Short Communication
Characterization of gyrA and gyrB mutations associated with fluoroquinolone resistance in Mycobacterium tuberculosis isolates from Morocco

https://doi.org/10.1016/j.jgar.2017.10.003Get rights and content

Highlights

  • Fluoroquinolones (FQs) are the cornerstone of treatment for drug-resistant TB.

  • 30% of multidrug-resistant TB (MDR-TB) isolates harboured FQ resistance mutations in gyrA.

  • All gyrA resistance mutants belonged to the LAM lineage, raising the possible emergence of a specific clone.

  • The results highlight the high prevalence of FQ resistance among MDR-TB isolates in Morocco.

  • Rapid detection of FQ resistance once MDR-TB is confirmed is critical to adjust treatment promptly.

Abstract

Objectives

Fluoroquinolones (FQs) are the cornerstone of treatment for drug-resistant tuberculosis (TB). They are the most effective second-line antimycobacterial drugs and are recommended for the treatment of multidrug-resistant TB (MDR-TB). However, it is widely accepted that FQ resistance is high among MDR-TB isolates. Thus, characterisation of mutations conferring resistance to FQs will be of a great interest for effective and efficient management of TB resistance in Morocco.

Methods

A laboratory collection of 30 Mycobacterium tuberculosis isolates previously characterised as phenotypically and genotypically MDR as well as 20 randomly selected pan-susceptible isolates were included in this retrospective study. The mutation profiles associated with resistance to FQs were assessed by PCR and DNA sequencing. Target sequences for two genes (gyrA and gyrB) were examined. All strains had their fingerprint previously established by spoligotyping.

Results

Molecular analyses showed that 30% of the MDR-TB isolates harboured FQ resistance mutations in gyrA, with the most prevalent being an alanine to threonine at position 90 (Ala90Thr) (56%; 5/9). None of the isolates harboured mutations in gyrB. All gyrA resistance mutant strains belonged to the LAM lineage, mostly LAM9, raising the possible emergence of a specific clone (gyrA mutant/LAM9).

Conclusion

The results of this preliminary study highlight the high prevalence of FQ resistance among MDR-TB isolates in Morocco and consequently the need for rapid detection of FQ resistance once MDR-TB is confirmed to adjust treatment in a timely manner and to interrupt the propagation of more severe forms of M. tuberculosis drug resistance.

Introduction

During the last decades, the emergence of drug resistance in Mycobacterium tuberculosis has made an effective control strategy for tuberculosis (TB) indispensable. TB control is hampered by the rapid spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB, both in new and previously treated cases, which warrants the need for decisive action to adjust therapies and to prevent further resistance [1].

Fluoroquinolones (FQs) are the most effective antimicrobial agents used to treat MDR-TB. Unfortunately, FQs have been widely prescribed in the treatment of undiagnosed respiratory bacterial infections [2], hence their uncontrolled and inappropriate use may contribute to FQ resistance in M. tuberculosis, which may influence the clinical outcome for MDR-TB patients. Thus, recognising the sensitive/resistant FQ status will provide new insights to develop appropriate regimens for MDR-TB patients.

Resistance to FQs such as ofloxacin, commonly used to treat MDR-TB, is thought to be mediated by mutations in the target genes gyrA and, less frequently, gyrB, which encode the respective subunits of DNA topoisomerase gyrase [3]. Most mutations conferring resistance to FQs are known to be accumulated in two short discrete regions of gyrA and gyrB termed the quinolone resistance-determining regions (QRDRs) [4], [5].

FQ resistance is increasingly reported worldwide. Hence, the present preliminary study was planned to assess the mutational status of gyrA and gyrB genes both in pan-susceptible and MDR-TB isolates from Morocco and to analyse the relationship between genotypes and FQ drug resistance patterns.

Section snippets

Sample collection

A collection of 30 MDR-TB and 20 pan-susceptible M. tuberculosis strains selected from our laboratory collection was included in this work [6], [7]. These strains were isolated from patients with pulmonary TB. The clinical status of patients revealed that 18 (36%) were new cases, 15 (30%) were relapses, 10 (20%) had failed treatment and 7 (14%) had defaulted.

It should be underlined that: (i) only good quality bacterial lysis was subject to the present study: in fact, many samples were damaged

Results

Both susceptible (n = 20) and MDR-TB (n = 30) isolates were subject to DNA sequencing of the gyrA and gyrB genes. None of susceptible isolates harboured any single nucleotide polymorphisms (SNPs) in the respective genes. Among the 30 genotypically MDR-TB strains, 9 (30%) had FQ resistance mutations in the gyrA gene but none harboured any SNPs in the gyrB gene. Genotyping results regarding resistance to FQs are reported in Table 1. The results showed that the most prevalent resistance mutation in

Discussion

Early diagnosis of MDR/XDR-TB is crucially important for a rational treatment regimen to prevent further transmission of drug-resistant TB. The present study focused on FQ resistance. The results showed that 30% (9/30) of MDR-TB isolates carried resistance mutations in gyrA at codons 90 (56%; 5/9) and 94 (44%; 4/9), with Ala90Thr being the most common (n = 5). These findings are in agreement with previously reported data, although it is suggested that mutations at codon 94, rather than codon 90,

Conclusion

The results of this study demonstrate the utility of detection of mutations associated with resistance to FQs, which is crucial for predicting phenotypic resistance to FQs, for optimising TB treatment and for preventing further transmission of drug-resistant M. tuberculosis strains. These findings emphasise the need for implementation of DST to SLDs in routine practice along with accurate and rapid molecular tests for the detection of FQ resistance mutations once MDR-TB is diagnosed.

Funding

This study was funded in part by the International Atomic Energy Agency under the RAF6040 project and the Regional Office for the Eastern Mediterranean/World Health Organization under the RPC/RAB&GH 10/11-03 project.

Competing interests

None declared.

Ethical approval

Not required.

References (18)

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