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210 Ji Youn Sung. PMQR Genes among CTX-M-14 Producing E. coli ORIGINAL ARTICLE Korean J Clin Lab Sci. 2016, 48(3):210-216 http://dx.doi.org/10.15324/kjcls.2016.48.3.210 pissn 1738-3544 eissn 2288-1662 The Prevalence of Plasmid-Mediated Quinolone Resistance Genes among CTX-M-14 Producing Escherichia coli Strains Isolated from a University Hospital in the Chungcheong Province Ji Youn Sung Department of Biomedical Laboratory Science, Far East University, Eumseong 27601, Korea 충청지역에위치한일개의대학병원에서분리된 CTX-M-14 형 ESBL 생성대장균을대상으로 PMQR 유전자빈도조사 성지연 극동대학교임상병리학과 Recently, there has been a considerable increase in the prevalence of CTX-M type extended-spectrum -lactamase (ESBL)-producing E. coli isolates worldwide, including Korea. To investigate the ESBL genes in the E. coli strains isolated from a university hospital in the Chungcheong area, a study was conducted using PCR amplification and nucleotide sequence analysis of the amplified products to detect the plasmid mediated quinolone resistance (PMQR) genes in ESBL producing E. coli isolates. The number of CTX-M-14 producing isolates was 25 (16.0%) isolates, and of them, 9 (5.8%) isolates also produced CTX-M-15. All CTX-M type ESBL producing E. coli isolates showed resistance to cefotaxime. Twelve (48%) CTX-M type ESBL producing E. coli isolates contained the PMQR genes, 8 contained qnrs1, and 8 contained aac(6 )-Ib-cr. Four isolates harbored both qnrs1 and aac(6 )-Ib-cr genes. In our study, we confirmed that the plasmid mediated antimicrobial resistant determinants the ESBL and PMQR genes were distributed in the E. col isolates. To prevent further spreading of the resistant genes among the E. coli isolates, consistent effort is required to investigate and monitor the resistant genes. Key words: CTX-M type, Extended-spectrum -lactamase, Plasmid mediated quinolone resistance, E. coli Corresponding author: Ji Youn Sung Department of Biomedical Laboratory Science, Far East University, Wangjang-ri, Gamgok-myeon, Eumseong 27601, Korea Tel: 82-43-879-3668 Fax: 82-43-880-3876 E-mail: azaza72@naver.com This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2016 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: July 30, 2016 Revised: August 29, 2016 Accepted: September 5, 2016 서론대부분의대장균 (Escherichia coli) 은사람과동물의장내에정상균무리로존재하지만때로는사람에게심한설사를동반한질환을일으키기도한다. 이러한대장균은병독성에따라크게 diffusely adherent E. coli (DAEC), enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), 및 enterotoxigenic E. coli (ETEC) 등의 6개의그룹으로분류되는데이중 ETEC는개발도상국가의어린이와여행자에게설사를일으키며 EAEC는개발

