J. Exp. Biomed. Sci. 11 (2005) 465 471 gyra Mutations Found Among Ofloxacin-resistant Mycobacterium tuberculosis is Isolated from Korea Junho Kim 1, Yeun Kim 1, Kiho Bae 2, Taek-Sun Song 3, Sang-Nae Cho 3 and Hyeyoung Lee 1 1 Department of Biomedical Laboratory Science, College of Health Sciences, 2 Department of Life Science, College of Liberal Arts and Sciences, Yonsei University, Wonju 220-710, 3 Department of Microbiology, Yonsei University College of Medicine, Seoul 120-752, Korea Ofloxacin has antimycobacterial activity that possibly contributes a pivotal role in the second-line drug regimens that are used for the treatment of multidrug-resistant tuberculosis. However, in some communities, the resistance rate of Mycobacterium tuberculosis to this agent is surging. Therefore, a rapid and accurate method that can be used to determine the resistance of M. tuberculosis to the ofloxacin can be very useful for effective treatment of the patients. As an effort to develop such a method, this study was set up to reveal general types of mutations that are related to ofloxacin resistance of M. tuberculosis. From previous studies, it has been well known that ofloxacin resistance is associated with mutations in a gene encoding the gyrase A subunit protein. In this study, we obtained 43 ofloxacin-resistant and 50 ofloxacin-susceptible M. tuberculosis clinical isolates from Masan National TB Hospital, and sequences of DNA fragment of 320 bp, region of gyra corresponding to the ofloxacin resistance-determining region were analyzed. In brief, the results showed that a total of seven mutation types were found at gyra. Theses mutations were all clustered within nucleotides 2574 to 2586 of the gyra gene (codons 88 to 94). Codon 94 was the most frequently substituted site. Twenty-four of the 43 isolates had mutations at this position resulting in a total of five different types of amino acid changes (Asp Ala, Asp Gly, Asp His, Asp Tyr, and Asp Asn). Five isolates contained a mutation at codon 90 resulting Ala Val change. Four isolates had mutations at codon 91 causing a Ser Pro change at this site. Two isolates contained a mutation at codon 88 and each of them resulted in different types of amino acid changes (Gly Cys, Gly Ala). On the other hand, polymorphic site at codon 95 was found in both ofloxacin-resistant and ofloxacin-susceptible isolates. From these results, we concluded that the rate of mutations present in gyra among ofloxacin-resistant M. tuberculosis in Korea is similar to the general rates of mutations found throughout the world. Subsequently, an oligonucleotide probe was designed based on the results of sequence analysis and was used to develop a dot