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Korean J Clin Microbiol Vol. 11, No. 2, October, 2008 Detection of CTX-M-Type Extended-Spectrum β-lactamase in Clinical Isolates of Chromosomal AmpC β-lactamase-producing Enterobacteriaceae from Korea and Their Molecular Characteristics Chang-Ki Kim 1, Jong Hwa Yum 2, Dongeun Yong 3,4, Seok Hoon Jeong 3,4, Kyungwon Lee 3,4, Yunsop Chong 3,4 1 Korean Institute of Tuberculosis, The Korean National Tuberculosis Association, Seoul; 2 Department of Biomedical Laboratory Science, Dong-Eui University College of Natural Science, Busan; 3 Department of Laboratory Medicine and 4 Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea Background: Clinical isolates of AmpC β-lactamaseproducing Enterobacteriaceae were evaluated to determine the prevalence of CTX-M extended-spectrum β-lactamases (ESBLs) and their genetic environments. Methods: A total of 250 non-duplicate isolates of Eneterobacter aerogenes, E. cloacae, Citrobacter freundii, Serratia marcescens and Morganella morganii were collected at a Korean hospital. ESBL production was determined by double disk synergy test. For ESBL producers, bla genes were sequenced and bla CTX-M environment was characterized by PCR mapping and sequencing. Results: Among the 250 isolates 29 (11.6%) produced ESBL, and 14 of the 29 isolates produced CTX-M ESBLs, including CTX-M-9 by 8 isolates, CTX-M-3 by 4 isolates, CTX-M-12 by 1 isolate, and CTX-M-14 by 1 isolate. ISEcp1 was present upstream of bla CTX-M-3, 12, and 14. Three of the four CTX- M-3 producers had the same genetic environment (pemk-isecp1-bla CTX-M-3-orf477-mucA). An IS903-like element was found downstream of bla CTX-M-14. ISCR1 was identified upstream of bla CTX-M-9 and ISCR1 and bla CTX-M-9 were located on sul1-type class 1 integron. The variable region between the 5 -CS and the first 3 -CS contained dfra16 and aada2. Its structure was similar to that of In60, but our isolates did not have IS3000 or second 3 -CS. Conclusion: CXT-M type ESBL was prevalent in AmpC β-lactamase-producing Enterobacteriaceae, particularly E. cloacae. bla CTX-M genes were associated with ISEcp1 or ISCR1. This is the first report on the genetic environment of bla CTX-M in Korean isolates. (Korean J Clin Microbiol 2008;11:90-97) Key Words: Extended-spectrum β-lactamase, CTX- M, Genetic characteristics 서 1983 년 extended-spectrum β-lactamase (ESBL) 를생성하는 Klebsiella pneumoniae가발견된이래로, 전세계적으로여러균종에서 ESBL 생성균주가증가하고있다 [1]. 