Korean J Clin Lab Sci. Vol. 48, No. 3, September 2016 211 도상국가및선진국가에서설사질환을일으키는원인균으로작용한다 [1]. 이러한대장균을포함한장내병원성세균에의한감염증치료를위해 3세대 cephalosporins 및 fluoroquinolones 이일반적으로사용되어왔다. 그러나대장균을비롯한장내세균과에속한세균들은빈번하게 extended-spectrum -lactamase (ESBL) 를생성하여 penicillins, cephalosporins, 및 monobactams 등 -lactam 계열의항균제에내성을나타내게되었다. 특히 Ambler class A에속하는 CTX-M형 ESBL은최근들어설사를유발하는대장균들사이에빠르게확산되었고전세계적으로대장균에서가장빈번하게검출되는 ESBL이되었다 [2]. CTX-M형 ESBL은최근까지약 50여종이상이보고되었는데아미노산서열에기초하여 CTX-M-1like (CTX-M-1, -3, -10, -11, -12, -15, -28, 및 FEC-1), CTX-M-2 like (CTX-M-2, -4, -5, -6, -7, -20, 및 TOHO-1), CTX-M-8 like (CTX-M-8), CTX-M-9 like (CTX-M-9, -13, -14,-16, -17, -19, -21, -24, -27 및 TOHO-2), 및 CTX-M-25 등크게 5개의그룹으로분류되고있다 [3]. 국내에서도 1990년대후반까지는장내세균에서분리되는주된 ESBL형은 TEM-52, SHV-2a, 및 SHV-12형이었으나최근들어 CTX-M 형이그리고그중에서도특히 CTX-M-14 및 CTX-M-15형이가장빈번하게분리되고있다 [4]. 한편 CTX-M형을포함한다양한 ESBL 유전자는플라스미드, transposons 및 integrons 같은 mobile genetic elements에의해세균사이에전파되는데이는 ESBL 생성세균의광범위한확산을유발하는원인이된다 [2]. 특히 mobile genetic elements는 ESBL 유전자뿐아니라다양한항균제내성유전자를운반할수있는데그대표적인것이 plasmid mediated quinolone resistance (PMQR) 유전자이다. 대장균이 PMQR 유전자를 mobile genetic elements 를통해획득할경우 quinolone 계열의항균제에도내성을나타낼수있게된다 [5]. Quinolone 계열의항균제는 1960년대부터세균감염을치료하기위해보편적으로사용되어온항균제이나최근 quinolone 계열의항균제에내성을나타내는장내세균이광범위하게확산되고있다. 세균이 quinolone 계열의항균제에내성을나타내는주된기전은염색에위치한 DNA gyrase 및 topoisomerase IV 유전자의돌연변이, 유출펌프의과량발현, 및외막포린단백감소등염색체상의돌연변이에의한다 [6]. 그러나최근 qnra, qnrb, qnrs, aac(6 )-Ib-cr, 및 qepa 등과같은 PMQR 유전자가장내세균과에속한세균들에서빈번하게검출되고있다 [7]. PMQR 유전자를획득한세균은 quinolone 계열의항균제가 DNA gyrase 및 topoisomerase IV의기능을억제하는것을방해하거나, quinolone 계열의항균제를변형시키거나세포밖으로능동적으로유출시킴으로써내성을나타내게된다 [8]. 세균간의 mobile genetic elements를매개로한 ESBL 및 PMQR 유전자들의수평적확산은다제내성세균의출현을야기할뿐아니라다제내성세균을빠르게확산시킬수있어문제가되고있다. 본연구에서는충청지역에위치한일개의대학병원에서분리된대장균을대상으로 ESBL의생성여부와그유전형을조사하였으며아울러 ESBL 생성대장균사이에확산되어있는 PMQR 유전자의빈도및그종류를확인하였다. 또한 repetitive extragenic palindromic sequence-based PCR (REP-PCR) 을이용하여 ESBL생성대장균의역학적연관성을조사하였다. REP-PCR은 pulsed-field gel electrophoresis (PFGE) 와같이세균감염확산의역학적연구를위해광범위하게사용되는분자생물학적방법이다. PFGE는고도의분별력을가지고있어일반적으로 gold standard 로불리지만시간과비용이많이소요되는단점이있으며고도의숙련도가필요하다. 