다제내성결핵균에서 유전자 변이분석을 이용한 퀴놀론 내성검출의 유용성 평가
Molecular Detection of Fluoroquinolone Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates
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배경: Fluoroquinolone (FQ)는 다제내성결핵 치료에 있어서 중요한 약제이다. 근래 광범위한 FQ의 사용으로 인해 결핵균에서의 내성이 증가하고 있어 신속하고 정확한 FQ 내성검사의 필요성이 커지고 있다. 본 연구에서는 분자생물학적 방법을 이용한 결핵균 FQ 내성검출의 유용성을 평가하고자 하였다. 방법: 2009년 1월부터 10월까지 수집된 95개의 다제내성결핵균을 대상으로 하였다. Ofloxacin (OFL), levofloxacin 그리고 moxifloxacin에 대한 감수성은 Lowenstein-Jensen배지 절대농도법으로 시험하였고 내성기준 농도는 동일하게 2.0 μg/mL을 이용하였다. 액체배지 미량희석법으로 세 약제의 최소억제농도를 시험하였으며 gyrA와 gyrB 유전자의 quinolone-resistance determining region (QRDR) 염기서열을 분석하였다. 결과: 95주 중에서 79주가 하나 이상의 FQ에 내성이었고, 이중 71 (89.9%)주에서 gyrA 혹은 gyrB의 QRDR에 변이가 발견되었다. 반면 FQ에 감수성인 균주에서는 변이가 관찰되지 않았다. gyrA 유전자의 codon 94 위치에 변이가 가장 흔하였고 2주에서 gyrB 변이만 관찰되었다. gyrA 유전자 변이가 확인된 균주에 대한 OFL 최소억제농도는 1-32 μg/mL이었으나, FQ 감수성 균주에 대한 OFL 최소억제농도는 ≤0.06-0.5 μg/mL로 차이가 있었다. 결론: gyrA와 gyrB QRDR 변이분석의 FQ 내성검출 민감도와 특이도가 89.9%와 100%로 우수하여 다제내성결핵균에서 신속한 FQ 내성검출에 유용할 것으로 판단되었다. [Ann Clin Microbiol 2014;17:80-85]
Abstract
Background: Fluoroquinolones (FQs) are important drugs for treating multidrug-resistant tuberculosis (MDR-TB). However, due to widespread use of FQs, the resistance rates to FQs have been increasing among Mycobacterium tuberculosis. Rapid and reliable FQ drug susceptibility testing (DST) is crucial for successful treatment of MDR-TB. In this study, the feasibility of molecular detection of FQ resistance was evaluated. Methods: A total of 95 MDR-TB isolates were collected from Jan through Oct 2009 at the Korean Institute of Tuberculosis. DST for ofloxacin (OFL), levofloxacin, and moxifloxacin was performed using the Lowenstein-Jensen media absolute concentration method. Minimum inhibitory concentrations (MIC) of these were determined using the broth microdilution method. DNA was extracted from cultured isolates using bead beating method. The quinolone resistance-determining region (QRDR) of gyrA and gyrB were amplified and those sequences were analyzed. Results: Of 95 isolates, 79 were resistant to at least one of FQs. Of these, 71 (89.9%) harbored mutation in the QRDR of gyrA or gyrB. None of FQ susceptible strains possessed any mutation in gyrA or gyrB. Mutations in codon 94 of gyrA were most common; only two isolates had mutation in only the gyrB gene. OFL MICs for isolates with gyrA mutation ranged from 1 to 32 μg/mL, but FQ susceptible isolates showed MICs ranging from ≤0.06 to 0.5 μg/mL. Conclusion: Mutation analysis of QRDR of gyrA and gyrB showed 89.9% sensitivity and 100% specificity for detecting FQ resistance in MDR-TB. Therefore, molecular DST can be useful for rapid detection of FQ resistance in MDR-TB. (Ann Clin Microbiol 2014; 17:80-85)
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