과거에는대부분의 ESBL이 TEM 혹은 SHV형이었으나최근새로운종류의 ESBL인 CTX-M, VEB, GES 등이출현하였다 [2-4]. CTX-M 형 ESBL은 ceftazidime에비해 cefotaxime을가수분해활성이더강한특성이있다 [5]. 이효소는 1980년대후반일본, 유럽그리 Received 4 February, 2008, Accepted 20 August, 2008 Correspondence: Kyungwon Lee, Department of Laboratory Medicine, Yonsei University College of Medicine, 250, Seongsan-ro, Seodaemun-gu, Seoul 120-752, Korea. (Tel) 82-2-2228-2446, (Fax) 82-2- 313-0908, (E-mail) leekcp@yuhs.ac 론 고아르헨티나에서발견되기시작하였고 [6], 1990년대중반부터변이형이급속하게증가하여 [7], 현재까지 60개이상의 CTX-M 효소가보고되었다 (http://www.lahey.org/studies). CTX-M형 ESBL은 Escherichia coli, Klebsiella spp., Proteus mirabilis, Salmonella typhimurium 등의 Enterobacteriaceae에흔한것으로알려져있다 [5]. bla CTX-M 유전자의전이와발현에는여러유전요소가관여한다 [7]. 즉, ISEcp1-like insertion sequence (IS) 는두개의불완전역방향반복서열과 transposase 유전자로추정되는 ORF로구성되어있는데, CTX-M-1, -3, -14, -15 등의 bla CTX-M 유전자상류에있음이보고되었다 [7]. 반면 CTX-M-2와 -9에서는 ISCR1이 bla CTX-M 유전자상류에있으며 sul1 type의 complex integron에존재하는것으로알려져있다 [5,7-10]. 90

Chang-Ki Kim, et al. : CTX-M ESBLs AmpC β-lactamase-producing Enterobacteriaceae 91 국내에서는 2001년 CTX-M-14 을생성하는 Shigella sonnei, E. coli 및 K. pneumoniae가처음으로보고되었고 [11], 그후 CTX-M-3, -9, -12, -15 등이여러그람음성간균에서보고되었다 [12-15]. 그러나종특이적염색체성 AmpC β-lactamase 생성 Enterobacteriaceae에서의 CTX-M 효소생성에관한국내연구가적고, 국내균주의 bla CTX-M 유전자주변구조에대해알려진바가거의없다. 본연구에서는종특이적염색체성 AmpC β-lactamase를생성하는 Enterobacteriaceae 균종을대상으로 bla CTX-M 유전자를검출하고, 이유전자의주변구조를규명하고자하였다. 재료및방법 1. 균주수집서울의한대학병원에서 2006년 2월부터 9월에분리된 C. freundii, E. aerogenes, E. cloacae, S. marcescens 및 M. morganii 일련균주를 50주씩수집하였다. 균종동정은전통적인생화학적방법과 Vitek GNI card (biomérieux Vitek, Marcy l'étoile, France) 를이용하였다. 2. 항균제감수성시험항균제감수성은 Clinical and Laboratory Standards Institute (CLSI) 디스크확산법으로시험하였으며 [16], 정도관리를위해 E. coli ATCC 25923과 Pseudomonas aeruginosa ATCC 27853 를함께시험하였다. 3. ESBL 생성시험 Ceftazidime 및 cefotaxime에내성혹은중등도감수성인균주를선택하여, double disk synergy (DDS) 시험으로 ESBL 생성을확인하였다. 즉, 세균을생리식염수에 0.5 McFarland 농도로부유시킨후 Mueller-Hinton 한천에고르게접종하였고, 배지중앙에는 amoxicillin-clavulanic acid 디스크 (Becton Dickinson Microbiology Systems, Sparks, MD, USA) 를놓고주위에 ceftazidime, cefotaxime 및 cefepime 디스크 (Becton Dickinson) 를 1.5 cm ( 디스크가장자리에서가장자리 ) 거리를두고놓았다. 