이에비해 REP-PCR은아주간단하지만강력한 molecular typing 방법이어서최근많이이용되고있다 [9]. 본연구에서는 REP-PCR을이용하여 ESBL생성대장균의 clonality 를분석하였는데이결과는다제내성세균의출현및전파양상을제고하고확산방지책을마련하는데필요한기초자료가될것으로사료된다. 재료및방법 1. 균주의수집 2013년 8월부터 2014년 6월까지충청지역에위치한일개의대학병원에의뢰된임상검체로부터분리된대장균 156균주를대상으로하였다. 항균제내성에상관없이분리된순서대로균주를수집하였으며, 동일환자에서반복분리된균주는수집대상에서제외하였다. 분리된균주를 VITEK GNI card (biomerieux Vitek Inc., Hazelwood, MO, USA) 를이용하여생화학적방법으로동정하였다. 2. ESBL 생성확인시험및항균제감수성시험대장균이 ESBL을생성하는지알아보기위해 Clinical and Laboratory Standards Institute (CLSI) 지침에따라 ESBL 생성확인시험을시행하였다 [10]. Mueller-Hinton 한천배지 (Difco, Cockeysville, MD, USA) 에 McFarland 0.5 탁도의균액을바른후 cefotaxime (BBL, Cockeysville, MI, USA, 30 g) 과 cefotaxime/clavulanic acid (CTC) (BBL, 30/10 g), ceftazidime (BBL, 30 g) 과 ceftazidime/clavulanic acid (CZC) (BBL, 30/10 g) 디스크를놓고 35 o C에서 16 18 시간배양후억제대를측정하여 CTC 또는 CZC에의한억제대가 cefotaxime과 ceftazidime에의한억제대보다 5 mm 이상클경우 ESBL 생성양성균주로판정하

212 Ji Youn Sung. PMQR Genes among CTX-M-14 Producing E. coli 였다. ESBL 생성확인시험에서양성으로판정된균주를대상으로 CLSI 지침에따라 ampicillin cefotaxime, ceftazidime, tobramycin, amikacin, gentamicin, nalidixic acid, ciprofloxacin, 및 levofloxacin (BBL) 에대한감수성을 Mueller-Hinton 한천 (Difco) 을사용하여디스크확산법으로확인하였다 [8]. 정도관리를위해서 Escherichia coli ATCC 25922를동시에시험하여허용범위내에있는지를확인하였다. 3. 항균제내성유전자검출 ESBL 생성확인시험에서양성으로판정된균주를대상으로 ESBL 유전자의유전형확인과 PMQR 유전자의검출을위해기존의시발체 (Table 1) 를사용하여중합효소연쇄반응을수행하였다 [11-14]. 대상균주를 brain heart infusion broth (Difco) 에접종하여 37 o C에서하룻밤진탕배양한후배양액으로부터 DNA 추출시약 ( 솔젠트, 대전, 한국 ) 을사용하여 chromosomal DNA를추출하였다. DNA 추출액 (5 L), 10 Taq buffer (2.5 L), 10 mm dntp mix (0.5 L), primer 각 10 pmol, 0.7U Taq DNA 중합효소 ( 솔젠트 ) 및증류수를혼합하여총부피 25 ml의반응용액을만들었다. Gene Amp PCR System 9600 (Perkin-Elmer Cetus Corp., Norwalk, CT, USA) 으로 95 o C에서 5분간반응시킨후, 95 o C에서 20초, 59 o C에서 40초, 72 o C에서 30초씩 30회증폭반응시키고, 72 o C에서 5분간연장반응시켰다. 각각의 PCR 생산물을 ethidium bromide가포함된 1% agarose gel에서 40 분간전기영동하여 band를확인하였다. 증폭산물을 DNA 추출시약 ( 솔젠트 ) 로분리후, BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA, USA) 와 ABI PRISM 3730xl DNA analyzer (PE Applied Biosystems) 를이용하여염기서열을분석하였다. 4. Repetitive extragenic palindromic sequence-based PCR (REP-PCR) 을이용한 ESBL생성대장균의역학적연관성조사 ESBL 유전자를포함하고있는균주를대상으로 REP1 (5'- IIIGCGCCGICATCAGGC-3') 과 REP2 (5'-ACGTCTTATCAGG- CCTAC-3') 로명명된장내세균의반복서열을시발체로하여 REP-PCR을수행하였다 [15]. 