생성하고있었으며, 이들중 K. pneumoniae 2주는 PMABL를동시에생성하고있었다. 한편 Lee 등의보고 (Lee et al., 2010) 에의하면국내 arma 양성장내세균 14주 (K. pneumoniae와 E. coli) 중 9주 (64%) 가 PMABL를생성하고있다는보고와유사한결과를보인다. 결과적으로 16S rrna methylase 생성장내세균은 ESBL을흔히생성하며, K. pneumoniae 와 E. coli 에서는 PMABL도동시에생성하므로흔히다제내성을보일수있을것으로추측된다. 16S rrna methylase 생성포도당비발효그람음성간균에서 carbapenemase를동시에생성하는균주가보고되고있는데, Yu 등 (Yu et al., 2007) 은중국 19개병원에서수집한 carbapenem 내성 A. baumannii 342주중에서 arma 을생성하는균주가 221주 (61%) 였다고보고하였으며, 미국 Pennsylvania에서분리된 5주의 arma을생성하는 균주중에서 2주가 OXA-23 carbapenemase를동시에생성하여 carbapenem에내성을보였다고하였다 (Adams- Haduch et al., 2008). 또한브라질에서는 SPM-1형 MBL 과 rmtd 16S rrna methylase 를동시에생성하는 P. aeruginosa의집단감염이보고되기도하였다 (Doi et al., 2007b). 본연구에서도 arma 양성 A. baumannii 43주가모두 imipenem에대한 MIC가 16 μg/ml 이상으로내성이었으며, modified Hodge test에서양성, EDTA-DDS에서음성을보여 class D carbapenemase의생성을시사하였다. 그리고 class D carbapenemase 중 A. baumannii에서가장흔한 bla OXA-23 에대한유전자검사에서모두양성을보였다. 한편 arma와 OXA-23 carbapenemase 양성균주에의한감염의창궐 (outbreak) 이 2007년국내의서로다른지역의병원에서보고되었는데 (Kim et al., 2008; Jeong et al., 2011), 본균주의수집병원은기존보고된병원들과서로다른지역이므로국내에서 arma와 OXA-23 carbapenemase 동시생성 A. baumannii이널리확산되었음을시사한다. 16S rrna methylase 생성균주는 quinolone에대해서도높은내성을보이는데 (Lee et al., 2010) 본연구에서도 arma 생성장내세균및 A. baumannii는 ciprofloxacin 에 100% 의내성을보였다. 한편 quinolone에대한내성기전으로 quinolone-resistance determining region의변화 (Hooper, 2003) 와 efflux pump의증가 (Poole, 2007) 및 porin의감소 (Ruiz, 2003) 등이알려져있다. 최근에는플라스미드를통하여다른균주로전파가되는 Qnr 단백질에의한내성기전과 qepa gene에의한내성이알려져있는데, 본연구에서이들유전자를검사한결과 E. coli 1균주와 K. pneumoniae 5주에서 qnrb 유전자가검출되었으며, arma 양성 K. pneumoniae 2주는모두 qnrb 유전자를포함하고있었다. 이는국내에서 arma 생성 K. pneumoniae의 91% 가 qnrb가검출되어높은연관성을보였다는보고와일치한다 (Lee et al., 2010). 결국 arma 양성 K. pneumoniae는 2주모두 ESBL, PMABL, qnrb를동시에보유하고있었는데, 이들은항균제선택압력이높은병원환경에서 16S rrna methylase, PMABL 및 qnrb 내성유전자가동시에수평-확산되었을가능성을시사하므로감염관리에철저한주의가필요할것으로사료된다. 결론적으로국내에서 16S rrna methylase에의한 aminoglycoside 고도내성인그람음성간균의양성율은 5.7% 로높았으며특히 A. baumannii에서양성율이 53.6% 로외국의보고뿐만아니라다른국내보고보다월등히

7 높았다. 그리고다양한균종으로 16S rrna methylase의내성유전자가전파되고있었으며유전자형은모두 arma 형이었다. 마지막으로 16S rrna methylase 생성세균에서 ESBL, PMABL, qnrb ( 장내세균 ) 및 carbapenemase (A. baumannii) 와같은다양한내성기전을동시에포함하고있었다. REFERENCES Adams-Haduch JM, Paterson DL, Sidjabat HE, Pasculle AW, Potoski BA, Muto CA, Harrison LH, Doi Y. Genetic basis of multidrug resistance in Acinetobacter baumannii clinical isolates at a tertiary medical center in Pennsylvania. Antimicrob Agents Chemother : Bogaerts P, Galimand M, Bauraing C, Deplano A, Vanhoof R, De Mendonca R, Rodriguez-Villalobos H, Struelens M, Glupczyski Y. Emergence of arma and rmtb aminoglycoside resistance 16S rrna methylase in Belgium. J Antimicrobial Chemotherapy : Cattoir V, Poirel L, Rotimi V, Soussy CJ, Nordmann P. Multiplex PCR for detection of plasmid-mediared quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. J Antimicrob Chemother : Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: twenty two informational supplement (M100-S22). Wayne, PA: CLSI, Davis MA, Baker KN, Orfe LH, Shah DH, Besser TE, Call DR. Discovery of a gene conferring multiful-aminoglycoside resistance in Escherichia coli. Antimicrob Agents Chemother : Doi Y, Arakawa Y. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycoside. Clin Infect Dis. 2007a. 45: Doi Y, de Oliveira GD, Adams J, Paterson DL. Coproduction of novel 16S rrna methylase rmtd and metallo-β-lactamase SPM-1 in a panresistant Pseudomonas aeruginosa isolate from Brazil. Antimicrob Agents Chemother. 2007b. 51: Doi Y, Yokoyama, K. Yamane, K, Wachino J, Shibata N, Yagi T, Shibayama K, Kato H, Arakawa Y. Plasmid-mediated 16S rrna methylase in Serratia marcescens conferring high level resistance to aminoglycosides. Antimicrobial Agents Chemother : Galimand M, Courvalin P, Lambert T. Plasmid-mediated highlevel resistance to aminoglycosides in Enterobacteria due to 16S rrna methylation. Antimicrob Agents Chemother : Hooper DC. Mechanisms of quinolone resistance. In: Hooper DC and Rubenstein E, eds. Quinolone antimicrobial agents. 3 rd ed, pp American Society for Microbiology Press. Washington DC, USA. Jarlier V, Nicolas M, Fournier G, Philippon A. Extended spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: Hospital prevalence and susceptibility patterns. Rev Infect Dis : Jeong HW, Son BR, Shin DI, Ryu D, Hong SB, Hand K, Shin KS. Characterization of Acinetobacter baumannii co-producing carbapenemase OXA-23 and OXA-66, and arma 16S ribosomal RNA methylase at a university hospital in South Korea. Korean J Clin Microbiol : Kim JW, Heo ST, Jin JS, Choi CH, Lee YC, Jeong YG, Kim SJ, Lee JC. Characterization of Acinetobacter baumannii carrying bla(oxa-23), bla(per-1) and arma in a Korean hospital. Clin Microbiol Infect : Lee H, Koh EM, Kim CK, Yum JH, Lee K, Chong Y. Molecular and phenotypic characteristics of 16S rrna methylaseproducing Gram-negative Bacilli. Korean J Clin Microbiol : Lee H, Young D, Yum JH, Roh KH, Lee K, Yamane K, Arakawa Y, Chong Y. Dissemination of 16S rrna methylase-mediated highly amikacin resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea. Diagn Microbiol Infect Dis : Lee K, Kim MN, Kim JS, Hong HL, Kang JO, Shin JH, Park YJ, Yong D, Jeong SH, Chong Y, KONSAR Group. Further increases in carbapenem, amikacin and fluroquinoloneresistant isolates of Acinetobacter spp. and P. aeruginosa in korea: KONSAR study Yonsei Med J : Lee K, Lim YS, Yong D, Yum JH, Chong YS. Evaluation of the Hodge test and the imipenem-edta double disk synergy test test for differentiating metallo-β-lactamase producing isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol : Lee K, Park KH, Jeong SH, Lim HS, Shin JH, Yong D, Ha GY, Chong Y, KONSAR Group. Further increase of vancomycinresistant Enterococcus faecium, amikacin- and fluoroquinolone -resistant Klebsiella pneumoniae, and imipenem-resistant Acinetobacter spp. in Korea: 2003 KONSAR surveillance

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