blot hybridization assay system to determine ofloxacin-resistance of M. tuberculosis. To evaluate this probe, dot-blot hybridization was carried out using other 57 clinical isolates, and the results showed that the dot-blot hybridization assay is good for detecting sequence alterations at gyra gene. Key Words: Mycobacterium tuberculosis, Ofloxacin resistance, gyra 서 세계보건기구 (The World Health Organization) 에의해 'global emergency' 로선포되었을만큼결핵은아직까지도퇴치되지않고있는세계적보건문제이다. 현재세계인구의 * 논문접수 : 2005년 11월 12일수정재접수 : 2005년 12월 2일 교신저자 : 이혜영, ( 우 ) 220-710 강원도원주시흥업면매지리 234, 연세대학교보건과학대학임상병리학과 Tel: 033-760-2740, Fax: 033-760-2195 e-mail: hylee@dragon.yonsei.ac.kr 론 1/3이결핵의원인균인 Mycobacterium tuberculosis에감염되어있고매년 3백만이상의사람들이결핵에의해죽어가고있다고보고된다. 결핵은선진국보다는개발도상국이나후진국에서더큰문제인질병인데, 이는열악한환경이나보건정책의부재에그원인이있다고볼수있을것이다 (Canetti et al., 1969). 하지만최근들어서는에이즈처럼면역력이약해진환자들이점점증가하고에이즈환자들은결핵균에더쉽게감염되므로에이즈와 M. tuberculosis에동시에감염된환자의수는증가하는추세에있다. 따라서결핵환자의수도전세계적으로더욱증가되고있는실정이며이로인해다제내성결핵 (Multidrug-resistant tuberculosis: MDR-TB) - 465 -
역시증가하는추세에있다 (Yew et al., 2000). 다제내성결핵이란 1차결핵약제가운데리팜핀 (rifampin) 이나아이나 (isoniazid) 에동시에내성을갖거나 (Meier et al., 1996) 리팜핀을포함한두개이상의약제에내성을가지는결핵균에의한결핵을일컫는다 (Moghazeh et al., 1996). MDR-TB의증가에따라 1차결핵약제에의해치료가어려운환자를치료하기위한 2차결핵약제의중요성이커지고있다 (Jesus Ruiz-Serrano et al., 2000). 2차항결핵약제가운데 Fluoroquinolone (FQ) 계통의약제들이가장유용한것으로알려져있고 3세대퀴놀론항균제로임상에서널리사용되고있다 (Dong et al., 1998). FQ의계통의약제들중결핵치료에는 ofloxacin 이가장많이사용되고있다 (Fattorini et al., 1999). FQ의세포내로의유입은세포막의지질이중층을통해유입되거나세포외막에존재하는 porin 단백질에의해일어나는것으로알려져있다 (Kocagoz et al., 1996). 세포내로들어온 FQ의작용부위는 DNA의나선구조를유지하면서 DNA의잘려진부위를봉합하는 Topoisomerse II로알려져있다. Topoisomerse II는 gyra에서만드는 A subunit과 gyrb에서만드는 B subunit이각각두개씩합쳐져이루어져있다 (Lu et al., 1999). DNA가복제되기위해서는수소결합을통하여붙어있는두개의가닥이분리되어야하는데이때가닥을끊어주고다시결합시키는과정은 subunit A가하며이때필요한에너지를제공하는 ATPase 의기능을 subunit B가담당한다. FQ는 gyrase subunit A에작용하는것으로알려져있다 (Guillemin et al., 1998). 지금까지알려져있는 FQ에대한내성기전으로는 DNA gyrase의친화력감소나 efflux pump에의한 FQ의배출등이있다 (Takiff et al., 1999; Xu et al., 1996). 결핵균에서 FQ에내성을일으키는돌연변이는 DNA gyrase A에서일어난다고알려져있고, 특히 codon 88~94 부위에 FQ 내성과관련된돌연변이가집중적으로존재하는것으로알려져있으며, 이부위를 QRDR (Fluoroquinolone Resistant Determining Region) 이라고한다 (Grimaldo et al., 2001). 이연구는한국에서분리된결핵균주가운데 ofloxacin에내성인결핵균주를대상으로 gyra 유전자변이와 ofloxacin 내성과의연관관계를알아보기위해수행되었다. 재료및방법 1. 균주국립마산결핵병원에내원한결핵환자들로부터채취한샘플가운데결핵균배양검사를통해결핵으로판명된균주를대상으로 Löwenstein-Jensen 배지에서절대농도법을이용하여약제감수성검사 (Drug susceptibility test: DST) 를수행하 였다. 배지에포함된각약제의내성기준농도는다음과같다 : isoniazid 0.2 µg/ml; rifampin 40 µg/ml; ofloxacin 2 µg/ml; para-aminosalicylic acid 1 µg/ml; streptomycin 10 µg/ml; ethambutol 2 µg/ml; ethionamide 2 µg/ml; cycloserine 30 µg/ml; kanamycin 40 µg/ml; pyrazinamide는 pyrazinamidase test로내성여부를검사하였다. 표준균주로는결핵균주 H37Rv를사용하였다. 