증폭반응은 DNA 추출액 (5.0 L), 10 Taq buffer (5.0 L), 10 mm dntp mix (1.0 L), primer 각 20 pmol, 1.4 U Taq DNA polymerase ( 솔젠트 ) 및증류수를혼합하여 50 L의혼합액으로시행하였다. Gene Amp PCR System 9600 (Perkin-Elmer Centus) 으로 95 o C에서 5분간반응시킨후, 90 o C에서 40초, 42 o C에서 1분, 68 o C에서 7분씩 35회증폭반응시키고, 68 o C에서 15분간연장반응시켰다. 증폭산물 (10 L) 은 ethidium bromide가포함된 2% agarose gels에전기영동한후 Table 1. Oligonucleotides used in this study for detection of plasmid mediated antimicrobial resistant genes Group Primer Sequence (5' 3') Gene Reference ESBL genes TEM-F ATAAAATTCTTGAAGACGAAA bla TEM [11] TEM-R GACAGTTACCAATGCTTAATCA SHV-F GGGTTATTCTTATTTGTCGC bla SHV [11] SHV-R TTAGCGTTGCCAGTGCTC CTX-M-1F GACGATGTCACTGGCTGAGC bla CTX-M1-like [11] CTX-M-1R AGCCGCCGACGCTAATACA CTX-M-2F GCGACCTGGTTAACTACAATCC bla CTX-M2-like [11] CTX-M-2R CGGTAGTATTGCCCTTAAGCC CTX-M-8F AGACCTGATTAACTACAATCCCATTA bla CTX-M8-like [11] CTX-M-8R ACTTTCTGCCTTCTGCTCTGGC CTX-M-9F GCTGGAGAAAAGCAGCGGAG bla CTX-M9-like [11] CTX-M-9R GTAAGCTGACGCAACGTCTG Quinolone resistant genes QnrA-F AGAGGATTTCTCACGCCAGG qnra1 to qnra6 [12] QnrA-R TGCCAGGCACAGATCTTGAC QnrB-F GGMATHGAAATTCGCCACTG qnrb1 to qnrb6 [12] QnrB-R TTTGCYGYYCGCCAGTCGAA- QnrS-F GCAAGTTCATTGAACAGGGT qnrs1 to qnrs2 [12] QnrS-R TCTAAACCGTCGAGTTCGGCG AAC(6')-IbF TGACCAACAGCAACGATTCC aac(6')-ib cr [13] AAC(6')-IbR TTAGGCATCACTGCGTGTTC QepA-F GCAGGTCCAGCAGCGGGTAG qepa [14] QepA-R CTTCCTGCCCGAGTATCGTG Abbreviations: F, sense primer; R, antisense primer.

Korean J Clin Lab Sci. Vol. 48, No. 3, September 2016 213 BioDoc-14 Imagingsystem (UVP, Cambridge, UK) 을이용하여분석하였다. Band의강도와상관없이 band의분자량과개수로각균주를비교하며, 두개이상의밴드차이가있으면역학적상관관계가없는것으로판단하였다 [15]. 결과 1. ESBL 생성균주의분리및항균제감수성양상임상검체에서분리된대장균 156균주를대상으로 CLSI 디스크확산법에의한 ESBL 생성확인시험을시행한결과 20.5% 에해당하는 32균주가양성반응을보였다. 양성결과를보인 32균주를대상으로 Ambler class A에속하는 ESBL (bla TEM, bla SHV, 및 bla CTX-M) 유전자검출을위한중합효소연쇄반응을시행한결과 bla CTX-M9-like 를검출하기위한반응에서 25균주가양성반응을보였다. 그리고 25균주중 9균주는 bla CTX-M1-like 를검출하기위한반응에도동시에양성반응을나타냈다. 유전자의유전형을결정하기위해염기서열분석을실시한결과 bla CTX-M9-like 반응에서생성된 PCR 산물은모두 bla CTX-M-14 로, 그리고 bla CTX-M1-like 반응에서생성된 PCR 산물 은모두 bla CTX-M-15 로확인되었다 (Table 2). 