접종한배지는 35 o C에서하룻밤배양한후, 두디스크사이 Table 1. Sequences of the primers for bla genes and bla CTX-M genetic environment Target Primer name Primer sequence Reference bla CTX-M CTX-M 1F 5 -CCGTCACGCTGTTGTTAGG-3 [27] CTX-M 1R 5 -GACGATTTTAGCCGCCGAC-3 [27] CTX-M 2F 5 -CGGTGCTTAAACAGAGCGAG-3 [27] CTX-M 2R 5 -CCATGAATAAGCAGCTGATTGCCC-3 [27] CTX-M 8F 5 -ACGCTCAACACCGCGATC-3 [27] CTX-M 8R 5 -CGTGGGTTCTCGGGGATAA-3 [27] CTX-M 9F 5 -GATTGACCGTATTGGGAGTTT-3 [27] CTX-M 9R 5 -CGGCTGGGTAAAATAGGTCA-3 [27] CTXM-3A 5 -CCCATGGTTAAAAAATCACT-3 This study CTXM-3B 5 -CCGTTTCCGCTATTACAAAC-3 This study bla TEM TEM F 5 -ATGAGTATTCAACATTTCCGT-3 [27] TEM R 5 -TTACCAATGCTTAATCAGTGA-3 [27] bla SHV SHV F 5 -CCGGGTTATTCTTATTTGTCGCT-3 [27] SHV R 5 -TAGCGTTGCCAGTGCTCG-3 [27] bla GES GES/IBC F 5 -GTTAGACGGGCGTACAAAGATAAT-3 [27] GES/IBC R 5 -TGTCCGTGCTCAGGATGAGT-3 [27] bla VEB VEB1-F 5 -ACGAAGAACAAATGCACAAGG-3 This study VEB1-R 5 -GAACAGAATCAGTTCCTCCG-3 This study orf477 orf477 reverse 5 -ACTTCAAAAATTATGCCACC-3 [7] muca muca lower 5 -GGCATCAGGCAGGGGTAAGG-3 [7] orf513 orf513 5 -TGGAAGAGGGCGAAGACGAT-3 [7] orf513 rev 5 -GCGTTTTATCGGTAGTCGTC-3 [7] 3 CS qaced1 5 -TCGCAACATCCGCATTAAAA-3 [7] qaced1 reverse 5 -TTTTAATGCGGATGTTGCGA-3 [7] suli rev 5 -GCTCAAGAAAAATCCCATCCCC-3 [7] ISEcp1 tnpa1 ISEcp1 5 -AATACTACCTTGCTTTCTGA-3 [7] ISEcp1 5 5 -TTCAAAAAGCATAATCAAAGCC-3 [7] ISEcp1 reverse 5 -CAACCACCTTTCAATCATTTTT-3 [7] IS903 IS903 Rev 5 -CATCATCCAGCCAGAAAGTT-3 [7] tnpa1 IS903 Rev 5 -CGGTTGTAATCTGTTGTCCA-3 [7] pemk pemk F 5 -AGGGGAAATCTGGCTTGTCT-3 [27] pemk R 5 -TCTGTCGAAACAATGGGTCA-3 [27]

92 Korean J Clin Microbiol 2008;11(2):90-97 에억제대가커지면 DDS 양성으로해석하였다. 4. ESBL 유전자검출및 CTX-M 유전자주변구조분석 TEM, SHV, CTX-M, GES 및 VEB형의 ESBL 유전자를 PCR 로검출하기위해사용된시발체는 Table 1과같다. 시험세균을증류수 100μL 에부유시킨후 100 o C에서 10분간가열하여 DNA를추출하였고, 원심분리후상청액 1μL를 template로사용하였다. PCR 반응은시발체의 melting temperature와증폭산물의크기에따라시행하였고, PCR 반응을위해서는 PCR Premix (Bioneer, Chongwon, Korea) 20μL tube를사용하였다. PCR 산물은 1% agarose (Qbiogene Inc., Carlsbad, CA, USA) 에전기영동하여관찰하였고, 유전자증폭이확인된경우 DNA extraction kit (QIAGEN, Hilden, Germany) 을이용하여전기영동 gel에서 DNA를추출하여염기서열분석이사용하였다. 염기서열은양방향모두분석하였다. CTX-M 유전자의주변구조는 PCR mapping 과염기서열분석을통해분석하였다. ISEcp1, IS903 및 ISCR1 등의 bla CTX-M 주 변구조를분석하는데사용한시발체는 Table 1과같다. 5. Pulsed-field gel electrophoresis (PFGE) 분석시험세균을제한효소 XbaI (Takara Biotechnology, Shiga, Japan) 으로처리하여, contour-clamped homogeneous field machine (CHEF-DR II, Bio-Rad Laboratories, Hercules, CA, USA) 으로전기영동하였으며, 결과의해석은 Tenover 등 [17] 의방법에따랐다. 