이가운데 ofloxacin에내성인 43개의결핵균주와감성인 50 균주를대상으로 1차적으로염기서열분석실험을수행하였다. 그리고, dot blot hybridization을위해, 국립마산결핵병원으로부터추가로 ofloxacin에내성인 48개의결핵균주와감성인 9개의결핵균주를얻었다. 2. 결핵균 DNA의분리결핵균 DNA는 cetyltrimethylammonium bromide (CTAB) 를사용한방법에의해준비되었다 (Telenti et al., 1993). Ogawa 배지에서배양된균은생리식염수에현탁하여 70 에서 20 분간불활성화시키고원심분리하여모은뒤상층액을버리고 400 µl TEN 완충액 (0.1 M Tris-Cl ph 8.0, 0.01 M EDTA ph 7.5, 1 M NaCl) 에부유시킨후, 동량의 saturated phenol과지름 0.1 mm의 zilconium bead를넣고 2분 40초동안 minibeadbeater에서저속으로진탕하고 4, 12,000 rpm에서 10분간원심분리하였다. 상층액 400 µl에 120 µl의 5 M NaCl과 100 µl의 CTAB (cethyltrimethylammonium bromide)/nacl (10% CTAB, 0.7 M NaCl) 을첨가하고혼합한후 70 에서 20분간가온기에서처리하였다. 가온후 200 µl의 chloroform/ isoamylalcohol (24:1) 을가하여섞은후 4, 12,000 rpm에서 10분간원심분리하였다. 상층액에동량의 chloroform/soamylcohol (24:1) 을가하고잘혼합한후 4, 12,000 rpm에서 5 분간원심분리하였다. 상층액에 3배의무수냉에탄올을첨가하고잘흔든후 -20 에하룻밤방치하고다음날 4, 12,000 rpm에서 15분간원심분리하여 DNA를침전시켰다. 침전된 DNA를 70% 에탄올로세척한다음물기를제거하고공기중에건조시켰다. 30 µl 멸균증류수에 DNA를녹인후정량하고 -70 에보관하면서사용하였다. 3. PCR gyra 중에서 FQ에대한내성을결정하는부위가포함되는 320 bp 부위를증폭하기위한 PCR은 Thermal Cycler (Gene- Amp PCR System 2700, Perkin-Elmer Cetus, Boston, MA, USA) 를사용하였다. 전체반응부피는 50 µl로하여 PCR Accu- Power premix (Bioneer Co., Daejeon, Korea) 제품을사용하였다. PCR에사용된 Genomic DNA의양은반응당 50 ng이었고 PCR 과정에서주형 DNA대신멸균된증류수를첨가하여 negative control 반응을실시하였다. 이때사용된 PCR primer는 5'-CAGCTACATCGACTATGAGA-3' 와 5'-GGGCT- - 466 -
TCGGTGTACCTCAT-3' 이었다. 증폭반응은 denaturation을 94 에서 5분동안수행한후 94 에서 30초, 50 에서 20 초, 72 에서 30초과정을 35 cycle 한후마지막으로 72 에서 7분동안수행하였다 (Takiff et al., 1994). 이증폭산물을 2% TBE agarose gel에확인한후 PCR 산물로염기서열분석을수행하였다. 4. 염기서열분석 PCR 산물의염기서열을분석하기위해 PCR 산물 extraction kit (Bioneer Co., Daejeon, Korea) 를이용하여 PCR 산물을정제한뒤염기서열분석은제노텍 (Genotech, Daejeon, Korea) 에의뢰하였다. 5. Dot blot hybridizaiton 증폭한 PCR 산물 10 µl를 dot blot buffer (per 100 ml; 0.4 M NaOH, 25 mm EDTA) 190 µl와혼합하여 95 에서 10분동안반응시켜두가닥의 DNA를한가닥으로변성시켰다. 변성된 DNA 용액 200 µl 모두를 Easy-Titer TM ELIFA System (770000, Pierce Biotechnology, USA) dot blot apparatus를이용하여 Hybond-N + membrane (Amersham Life Science, Anersham, UK) 에 blot하였다. Blot된 membrane을기기로부터분리하고 80 에서 2시간동안고열건조하여 blocking하였다. Oligonucleotide labeling은 Image 3'-oligolabelling module kit (RPN5770, Amersham Bioscience, UK) 을사용하였다. 이실험에사용된 probe의염기서열은 5'-GGCGACGCGTCG ATCT- ACGAC-3' 이었다. 표지된 oligonucleotide probe를 Hybridization buffer (7.2 ml 5 SSPE, 0.4 ml 10% SDS, 0.4 ml 100% Denhardt's solution) 에첨가하여 56, 90 rpm 항온수조에서 1시간동안 hybridization 시킨후 0.1% SDS가포함된 2 SSPE로실온에서 30분동안 2회세척하고, 0.1% SDS가포함된 1 SSPE로 62 에서 10분동안 2회세척하였다. Membrane에고정된 PCR product의염기서열과결합된 oligonucleotide probe의 3' 말단의 Fluorescein-11-dUTP label를검출하기위한 chemiluminescent detection 은 Image ECL Detection Kit (RPN3130, Amersham pharmacia biotech, UK) 를사용하였다. Hyperfilm TM (Amersham Bioscience, UK) 에 30분동안노출시키고 X-ray developer로현상하였다. 