한편 bla CTX-M1-like 및 bla CTX-M9-like 를제외한나머지 bla CTX-M2-like, bla CTX-M8-like, bla TEM, 및 bla SHV 를검출하기위한중합효소연쇄반응에서는 PCR 산물이하나도생성되지않았다. CTX-M-14 및 CTX-M-15 를생성하는대장균 25균주를대상으로항균제감수성시험을한결과 ceftazidime에대한감수성이 76.0% 로가장높았고 gentamicin (48.0%) 이그다음으로높았다. 한편본연구에서검사한 9종류의항균제중 ceftazidime 및 gentamicin을제외한 7종류의항균제에대해서는모든균주가 40% 이하의낮은감수성률을보였다 (Table 3). 2. PMQR 내성유전자검출 CTX-M형 ESBL 유전자를포함하고있는대장균 25균주를대상으로 PMQR 유전자 (qnra, qnrb, qnrs, aac(6 )-Ib-cr, 및 qepa) 의빈도를조사하기위해중합효소연쇄반응을수행한결과 12균주 (48%) 가 PMQR 유전자를가지고있는것으로나타났다. qnrs 유전자를검출하기위한반응에서 8균주 (32%) 그리고 aac(6 )-Ib-cr 유전자를검출하기위한반응에서 8균주 (32%) 가각각양성반응을 Table 2. Characterizations of 25 CTX-M type ESBL producing Escherichia coli Isolates Specimens Antimicrobial resistance genes Antimicrobial susceptibilities ESBL genes PMQR gens AM CTX CAZ NN AK CN NA CIP LEV CD1 Urine bla CTX-M14 qnrs1 R R S R R I R R R CD2 Urine bla CTX-M14 qnrs1 R R S S S S R R R CD3 Urine bla CTX-M14 bla CTX-M15 aac6-1b-cr qnrs1 R R R R R I R R R CD5 Body Fluid bla CTX-M14 R R R S S S R R R CD7 Urine bla CTX-M14 qnrs1 R R S S I S R S S CD9 Urine bla CTX-M14 bla CTX-M15 aac6-1b-cr qnrs1 R R S S S S I R I CD11 Sputum bla CTX-M14 bla CTX-M15 aac6-1b-cr R R R R I R R R R CD17 Urine bla CTX-M14 bla CTX-M15 R R S R I R I S S CD19 Urine bla CTX-M14 aac6-1b-cr qnrs1 R R S S I S R R R CD20 Urine bla CTX-M14 bla CTX-M15 aac6-1b-cr R R S R I R S S S CD24 Blood bla CTX-M14 aac6-1b-cr qnrs1 R R R S S S I R I CD27 Blood bla CTX-M14 R R S S I S R R R CD33 Urine bla CTX-M14 R R S R R S S S S CD34 Urine bla CTX-M14 bla CTX-M15 R R S S I S R I I CD35 Urine bla CTX-M14 R R S I I R R S S CD36 Urine bla CTX-M14 qnrs1 R R S R I R R R R CD37 Urine bla CTX-M14 bla CTX-M15 R R I R I R R R R CD38 Urine bla CTX-M14 bla CTX-M15 R R S R I R R R R CD39 Urine bla CTX-M14 R R S R I R S S S CD42 Urine bla CTX-M14 R R S R I R R R R CD46 Urine bla CTX-M14 bla CTX-M15 R R I I I S R R R CD49 Urine bla CTX-M14 R R S S I S R S S CD50 Urine bla CTX-M14 R R S R S R S S S CD53 Urine bla CTX-M14 aac6-1b-cr R R S R I R R R R CD55 Urine bla CTX-M14 aac6-1b-cr R R S S I S S S S Abbreviations: ESBL, extended-spectrum β-lactamase PMQR, plasmid-mediated quinolone resistance; AM, ampicillin; CTX, cefotaxime; CAZ, ceftazidime; NN, tobramycin; AK, amikacin; CN, gentamicin; NA, nalidixic acid; CIP, ciprofloxacin; LEV, levofloxacin; S, susceptible; I, intermediate resistant, R, resistant.