결과 1. ESBL 생성현황시험균주 250주중 ceftazidime 및 cefotaxime에내성이거나중등도감수성인균주는 90주 (36%) 이었고, 이중에서 29주 (12%) 가 DDS 양성이었다. 균종별로는 E. cloacae 50주중 10 주 (20%) 가 ESBL을생성하여가장높은비율을보였다 (Table 2). 한편 M. morganii 50주중에서는단한주만이 ESBL을생 Table 2. Types of ESBL produced by species Species (No. tested) No. (%) of DDS positive ESBL (No. of isolates) CTX-M type SHV type TEM type Mixed ESBLs E. aerogenes (50) 6 (12) - SHV-12 (3) TEM-52 (2) CTX-M-9+SHV-12 (1) E. cloacae (50) 10 (20) CTX-M-3 (1), -9 (1) SHV-12 (3) - CTX-M-9+SHV-12 (5) S. marcescens (50) 8 (16) CTX-M-3 (2) SHV-12 (3) TEM-52 (3) - C. freundii (50) 4 (8) CTX-M-3 (1), -9 (1), -12 (1) SHV-2a (1) - - M. morganii (50) 1 (2) CTX-M-14 (1) - - - Total (250) 29 (12) 8 10 5 6 Abbreviation: DDS, double disk synergy. Table 3. Characteristics of CTX-M type ESBLs in clinical isolates Upstream of bla CTX-M Other CTX-M Isolate Specimen Ward Associated resistances type β-lactamases ISEcp1 ISCR1 C. freundii 06-5-U664 Urine Hematology CTX-M-12 TEM-1 + - GEN TOB C. freundii 06-5-U1734 Urine Medical ICU CTX-M-9 - - + AMK GEN NET TOB COT C. freundii 06-5-P448 Wound Oncology CTX-M-3 TEM-1 + - AMK GEN NET TOB COT E. aerogenes 06-6-R756 Sputum Neurolgogy CTX-M-9 TEM-1, SHV-12 - + GEN NET TOB LEV COT TET E. cloacae 06-2-R1363 Sputum Cardiosurgery CTX-M-9 SHV-12 - + AMK NET TOB LEV COT TET E. cloacae 06-5-R1137 Sputum Neurology ICU CTX-M-9 SHV-12 - + NET TOB COT TET E. cloacae 06-5-R468 Sputum GI CTX-M-9 SHV-12 - + NET TOB COT TET E. cloacae 06-5-R1186 Sputum GI CTX-M-9 SHV-12 - + NET TOB COT TET E. cloacae 06-5-R1124 Sputum GI CTX-M-9 - - + AMK GEN NET TOB LEV COT TET E. cloacae 06-5-C242 Pleural fluid CCU CTX-M-9 SHV-12 - + AMK GEN TOB LEV COT TET E. cloacae 06-4-P445 Wound Neurosurgery CTX-M-3 - + - AMK NET TOB LEV COT TET M. morganii 06-8-P334 Bile Oncology CTX-M-14 TEM-1 + - S. marscecens 06-6-R1088 Sputum Pediatrics CTX-M-3 - + - AMK GEN NET TOB LEV COT TET S. marscecens 06-7-B4288 Discharge ENT CTX-M-3 - + - AMK GEN NET TOB LEV COT TET Abbreviations: GEN, gentamicin; TOB, tobramycin; AMK, amikacin; NET, netilmicin; COT, cotrimoxazole; LEV, levofloxacin; TET, tetracycline.