결과 1. gyra 유전자분석결과 Ofloxacin 감성을보이는 50 결핵균주와 ofloxacin에내성을보이는 43 결핵균주로부터 genomic DNA를분리하여 ofloxacin 내성에관련된것으로알려진 gyra 부위를 PCR을이용하여증폭하였다. 그결과 ofloxacin에감성과내성을보이는총 93개의결핵균주중에서 3 균주 ( 감성 1 균주, 내성 2 균주 ) 를제외한 90 균주에서 codon 95 부위에아미노산이 Ser에서 Thr으로변화되는점돌연변이가존재하였다. 즉, codon 95 부위의점돌연변이는 ofloxacin 감성균주및내성균주모두에존재하며감성균주 50 균주중 49 균주와내성균주 43 균주중 41 균주에존재하였다. 한편, ofloxacin에내성인 43 균주에서는 codon 95 부위의 mutation을제외한총 4개의 codon 부분 (codon 88, 90, 91, 94) 에서총 9종류의돌연변이를발견하였다. 발견된모든종류의돌연변이는 gyra의핵산서열 2574와 2586 부위사이에존재하였다 (codons 88 to 94). 이들돌연변이중 codon 94 부위에일어난돌연변이의종류가가장많아아미노산을변화시키는총 5종류의돌연변이가있었으며 (Asp Ala, Asp Fig. 1. Mutations found in the clinical isolates of oflocxacin resistant M. tuberculosis. A total of nine mutation types were found. Theses mutations were all clustered with in nucleotides 2574 to 2586 of the gyra (codons 88 to 94). Missense mutations within the QRDR have been identified that are associated with ofloxacin resistance. Codon 95 contains a naturally occurring polymorphism. Underlined letters represent altered bases - 467 -
Table 1. Mutations found among 43 ofloxacin-resistant clinical isolates of M. tuberculosis Mutation 부위 Changed AA (Amino acid) No Total % No mutation 4 9 88 GGC (Gly) TGC (Cys) 1 GGC (Gly) GCC (Ala) 1 5 90 GCG (Ala) GTG (Val) 6 14 91 TCG (ser) CCG (Pro) 5 12 GAC (Asp) GGA (Gly) 1 2 GAC (Asp) GGC (Gly) 14 94 GAC (Asp) GCC (Ala) 4 GAC (Asp) TAC (Tyr) 2 GAC (Asp) CAC (His) 2 56 GAC (Asp) AAC (Asn) 2 94 90 GAC (Asp) GGC (Gly) GCG (Ala) GTG (Val) 1 Total 43 100 Table 2. Comparison of results between dot blot hybridization and conventioanl drug susceptible test DST Dot blot S S 7 2 R 25 23 R Gly, Asp His, Asp Tyr, and Asp Asn) (Fig. 1), 총 43개의내성균주중 24개균주가 codon 94 부위에돌연변이가있어가장높은비율의돌연변이를가지고있었다. Codon 94 부위에돌연변이를갖고있지않은 19개의균주중에서는, codon 90 부위의아미노산이 Ala에서 Val로바뀌는결과를가져오는점돌연변이가 5 균주가존재하였고, codon 91 부위의 Ser 을 Pro으로변화시키는돌연변이를가진 4 균주가존재했다. 한편, codon 88 부위에는두개의돌연변이종류가존재하였는데, 하나는아미노산을 Gly에서 Cys로다른하나는 Gly에서 Ala로변화시키는점돌연변이였다 (Fig. 1, Table 1). 결과적으로실험에사용한총 43개의 ofloxacin 내성결핵균주가운데 codon 95 부위에존재하는 polymorphism이아닌여타돌연변이를가지고있는균주의총수는 39 균주였고 4개의균주는이부위에돌연변이가없었다. 2. Dot blot hybridization을위한 probe 개발마산국립결핵병원에서 1차로받아염기서열을분석한결과 QRDR에돌연변이가있는지의여부를 dot blot hybridization을이용하면알아낼수있을것으로생각되었다. 따라서, 돌연변이가없는 QRDR에결합할수있는 wild type (WT) probe를고안하였다. 이 WT probe는 polymorphism 부위인 95 부위를제외한돌연변이부위 (codon 88~94) 에결합할수있는 21 mer 길이의 probe이다. 이 probe를이용하여돌연변이가없는 codon 88~94 부위에는결합하고돌연변이가존재하는부위에는결합하지않도록 hybridization condition을조절하였다. 