214 Ji Youn Sung. PMQR Genes among CTX-M-14 Producing E. coli Table 3. Antimicrobial susceptibilities of 25 CTX-M type ESBL producing Escherichia coli Numbers (%) of isolates Agent E. coli containing PMQR genes (n=12) E. coli not containing PMQR genes (n=13) Total E. coli (n=25) S I R S I R S I R AM 0 (0.0) 0 (0.0) 12 (100.0) 0 (0.0) 0 (0.0) 13 (100.0) 0 (0.0) 0 (0.0) 25 (100.0) CTX 0 (0.0) 0 (0.0) 12 (100.0) 0 (0.0) 0 (0.0) 13 (100.0) 0 (0.0) 0 (0.0) 25 (100.0) CAZ 9 (75.0) 0 (0.0) 3 (25.0) 10 (76.9) 2 (15.4) 1 (7.7) 19 (76.0) 2 (8.0) 4 (16.0) NN 6 (50.0) 0 (0.0) 6 (50.0) 4 (30.8) 2 (15.4) 7 (53.8) 10 (40.0) 2 (8.0) 13 (52.0) AK 3 (25.0) 7 (58.3) 2 (16.7) 2 (15.4) 10 (76.9) 1 (7.7) 5 (20.0) 17 (68.0) 3 (12.0) CN 6 (50.0) 2 (16.7) 4 (33.3) 6 (46.2) 0 (0.0) 7 (53.8) 12 (48.0) 2 (8.0) 11 (44.0) NA 2 (16.7) 1 (8.3) 9 (75.0) 3 (23.1) 1 (7.7) 9 (69.2) 5 (20.0) 3 (12.0) 17 (68.0) CIP 3 (25.0) 0 (0.0) 9 (75..0) 6 (46.2) 1 (7.7) 6 (46.2) 9 (36.0) 1 (4.0) 15 (60.0) LEV 3 (25.0) 2 (16.7) 7 (58.3) 6 (46.2) 1 (7.7) 6 (46.2) 9 (36.0) 3 (12.0) 13 (52.0) Abbreviations: PMQR, plasmid-mediated quinolone resistance; AM, ampicillin; CTX, cefotaxime; CAZ, ceftazidime; NN, tobramycin; AK, amikacin; CN, gentamicin; NA, nalidixic acid; CIP, ciprofloxacin; LEV, levofloxacin; S, susceptible; I, intermediate resistant, R, resistant. Fig. 1. Repetitive element sequence-based (REP)-PCR patterns of genomic DNA from twenty-five CTX-M type ESBL producing Escherichia coli isolates. Lane M is 1 kb DNA size marker. 보였으며그중 4균주는두개의반응모두에서양성결과를나타냈다. 생성된 PCR 산물을대상으로유전형을결정하기위해염기서열분석을실시한결과 qnrs와 aac(6 )-Ib-cr 유전자증폭산물은각각 qnrs1와 aac(6 )-Ib-cr 유전자로확인되었다. 본연구에서는 qnra, qnrb, 및 qepa 유전자는검출되지않았다 (Table 2). 3. CTX-M 형 ESBL 생성대장균의유전형분석시험기간중분리된 25균주의 CTX-M형 ESBL 생성대장균이같은클론에서유래되었는지를확인하기위하여 REP-PCR 을수행한결과총 9개의 band 패턴 (A I 형 ) 이확인되었다 (Fig. 1). 그중 A 형 band 패턴을보인균주가 12균주로가장많았으며, A형을제외한나머지 8개의 band 패턴 (B I 형 ) 을나타낸균주는각각 3균주이하였다. 고찰최근들어 CTX-M형 ESBL의종류가급격하게증가하고있으며 CTX-M형 ESBL을생성하는세균또한전세계적을빠르게확산되고있는추세에있다. 국내에서도 CTX-M 형 ESBL을생성하는대장균이빈번하게분리되고있는데그중에서도 CTX-M-14 또는 CTX-M-15 생성대장균이가장많이보고되고있다 [4,16]. 본연구에서도수집된대장균 156균주중약 16.0% 에해당하는 25균주가 CTX-M형 ESBL을생성하는것으로나타났다. 