Chang-Ki Kim, et al. : CTX-M ESBLs AmpC β-lactamase-producing Enterobacteriaceae 93 Fig. 1. Schematic representation of the genetic environment of bla CTX-M genes. (A) C. freundii 06-5-U1734 and E. cloacae 06-2-R1363 (CTX- M-9), (B) C. freundii 06-5-P448, S. marcescens 06-6-R1088 and 06-7- B4288 (CTX-M-3), (C) M. morganii 06-8-P334 (CTX-M-14). 성하였다. 2. ESBL 유전형및 CTX-M 생성균주의항균제감수성 DDS 양성균주 29주중에서유전자형별로 PCR 양성인균주수는 bla SHV 16주, bla CTX-M 은 14주가, bla TEM 은 5주이었다. bla GES 와 bla VEB 양성인균주는없었다. 염기서열분석에서 bla SHV 양성균주 16주중 15주는 bla SHV-12 와염기서열이일치하였고, 한주는 bla SHV-2a 와일치하였다. bla CTX-M 생성 14주중 8주가 bla CTX-M-9 와, 4주가 bla CTX-M-3 과일치하였다 (Table 2). bla CTX-M-12 양성 C. freundii와 bla CTX-M-14 양성 M. morganii도각 각한주씩검출되었다. 5주에서검출된 bla TEM 모두는 bla TEM-52 와일치하였다. bla CTX-M-9 유전자를지닌균주중 6주는 bla SHV-12 를동시에지니고있었다. bla CTX-M 유전자를지닌 14주중 7주는 levofloxacin, 12주는 cotrimoxazole, 10주는 tetracycline에내성이었다. 또한 bla CTX-M-14 유전자를지닌 M. morganii를제외한모든균주가다양한 aminoglycoside 항균제에내성이었다 (Table 3). 3. CTX-M 유전자의주변구조및 PFGE 분석결과 bla CTX-M 유전자상류에서 IS를검출하기위하여 PCR mapping과염기서열분석을시행하였다. 총 8주에서검출된 bla CTX-M-9 유전자모두의상류에서는 ISCR1, bla CTX-M-3 (4주), bla CTX-M-12 (1주) 및 bla CTX-M-14 (1주) 의상류에는 ISEcp1이검출되었다 (Fig. 1). bla CTX-M-9 유전자와연관된 ISCR1 모두의상류에는 dfra16과 aada2 유전자 cassette를가진 class 1 integron이확인되었다. 이들중 2주를임의로선택하여 bla CTX-M-9 하류구조를분석한결과 orf3-like sequence가확인되었다. 한편 bla CTX-M-3 유전자가검출된 4주중 3주의 ISEcp1 상류에서 pemk 유전자, bla CTX-M-3 의하류에서 muca 유전자가확인되었다. M. morganii 균주의 bla CTX-M-14 하류에서는 IS903-like element가검출되었다. 반면 bla CTX-M-12 유전자의상류에는 ISEcp1 Fig. 2. PFGE analysis of CTX-M-9 producing E. cloacae isolates. Lane 1, 06-2-R1363; Lane 2, 06-5-R1137; Lane 3, 06-5-R468; Lane 4, 06-5-C242; Lane 5, 06-5-R1186; Lane 6, 06-5-R1124. Lane; M, DNA molecular marker. 이있었으나 integron이나다른 IS 등의주변구조는검출되지않았다. bla CTX-M-9 유전자함유하는 E. cloacae 6주의염색체 DNA를 XbaI으로처리하여 PFGE를시행한바, banding pattern이서로상이하여이들균주간에역학적인연관성이없는것으로판단되었다 (Fig. 2). 고 ESBL 생성세균의비율은지역과균종에따라차이가큰데, 1997년에서 1999년까지수집된 SENTRY 프로그램균주중에서 K. pneumoniae의 4.9 45.4% 와 E. coli의 4.2 8.5% 가 찰