이어고안된 probe의유용성을알아보기위해, 염기서열분석을통해이미염기서열이확인된균주를이용하여 Fig. 2. Dot blot hybridization using wild type probe with clinical isolates of M. tuberculosis. No.1~5: Ofloxacin susceptible clinical isolates of M. tuberculosis that do not have mutations at QRDR region; No.6~14: Ofloxacin-resistant clinical isolates of M. tuberculosis that have various kinds of mutations: No.6; 88GGC TGC, No.7; 88GGC GCG, No.8; 90GCG GTG, No.9; 91TCG CCG, No.10; 94GAC AAC, No.11; 94GAC GGC, No.12; 94GAC GCC, No.13; 94GAC TAC, No.14; 94GAC CAC; No.16~45: Clinical isolates of M. tuberculosis their sequence alterations of QRDR are not known. dot blot hybridization을실시하였다. 그결과 WT probe는 gyra의 QRDR에돌연변이가없는균주의 PCR 산물에는결합하는반면어떤종류의돌연변이라도돌연변이가존재하는균주의 PCR 산물에는결합하지않음을알수있었다 (Data not shown). 이로써 design한 WT probe가돌연변이가없는 wild type 염기서열에만특이적으로결합반응을보인다는것을알수있었다. 3. Dot blot hybridization 을이용한임상결핵균주의 ofloxacin 내성검출 Dot blot hybridization을이용하여 2차적으로마산국립결핵병원에서제공받은 oflocxacin 감성 9 균주와 oflocxacin 내성 48 균주의총 57개의균주를가지고그유용성을확인하는실험을실시하였다 (Fig. 2). 실험을통해얻어진 dot blot hybridization 결과와 ofloxacin 감수성결과를비교해보니일치된결과를보이는균주가총 30주였다. 즉, DST 감수성이고 dot blot hybridization에붙는균주가 7 균주였고 DST 내성이고 dot blot hybridization에붙지않는균주가 23 균주였다. 반면, dot blot hybridization에붙고 DST 결과가내성인균주가 25주였으며 dot blot hybridization에붙지않았는데 - 468 -
DST 결과가감수성인균주도 2주있었다 (Fig. 2, Table 2). Dot blot hybridization 실험결과를확인하기위해, 이중 dot blot hybridization에의해 gyra 돌연변이가없는것으로확인된 10개균주와돌연변이가있는것으로확인된 10개균주, 총 20개균주를골라 ofloxacin 내성부위와관련된 gyra 부위를염기서열을분석하였다. Dot blot hybridization 결과와염기서열분석결과는 100% 일치하였다. 따라서, 2차적으로마산국립결핵병원에서제공받은 57개의균주중 gyra 부위의돌연변이에의한결핵균주의 ofloxacin 내성율은 52% 였다. 고찰본연구에서는한국에서분리된 M. tuberculosis 임상균주를대상으로 ofloxacin 내성과관련된것으로알려진 gyra의 QRDR에존재하는돌연변이분포양상을분석하였다. 그결과, gyra의 codon 88~94 부위가특히 ofloxacin 내성과밀접한연관관계가있음을알수있었다. 분자생물학적방법인 hybridization assay를이용하여신속하고정확하게 gyra의 QRDR을검출할수있는지를평가하였다. 이를위해, 마산국립결핵병원으로부터 1차적으로얻은 ofloxacin 감성을보이는 50 결핵균주와 ofloxacin에내성을보이는 43 결핵균주로부터 genomic DNA를분리하여 ofloxacin 내성에관련된것으로알려진 gyra의 QRDR 부위를 PCR을이용하여증폭하여염기서열분석을수행하였다. gyra의염기서열을분석한결과, 총 93개의결핵균주중 ofloxacin 감성균주나내성균주를막론하고 90 균주에서 codon 95 부위에돌연변이가존재하는것을알수있었다. 따라서 codon 95 부위의돌연변이는이전논문들에서도보고된바와같이, ofloxacin 내성과관련없는 polymorphism 부위임을알수있었다. Ofloxacin 내성을갖는 43 균주가운데 39 균주가 gyra에서총 4개의 codon 부분에서총 9종류의돌연변이를갖고있었다. 이들돌연변이는모두 QRDR로알려진서열 2568에서 2586 사이에모여있었다 (codon 88 to 94). 이들돌연변이중, codon 94 부위에일어난돌연변이의종류가 5가지로가장많았고, 염기서열이분석된 43개의내성균주중에서 24개결핵균주가이부위에돌연변이가있어서 (56%) 가장높은비율의돌연변이를가지고있었으며, 이들모두아미노산을변화시키는 missense 돌연변이였다 (Fig. 1). Codon 90 부위의돌연변이가 5개 (14%), codon 91 부위에서돌연변이가 4개 (12%), codon 88 부위에서의돌연변이가 2개 (5%) 를차지했다. 결과적으로실험에사용한총 43 균주의 ofloxacin 내성결핵균주가운데 95 polymorphism 이아닌내성관련돌연변이를가지고있는균주의총수는 39 균주였 고 4개의균주는이부위에돌연변이가없었기때문에 grya 부위의돌연변이에의한결핵균주의 ofloxacin 내성율은 91% 였다. 이비율은지금까지알려진연구결과 (40~80%) 와비교해볼때약간높은수치이다. 따라서더많은 ofloxacin 내성결핵균주의 QRDR에존재하는돌연변이를 PCR-직접염기서열분석이아닌더신속하고간편한방법으로 dot blot hybridization을위한 probe (WT probe) 를고안하였다. 고안된 probe와 dot blot hybridization을이용하여염기서열이확인된균주를대상으로 WT probe가돌연변이가없는균주를검출할수있는지의여부를확인하였다. 