Molecular characterization of fluoroquinolone resistance in mycobacterium tuberculosis

Abstract

The global emergence of drug resistance is posing increasing difficulties in

the public health control and treatment of tuberculosis (TB). Fluoroquinolones (FQs)

are regarded as having a pivotal role among the antimicrobial agents in multidrug

regimens against multidrug-resistant tuberculosis (MDR-TB). Thus, early diagnosis

of fluoroquinolone-resistant (FQr) MDR-TB and extensively drug-resistant

tuberculosis (XDR-TB) by molecular tests has predictive value for the guidance of

TB therapy.

The pharmacokinetic (PK) and pharmacodynamic (PD) indices are valuable

parameters to evaluate the activity and efficacy of fluoroquinolones (FQs) based upon

the bactericidal effect and prevention of the emergence of resistance. In the first part

of this study, the potencies of ofloxacin (OFX) and moxifloxacin (MXF) against

clinical isolates of MDR-TB in terms of their PK/PD indices (Cmax/MIC90,

AUC/MIC90, Cmax/MPC90 and AUC/MPC90) were investigated and compared. The

results revealed that MXF displays higher ratios of PK/PD in vitro and could serve as

a promising agent for the treatment of MDR-TB.

Molecular tests on resistance genes are reliable and rapid technology for

diagnosis of drug-resistant TB which facilitates timely patient management and

public health control of TB. In the second part of the study, the feasibility of a PCRsequencing

assay for the examination of mutations in the quinolone-resistance-determining-

region (QRDR) of the gyrase A (gyrA) gene in FQ-resistant (FQr)

Mycobacterium tuberculosis in direct clinical specimens was evaluated. As

determined by gyrA QRDR DNA sequencing analysis, complete concordance of

phenotypic and genotypic outcomes was demonstrated. The results indicate that the

molecular assay is an accurate and effective method for the diagnosis of FQr TB and

allows identification of mixed resistant variants in the same patient. GyrA mutations

that associated with FQr in clinical isolates of M. tuberculosis were clustered in

hotspot codons 88, 90, 91 and 94, corroborating other reports. We also detected a

novel gyrA Ala74Ser mutation in M. tuberculosis directly from the respiratory

specimens by using the PCR-DNA sequencing assay.

In the third part of this study, the functional effect of the Ala74Ser mutant was

verified through study of the DNA supercoiling inhibitory activities of OFX and

MXF against the recombinant DNA gyrase. Fifty percent inhibitory concentrations

(IC50) of FQs against the DNA supercoiling activities of the recombinant DNA gyrase

complex reconstituted with gyrA Ala74Ser were eight-fold and 14-fold greater than

the wild-type H37Rv reference strain, and results correlated well with their

phenotypic drug susceptibilities. Besides, a combination of gyrA mutations

(Glu21Gln, Ser95Thr and Gly668Asp) was also characterized to be non-functional

polymorphisms. The impact of the gyrA Ala74Ser mutation on drug binding affinity

was elucidated through a crystal structure model of the gyrA-MXF-DNA cleavage

complex. Alanine at position 74 of gyrA in M. tuberculosis, which corresponds to the

alanine at position 67 of gyrA in Escherichia coli, is an amino acid lying in the α3

helix domain which forms a hydrophobic interface between the gyrA-gyrA dimer.

Perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation

probably disturbs the putative drug binding pocket, and leads to the reduction of the

binding affinity of FQ due to the distance effect. This is the first report verifying that

gyrA Ala74Ser mutation alone is responsible for FQr in M. tuberculosis.