Ann Clin Microbiol.  2014 Sep;17(3):80-85. 10.5145/ACM.2014.17.3.80.

Molecular Detection of Fluoroquinolone Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates

• Kim CK
• Lee BS
• Choi MJ
• Kim HJ
• Lee K

• KMID: 1971099
• DOI: http://doi.org/10.5145/ACM.2014.17.3.80

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 microg/mL, but FQ susceptible isolates showed MICs ranging from < or =0.06 to 0.5 microg/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.

Keyword

Fluoroquinolone; gyrA; gyrB; Multidrug-resistant tuberculosis

MeSH Terms

Codon
DNA
Fluoroquinolones
Levofloxacin
Microbial Sensitivity Tests
Mycobacterium tuberculosis*
Ofloxacin
Tuberculosis
Tuberculosis, Multidrug-Resistant
Codon
DNA
Fluoroquinolones
Ofloxacin

Figure

• 

  Fig. 1. Distribution of fluoroquinolone minimum inhibitory concentration (MIC) of FQ-resistant multidrug-resistant tuberculosis (MDR-TB) isolates (n=71) and FQ-susceptible control isolates (n=25) according to gyrA and gyrB mutation patterns. This figure displays the number of isolates at each MIC and mutation patterns. The MICs of FQ-resistant isolates are close to but clearly discriminated from those of FQ-susceptible isolates. There is no apparent distinction in the MIC distribution between gyrA and gyrB mutation groups. (GyrA, GyrB and GyrA+B) (A: Ofloxacin, B: Levofloxacin, C: Moxifloxacin.) Abbreviations: SC, susceptible control; WT, wild type; gyrB, isolates with mutation in gyrB; gyrA, isolates with mutation in gyrB; gyrA+B , isolates with mutations in both gyrA and gyrB.

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