이 probe는 ofloxacin 내성에관여하는것으로잘알려진 QRDR 돌연변이부위 (codon 88~94) 를포함하지만 polymorphism 부위인 95 부위는포함하지않는 probe이다. 이어, dot blot hybridization을이용하여 2차적으로마산국립결핵병원에서제공받은 oflocxacin 감성 9개균주와 oflocxacin 내성 48개균주의총 57개균주를가지고그유용성을확인하는실험을실시하였다. 실험을통해얻어진 dot blot hybridization 결과와 ofloxacin 감수성결과를비교해본결과총 57개중 30개가일치하여약 52.6% 가일치성을보였다. 이중에서, DST 감수성이고 dot blot hybridization에붙는균주가 7 균주였고 DST 내성이고 dot blot hybridization에붙지않는균주가 23 균주였다. Dot blot hybridization 결과와 ofloxacin 감수성결과의일치를보이지않은 27 균주 (47.4%) 는 dot blot hybridization 에는붙고 DST 결과가내성인 25 균주와 dot blot hybridization에붙지않지만 DST 결과는감수성인 2 균주였다. Dot blot hybridization에붙고 DST 결과가내성인결과를보이는균주는 ofloxacin 대한내성의원인이 gyra의 QRDR 부위의돌연변이때문이아닌다른원인에의해생기는 ofloxacin 내성균주이기때문으로생각할수있다. 이전까지의연구결과를보면, 지금까지알려져있는 gyra에존재하는 QRDR 부위의돌연변이이외의 FQ에대한내성기전으로는 gyrb의돌연변이, DNA gyrase의친화력감소나 efflux pump에의한 FQ의배출이있다 (Takiff et al., 1999; Xu et al., 1996). Dot blot hybridization에붙지않았는데 DST 결과가감수성인균주도 2주존재하였는데이는 DST 실험이잘못되었거나감성균주와내성균주가섞인샘플인것으로생각되어진다. Dot blot hybridization 실험결과를확인하기이중 dot blot hybridization에의해 gyra 돌연변이가없는것으로확인된 10개균주와돌연변이가있는것으로확인된 10개균주, 총 20개균주를골라 ofloxacin 내성부위와관련된 gyra 부위를염기서열분석하였다. Dot blot hybridization 결과와염기서열분석결과는 100% 일치하여개발된 probe가유용함을알았다. - 469 -
2차로얻은결핵균주중약제감수성결과가내성인균은 48개였고 dot blot hybridization 결과에의해내성을보이는균은 25 균주였다. 즉, grya 부위의돌연변이에의한결핵균주의 ofloxacin 내성율은 52% 였다. 이는 1차로얻은 ofloxacin 내성균주가운데 QRDR에돌연변이를가지고있는비율과비교할때와는현격한차이를보이는것이었다. 따라서앞으로좀더많은균주를대상으로 ofloxacin 내성균주가운데 gyra에돌연변이가있는균주의비율을조사할필요가있을것으로생각된다. 감사의글이논문은 2002년도에선정된한국학술진흥재단의신진교수지원사업연구비 (KRF-2002-003-E00140) 지원에의해이뤄졌으며이에감사드립니다. REFERENCES Alangaden GJ, Manavathu EK, Vakulenko SB, Zvonok NM, Lerner SA. Characterization of fluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients. Antimicrob Agents Chemother. 1995. 39: 1700-1703. Canetti G, Wallace F, Khomenko A, Mahler H, Menon N, Mitchison D, Rist N, Smelev N. Advances in techniques of testing mycobacterial drug sensitivity and the use of sensitivity tests in tuberculosis control programmes. Bull WHO. 1969. 41: 21-43. Dong Y, Xu C, Zhao X, Domagala J, Drlica K. Fluoroquinolone action against mycobacteria: effects of C-8 substituents on growth, survival, and resistance. Antimicrob Agents Chemother. 1998. 42: 2978-2984. Dong Y, Zhao X, Domagala J, Drlica K. Effect of fluoroquinolone concentration on selection of resistant mutants of Mycobacterium bovis BCG and Staphylococcus aureus. Antimicrob Agents Chemother. 1999. 43: 1756-1758. Dong Y, Zhao X, Kreiswirth BN, Drlica K. Mutant prevention concentration as a measure of antibiotic potency: studies with clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2000. 