94 Korean J Clin Microbiol 2008;11(2):90-97 ESBL 생성이의심되었고, 남미와서태평양지역에서북미지역보다 ESBL 생성이더흔한것으로보고되었다 [18]. 국내에서시행된전국규모의연구에의하면 ESBL 생성 E. coli의비율은 9 10.4%, K. pneumoniae는 25 30% 로나타났다 [19,20]. E. coli와 K. pneumoniae 이외의균종에서도 ESBL 생성비율이증가하고있음이보고되었으나 [21-24], 종특이적염색체성 AmpC β-lactamase를생성하는 Enterobacteriaceae의 ESBL 빈도에대해서는알려진바가적다. 그이유는 CLSI에서 E. coli, Klebsiella spp. 및 P. mirabilis 에대해서만 ESBL 검출기준을제시하고있으며, AmpC β-lactamase가다량생산될경우이들균종에서 ESBL 생성을검출하기힘들기때문이다 [24]. 그러나 AmpC β-lactamase가 cefepime을분해하지못하므로 cefepime 디스크로 DDS 시행할경우이들균종에서 ESBL 검출율이향상됨이여러연구자들에의해보고되었다 [21,25,26]. 본연구에서도 ESBL 검출을위해 cefepime 디스크를포함하여 DDS를시행하였는데, 250주중에서 29주 (12%) 가 ESBL을생성하였고, 이중 2주는 cefepime 디스크만양성이었다. 한편본연구에서는 E. cloacae 중 20% (10/50) 가 DDS 양성으로가장흔히 ESBL 생성을생성하였는데, 이는박등 [22] 의보고와유사한결과였다. 국내에서검출되는 ESBL은과거 TEM 및 SHV형이대부분이었으나최근 CTX-M형의분리가증가함이보고되고있다 [12,13,27-31]. 오등 [15] 의연구에따르면국내주요병원분리 E. coli에서가장흔한 ESBL은 CTX-M형 ESBL이었으며, CTX-M-15, -14, -3, -9, 및 -12 등여러 CTX-M형 ESBL이검출되었다. 배등 [12] 은국내분리 K. pneumoniae에서가장흔한 ESBL은 SHV-12이었으며 CTX-M-3, -9, -12, -14, -15 등이검출되었음을보고하였다. 본연구에서는 SHV형 ESBL이가장흔하였으며, CTX-M 형 ESBL이그다음이었다. 단일유전자로는 SHV-12가 15주에서검출되어가장흔하였고, CTX-M-9 생성하는균주는 8주로두번째로흔하였다. 특징적으로 SHV-12 와 CTX-M-9를동시에생성하는균주가 6주있었는데 PFGE 분석결과유전적연관성은낮았다. 국내여러보고에서도 SHV-12와 CTX-M을동시에생성하는 E. coli와 K. pneumoniae 가보고되었는데 cefotaxime과 ceftazidime에모두고도내성이므로이들세균의전파가우려된다고하였다 [12,15]. 또한 CTX-M-3 생성균주가 4주있었고, CTX-M-12 생성 C. freundii 와 CTX-M-14 생성 M. morganii가각각한주씩분리되었다. CTX-M-12 는 2001년케냐에서처음발견된 ESBL로 2006년국내에서도 CTX-M-12 생성 E. coli와 K. pneumoniae가보고되었으나 [12,15,32], C. freundii에서검출된것은세계최초로생각된다. 본연구에서는 CTX-M을생성하는 14균주모두에서 bla CTX-M 유전자의주변구조를분석하였다. CTX-M-3, -12, -14 생성 6 주에서는 bla CTX-M 상류에 ISEcp1이있었고, ISEcp1에다른 IS 가삽입된경우는없었다. C. freundii 06-5-P44816, S. marcescens 06-6-R1088, 06-7-B4288 균주에서발견된 bla CTX-M-3 의주변구조는모두동일하였는데, ISEcp1 상류에는 pemk 유전자, bla CTX-M-3 하류에는 muca 유전자가있었다. pemk 유전자는 postsegregational killing system을 coding하며 plasmid를안정화하는데관여한다 [33,34]. muca는 LexA repressor에결합하는 protein 유전자이다 [33,34]. 이두유전자는 1993년 M. morganii 에서발견된 plasmid R446b 에일렬로존재하고있는데, bla CTX-M-3 를포함하는약 3 kb의 transposon이이들유전자사이에삽입된것으로추정되고있다 [33]. 