이수치는최근국내에서보고되고있는 CTX-M형 ESBL을생성하는대장균의비율과유사한것으로 CTX-M형 ESBL이국내에서분리되는대장균에서가장빈번하게검출되고있는 ESBL형임을의미한다 [17]. 한편, 일반적으로 CTX-M 형 ESBL 은 ceftazidime 보다는 cefotaxime 을더효과적으로분해하는것으로알려져있다. 본연구에서도 CTX-M형 ESBL을생성하는대장균 25균주가 cefotaxime 에는모두내성을보인반면 ceftazidime 에는 76.0% 가감수성을나타냈다. PMQR 유전자는 quinolone 계열의항균제에대해낮은수준의내성을나타냄에도불구하고 ESBL 유전자와마찬가지로 mobile genetic elements를통해다른세균으로전파될수있어내성확산의우려가있다. 본연구에서도 CTX-M 형 ESBL을생성하는대장균중 48% (12균주) 가 PMQR 유전자를가지고있는것으로나타났다. 특히 DNA gyrase를보호하는단백을암호화하는 qnrs1 유전자를포함하는대장균이 32% (8균주) 나되었다. 이전의중국과국내에서분리된 ciprofloxacin 내성대장균을대상으로한연구에서는각각 7.5% 와 5.6% 에해당하는대장균만이 qnr 유전자를가지고있는것으로보고한바있다 [7]. 본연구에서 qnr 유전자의분리빈도

Korean J Clin Lab Sci. Vol. 48, No. 3, September 2016 215 가월등하게높았던것은 CTX-M형 ESBL을생성하는대장균을대상으로조사했기때문인것으로사료되며이는 qnr 유전자가 CTX-M형 ESBL 유전자와연관되어있음을시사한다. 한편, 이전의연구자들은대장균에서 aac(6 )-Ib-cr 유전자의검출빈도는높은편으로미국의경우 32% 의대장균이그리고국내의경우 ESBL 생성대장균의 34.1% 가 aac(6 )-Ib-cr 유전자를포함하고있다고하였다 [7]. 본연구에서도 quinolone 계열의항균제를변형시켜항균제의기능을못하게하는 aac(6 )-Ib-cr 유전자를포함하는대장균이 32% (8균주) 로이전의보고와유사한결과를나타냈다. PMQR 유전자는 fluoroquinolone 항균제에비록낮은수준의내성을나타내지만치료를위한항균제의선택압력에놓일경우높은농도에도내성을유발할수있다고보고되고있다 [18,19]. 본연구에서는충청지역에위치한일개의대학병원에서분리된대장균을대상으로 ESBL 및 PMQR 유전자의빈도와유전형을조사하였다. 충청지역에서분리된대장균은상당히높은빈도로 ESBL 및 PMQR 유전자를포함하고있었다. ESBL 및 PMQR 유전자는빈번하게 mobile genetic elements를통해세균간에확산되어다재내성세균의출현을야기할수있다. 게다가 CTX-M형 ESBL 생성대장균의 48% (12균주) 가같은클론에서유래되었음을 REP-PCR을통해확인할수있었는데이는 CTX-M형 ESBL 생성대장균이수평확산되었음을의미한다. 따라서다제내성세균의출현및확산을방지하기위해서는지속적인항균제내성유전자에대한조사와모니터링이필요할것으로사료된다. 요약최근들어 CTX-M형 extended-spectrum -lactamase (ESBL) 생성대장균이국내는물론전세계적으로빠르게증가하고있다. 본연구에서는충청지역에위치한일개의대학병원에서분리된대장균을대상으로 ESBL 유전자를중합효소연쇄반응및염기서열분석방법을통해확인하였으며, 같은방법으로 ESBL 생성대장균으로부터 plasmid mediated quinolone resistance (PMQR) 유전자의빈도를조사하였다. 16.0% 에해당하는 25 균주가 CTX-M-14 를생성하였으며이중 9균주는 CTX-M-15도동시에생성하는것으로나타났다. 항균제감수성시험결과 CTX-M형 ESBL을생성하는대장균은모두 cefotaxime에내성을보였다. 한편 CTX-M형 ESBL을생성하는대장균의 48% (12균주) 가 PMQR 유전자를포함하고있음이확인되었는데 8균주가 qnrs1유전자를그리고 8균주가 aac(6 )-Ib-cr 유전자를포함하고있었다. 그중 4균주는두개의유전자를모두가지고있는것으로나타났다. 본연구에서는플라스미드를통해확산될수있는 ESBL 및 PMQR 유전자가대장균 사이에확산되어있음을확인하였다. 항균제내성유전자들의확산을막기위해서는지속적인내성유전자의모니터링과감시가필요할것으로사료된다. Acknowledgements: None Funding: None Conflict of interest: None References 1. Qadri F, Svennerholm AM, Faruque AS, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev. 2005;18:465-483. 