44: 2581-2584. Fattorini L, Iona E, Ricci ML, Thoresen OF, Orru G, Oggioni MR, Tortoli E, Piersimoni C, Chiaradonna P, Tronci M, Pozzi G, Orefici G. Activity of 16 antimicrobial agents against drugresistant strains of Mycobacterium tuberculosis. Microb Drug Resist. 1999. 5: 265-270. Fung-Tomc J, Minassian B, Kolek B, Washo T, Huczko E, Bonner D. In vitro antibacterial spectrum of a new broad-spectrum 8-methoxy fluoroquinolone, gatifloxacin. J Antimicrob Chemother. 2000. 45: 437-446. Gillespie SH, Billington O. Activity of moxifloxacin against mycobacteria. J Antimicrob Chemother. 1999. 44: 393-395. Grimaldo ER, Tupasi TE, Rivera AB, Quelapio MI, Cardano RC, Derilo JO, Belen VA. Increased resistance to ciprofloxacin and ofloxacin in multidrug-resistant Mycobacterium tuberculosis isolates from patients seen at a tertiary hospital in the Philippines. Int J Tuberc Lung Dis. 2001. 5: 546-550. Guillemin I, Jarlier V, Cambau E. Correlation between quinolone susceptibililty patterns and sequences in the A and B subunits of DNA gyrase in mycobacteria. Antimicrob Agents Chemother 1998. 42: 2084-2088. Jesus Ruiz-Serrano M, Alcala L, Martinez L, Diaz M, Marin M, Jose Gonzalez-Abad M, Bouza E. In vitro activities of six fluoroquinolones against 250 clinical isolates of Mycobacterium tuberculosis susceptible or resistant to first-line antituberculosis drugs. Antimicrob Agents Chemother. 2000. 44: 2567-2568. Ji B, Lounis N, Maslo C, Truffot-Pernot C, Bonnafous P, Grosset J. In vitro and in vivo activities of moxifloxacin and clinafloxacin against Mycobacterium tuberculosis. Antimicrob Agents Chemother. 1998. 42: 2066-2069. Kitamura A, Hoshino K, Kimura Y, Hayakawa I, Sato K. Contribution of the C-8 substituent of DU-6859a, a new potent fluoroquinolone, to its activity against DNA gyrase mutants of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1995. 39: 1467-1471. Kocagoz T, Hackbarth CJ, Unsal I, Rosenberg EY, Nikaido H, Chambers HF. Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra. Antimicrob Agents Chemother. 1996. 40: 1768-1774. Lalande V, Truffot-Pernot C, Paccaly-Moulin A, Grosset J, Ji B. Powerful bactericidal activity of sparfloxacin (AT-4140) against Mycobacterium tuberculosis in mice. Antimicrob Agents Chemother 1993. 37: 407-413. Lu T, Zhao X, Drlica K. Gatifloxacin activity against quinoloneresistant gyrase: allele-specific enhancement of bacteriostatic and bactericidal activities by the C-8-methoxy group. Antimicrob Agents Chemother. 1999. 43: 2969-2974. Lu T, Zhao X, Li X, Drlica-Wagner A, Wang JY, Domagala J, Drlica K. Enhancement of fluoroquinolone activity by C-8 halogen and methoxy moieties: action against a gyrase resistance mutant of Mycobacterium smegmatis and a gyrase- - 470 -
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