이와같은 bla CTX-M-3 의주변구조는프랑스, 폴란드에서분리된균주의 bla CTX-M-3 주변구조와동일하였다 [7,33,34]. bla CTX-M-12 유전자의경우 ISEcp1 이상류에있는것을확인하였으며, 주변구조의확인을위한추가적인연구가필요할것으로생각된다. bla CTX-M-14 상류에는 ISEcp1, 하류에는 IS903이있었는데프랑스, 중국및베트남에서검출된 bla CTX-M-14 유전자와동일한구조였다 [7,33]. IS903은 bla CTX-M-14 하류에서통상적으로관찰되지만, Poirel 등 [35] 은이 IS가 bla CTX-M 유전자의이동에는관여하지않는다고보고하였다. 한편 8주에서검출된 bla CTX-M-9 유전자의상류에서는 ISCR1 이관찰되었다. bla CTX-M-2 와 bla CTX-M-9 은 ISCR1을가지고있는 class 1 integron에존재하는것으로알려져있다. 이 complex class 1 integron에는 5 CS와 3 CS 사이에다양한 gene cassette 가존재하며, 첫번째 3 CS와두번째 3 CS 사이에 ISCR1과 bla CTX-M 유전자가위치하는것이특징이있다. bla CTX-M-2 는일반적으로 In35이나 InS21에삽입되어있으며, bla CTX-M-9 은 In60 이나그 variant에존재하는것으로보고되었다 [7,9]. 본연구의 bla CTX-M-9 보유균주에는 dfra16와 aada2 gene cassette를가진 integron이관찰되었으며이는 In60에있는 gene cassette와같았다. 이중 2주에서 bla CTX-M 주변구조를분석하였는데, bla CTX-M-9 하류에서 orf3-like sequence를확인하였으나 In60의구조인 IS3000 및두번째 3 CS는관찰되지않았다. Novais 등 [36] 은 CTX-M-9 생성균주를대상으로한연구에서 In60에다양한 variant가있음을보고하였으며, 본연구에서와같이 orf3-like sequence 하류가소실된경우도있었다. CTX-M-9, CTX-M-14, CTX-M-15 및 CTX-M-32 생성균주의전파는특정 plasmid와연관되어있음이보고되었다 [9]. bla CTX-M-15 의국제적인확산은 incompatibility group FII (IncFII) plasmid의전파와관련있는것으로알려져있다 [37]. Canton 등은 1996년부터 2003년에스페인한병원에서의 bla CTX-M-9 의확산은 narrow host-range plasmid인 IncH12의전파가원인이라고보고하였다 [9]. 본연구에서 CTX-M-9 생성 E. cloacae 는 PFGE 양상이서로상이하여, bla CTX-M-9 을보유하는동일 plasmid가전파되었을것으로생각된다. CTX-M ESBL 생성균주는다른계열항균제에내성인경우

Chang-Ki Kim, et al. : CTX-M ESBLs AmpC β-lactamase-producing Enterobacteriaceae 95 가많다 [9]. Golebiewski 등은폴란드에서확산된 plasmid pctx-m3에 bla CTX-M-3 와 16S rrna methylase 유전자인 arma 를비롯한여러내성유전자가함께있었다고보고하였다 [34]. 또한 CTX-M형 ESBL 생성균주중에는 fluoroquinolone에대한내성비율이높은데 [38], plasmid로전이되는 quinolone 내성유전자인 qnr이나 aac(6 )-Ib-cr 와밀접한연관성이있음이알려져있다 [39-41]. 본연구에서도 CTX-M 생성균주들은 aminoglycoside와 fluoroquinolone, tetracycline 및 cotrimoxazole 등에내성인경우가많았다. 따라서 bla CTX-M 을전달하는 plasmid 를분석할필요가있으며, 16S rrna methylase, qnr 및 aac(6 )-Ib-cr에대한추가연구가필요하다고판단된다. 본연구에서종특이적염색체성 AmpC-β-lactamase 생성 Enterobacteriaceae에서 CTX-M 효소를생성하는균주가흔하였고, CTX-M 유전자는 ISEcp1와연관되었거나 complex class 1 integron에존재함을확인하였다. Enterobacteriaceae에서급속하게증가하고있는 CTX-M ESBL에대한지속적인감시와전파에관련된유전환경및 plasmid에대한분석이필요할것으로생각된다. 