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. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/174523 |
| Date | January 2011 |
| Creators | Lau, Wing-tong, Ricky., 劉永棠. |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47849654 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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