2. Canton R, Gonzalez-Alba JM, Galan JC. CTX-M enzymes: origin and diffusion. Front Microbiol. 2012;3:110. 3. Poirel L, Kampfer P, Nordmann P. Chromosome-encoded Ambler class A beta-lactamase of Kluyvera georgiana, a probable progenitor of a subgroup of CTX-M extended-spectrum beta-lactamases. Antimicrob Agents Chemother. 2002;46:4038-4040. 4. Song W, Lee H, Lee K, Jeong SH, Bae IK, Kim JS, et al. CTX-M-14 and CTX-M-15 enzymes are the dominant type of extended-spectrum beta-lactamase in clinical isolates of Escherichia coli from Korea. J Med Microbiol. 2009;58: 261-266. 5. Seo MR, Park YS, Pai H. Characteristics of plasmid mediated quinolone resistance genes in extended-spectrum cephalosporin-resistant isolates of Klebsiella pneumoniae and Escherichia col in Korea. Chemotherapy. 2010;56:46-53. 6. Ruiz J. Mechanisms of resistance to quinolones: Target alterations, decreased accumulation and DNA gyrase protection. J Antimicrob Chemother. 2003;51:1109-1117. 7. Yang HY, Nam YS, Lee HJ. Prevalence of plasmid-mediated quinolone resistance genes among ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from blood cultures in Korea. Can J Infect Dis Med Microbiol. 2014;25:163-169. 8. Robicsek A, Strahilevitz J, Jacoby GA. Fluoroquinolone modifying enzyme: A new adaptation of a common aminoglycoside acetyltransferase. Nat Med. 2006;12:83-88. 9. Bae IK, Kim J, Sun JY, Jeong SH, Kim YR, Wang KK, et al. Comparison of pulsed-field gel electrophoresis & repetitive sequence-based PCR methods for molecular epidemiological studies of Escherichia coli clinical isolates. Indian J Med Res. 2014;140:679-685. 10. CLSI. Performance standards for antimicrobial susceptibility testing; sixteenth informational supplement. CLSI document M100-S20. Wayne, PA: Clinical and Laboratory Standards Institute; 2010, p52-53. 11. Lewis JS, Herrera M, Wickes B, Patterson JE, Jorgensen JH. First report of the emergence of CTX-M-type extended-spectrum beta-lactamases (ESBLs) as the predominant ESBL isolated in a

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