감사의글 본연구는 2006년도대한임상미생물학회지원으로이루어진것임. 참고문헌 1. Eckert C, Gautier V, Saladin-Allard M, Hidri N, Verdet C, Ould-Hocine Z, et al. Dissemination of CTX-M-type β-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. 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Chang-Ki Kim, et al. : CTX-M ESBLs AmpC β-lactamase-producing Enterobacteriaceae 97 = 국문초록 = 염색체성 AmpC β-lactamase 생성 Enterobacteriaceae 균종에서의 CTX-M 형 Extended-Spectrum β-lactamase 검출과분자유전학적특성규명 1 대한결핵협회결핵연구원, 2 동의대학교자연과학대학임상병리학과, 3 연세대학교의과대학진단검사의학교실, 4 세균내성연구소김창기 1, 염종화 2, 용동은 3,4, 정석훈 3,4, 이경원 3,4, 정윤섭 3,4 배경 : 세계여러지역에서 CTX-M형 extended-spectrum β-lactamase (ESBL) 를생성하는그람음성간균이증가하고있다. 본연구에서는 AmpC β-lactamase 생성 Enterobacteriaceae 균종에서 CTX-M형 ESBL유전자를검출하고, bla CTX-M 의주변유전구조를규명하고자하였다. 방법 : 2006년 4월부터 9월까지국내 1개대학병원에서분리된 Enterobacter cloacae, E. aerogenes, Citrobacter freundii, Serratia marcescens, Morganella morganii의일련균주를 50주씩수집하였다. ESBL생성은 ceftazidime, cefotaxime, cefepime 및 amoxicillin-clavulanic acid 디스크를이용한 double disk synergy로시험하였다. ESBL 유전자형규명을위해 bla TEM, bla SHV, bla CTX-M, bla GES 및 bla VEB 유전자를 PCR로증폭한후염기서열을분석하였다. CTX-M 이양성인경우 IS element와 integron 등의주변구조를분석하였고염색체를 XbaI으로처리해 pulsed-field gel electrophoresis를시행하였다. 결과 : 시험된 250주중 ESBL 생성주는 E. cloacae 10주, S. marsescens 8주, E. aerogenes 6주, C. freundii 4주, M. morganii 1주의 29주 (12%) 이었다. SHV-12와 CTX-M-9 유전자를동시에가진균주가 6주, SHV-12가 9주, SHV-2a 가 1주, TEM-52 가 5주, CTX-M-3 가 4주, CTX-M-9 가 2, CTX-M-14와 CTX-M-12가각각 1주이었다. 모든 bla CTX-M-9 는 ISCR1을포함하는 class 1 sul1-type integron에있었으며, bla CTX-M-3, bla CTX-M-12 및 bla CTX-M-14 유전자의상류에는 ISEcp1이있었다. CTX-M-3 양성주중 3주는 pemk-isecp1-bla CTX-M-3-mucA의유전자구조를갖고있었다. 결론 : 염색체성 AmpC β-lactamase 생성 Enterobacteriaceae 중에 ESBL 생성균주가흔하였으며가장흔한 ESBL형은 SHV-12이었고, 그다음은 CTX-M-9이었다. CTX-M유전자는 ISEcp1와연관되거나 complex integron에존재하였다. [ 대한임상미생물학회지 2008;11:90-97] 교신저자 : 이경원, 120-752, 서울시서대문구성산로 250 연세대학교의과대학진단검사의학교실 Tel: 02-2228-2446, Fax: 02-313-0908 E-mail: leekcp@yuhs.ac