Emergence of Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates from China as Determined by gyrA Mutation Analysis Using Denaturing High-Pressure Liquid Chromatography and DNA Sequencing

Abstract

A high rate of double point mutations in gyrA (56% of 87 ofloxacin-resistant Mycobacterium tuberculosis clinical isolates) indicates the emergence of fluoroquinolone resistance. This is the first report to describe denaturing high-pressure liquid chromatography analysis of mutations in gyrA of M. tuberculosis in a large number of clinical isolates.Up to the present, fluoroquinolones have been studied as a first-line treatment for tuberculosis (9). However, fluoroquinolone resistance among Mycobacterium tuberculosis strains is emerging, with important implications for treatment (4, 6). Fluoroquinolones have been widely used for tuberculosis treatment in China for more than 10 years and have been given routinely as monotherapy for the empirical treatment of numerous outpatient infections. Thus, China may be one of the countries with the highest rate of fluoroquinolone abuse and resistance in the world. The goal of this work was to identify quinolone resistance-determining regions (QRDRs) of gyrA in ofloxacin-resistant isolates from China by denaturing highpressure liquid chromatography (DHPLC) and DNA sequencing methods.( All the isolates contain a naturally occurring polymorphism, codon 95 AGC3ACC. Seventy-three (84%) of the 87 ofloxacin-resistant clinical isolates were found to carry a codon 94 mutation, and 49 (56%) were found to harbor a double point mutation. 4566on May 9, 2018 by guest

Mycobacterium tuberculosis Population Structure Determines the Outcome of Genetics-Based Second-Line Drug Resistance Testing

“…We propose that the relative proportions of resistant and susceptible clones or the number of different resistant clones present in a sputum isolate reflects different stages in the progression toward clonal resistance. This conclusion differs from a previous report which suggested that multiple mutations in the QRDR lead to higher levels of resistance (32). An alternative explanation could be that of laboratory crosscontamination.…”

A Systematic Review Of Gyrase Mutations Associated With Fluoroquinolone-Resistant Mycobacterium Tuberculosis And A Proposed Gyrase Numbering System

“…19,21,42,46 Heteropeaks in fluoroquinolone-resistant M. tuberculosis have been described in detail previously. 48 In contrast to most bacteria, M. tuberculosis acquisition of drug resistance does not occur as a result of horizontal transfer of resistance-bearing genetic elements.…”

“…c Substitutions GyrA Ala74Ser and Ala90Gly, and GyrB Gly470Ala, Asn499Lys and D644-645 were never observed independently. 18,21,26,33,42,46,47 Systematic review 827 JAC …”

“…37 b Substitutions Ala74Ser and Ala90Gly were never observed independently. 18,26,33,46,47 Systematic review 825 JAC an error in the molecular detection of resistance or the presence of a mixture of strains. For example, in a specimen that harbours mutations leading to GyrA Ala90Val, Asp94Ala and wild-type alleles, results of phenotypic susceptibility testing may have reflected only the wild-type strain.…”

Role of fluoroquinolones in the treatment of tuberculosis

“…Although some of these assays target both gyrA and gyrB genes, the majority of genotypic tests target only QRDR of gyrA gene for simplicity and acceptable levels of sensitivity. The genotypic tests that have been developed include PCR-DNA sequencing [83][84][85], PCR-single strand conformation polymorphism [101], temperature-mediated heteroduplex analysis by using denaturing high-pressure liquid chromatography (HPLC) [102], pyrosequencing [103], microchip array [104], locked nucleic acid probe real-time PCR [105], multiplex PCR amplimer conformation analysis [89] and both, in-house developed as well as commercially available line probe assays [92,96,97]. The FQs are an important component of the therapy regimens for patients with MDR-TB or XDR-TB since their inclusion greatly improves treatment outcome.…”