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Association of gyrA/B mutations and resistance levels to fluoroquinolones in clinical isolates of Mycobacterium tuberculosis.

ABSTRACT: To evaluate the association between mutations in the genes gyrA/B and resistance levels to fluoroquinolones in clinical isolates of Mycobacterium tuberculosis, a total of 80 ofloxacin-resistant isolates collected in 2009 by the Shanghai Municipal Centers for Disease Control and Prevention were studied. The minimum inhibitory concentration (MIC) of ofloxacin, moxifloxacin and gatifloxacin for each isolate was determined using the microscopic observation drug susceptibility assay. Sequencing was used to identify mutations in the quinolone resistance-determining region (QRDR) of the gyrA and gyrB genes. In total, 68 isolates had mutations in gyrA, three isolates had mutations in gyrB, six isolates had mutations in both gyrA and gyrB, and three isolates had no mutations. Two common mutations in gyrA, the D94G and D94N mutations, were associated with higher-level resistance to all three fluoroquinolones than two other common mutations (A90V and D94A). Understanding the relationship between MICs and mutations in ofloxacin-resistant isolates will facilitate the optimization of the use of new-generation fluoroquinolones to treat patients with ofloxacin-resistant tuberculosis (TB).

SUBMITTER: Li J

PROVIDER: S-EPMC3974338 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Association of gyrA/B mutations and resistance levels to fluoroquinolones in clinical isolates of Mycobacterium tuberculosis.

Li Jing J Gao Xu X Luo Tao T Wu Jie J Sun Gang G Liu Qingyun Q Jiang Yuan Y Zhang Yangyi Y Mei Jian J Gao Qian Q

Emerging microbes & infections 20140312 3

To evaluate the association between mutations in the genes gyrA/B and resistance levels to fluoroquinolones in clinical isolates of Mycobacterium tuberculosis, a total of 80 ofloxacin-resistant isolates collected in 2009 by the Shanghai Municipal Centers for Disease Control and Prevention were studied. The minimum inhibitory concentration (MIC) of ofloxacin, moxifloxacin and gatifloxacin for each isolate was determined using the microscopic observation drug susceptibility assay. Sequencing was u ...[more]

PMID: 26038513

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Project description:BackgroundNearly 5% of all Mycobacterium tuberculosis strains worldwide are resistant at least to rifampicin and isoniazid (multidrug-resistant tuberculosis, MDR-TB). Inclusion of a fluoroquinolone and an injectable agent (kanamycin, amikacin or capreomycin) in multidrug therapy is crucial for proper treatment of MDR-TB. The incidence of MDR-TB in Kuwait is ~1%. MDR-TB strains additionally resistant to fluoroquinolones and injectable agents are defined as extensively drug-resistant (XDR-TB) strains and have been detected in >55 countries. Infections with XDR-TB strains have very poor prognosis. This study detected the occurrence of gyrA mutations associated with fluoroquinolone resistance among MDR-TB strains in Kuwait.FindingsDirect DNA sequencing of quinolone resistance-determining region of gyrA gene was performed to detect fluoroquinolone resistance-associated mutations in 85 MDR-TB strains isolated from 55 TB patients and 25 pansusceptible M. tuberculosis strains. For isolates exhibiting gyrA mutations, 3'-end of rrs (16S rRNA) was sequenced for the detection of XDR-TB. Fingerprinting of fluoroquinolone resistant MDR-TB strains was performed by detecting mutations in three (81 bp hot-spot, N-terminal and cluster II) regions of rpoB, katG codon 315 and inhA-regulatory region, polymorphisms at gyrA codon 95 and katG codon 463 by DNA sequencing and by double-repetitive-element PCR for determining strain relatedness. None of the pansusceptible but six of 85 MDR-TB strains contained gyrA mutations. Only gyrA codon 94 was mutated in all six (D94A in one and D94G in five) strains. Three of six mutant strains were recovered from the same patient while three other strains represented individual patient isolates. Fingerprinting studies identified all individual patient isolates as epidemiologically distinct strains. All six strains with a gyrA mutation contained wild-type rrs sequence.ConclusionsAlthough fluoroquinolones are generally not used for chemotherapy of TB and drug susceptibility testing for second-line drugs is not carried out in Kuwait, four of 55 (7%) individual patient MDR-TB strains contained mutations in gyrA gene. The data advocate routine drug susceptibility testing for this important second-line drug for proper management of MDR-TB in Kuwait. Lack of mutations in 3'-end of rrs gene that confer resistance to injectable agents reduce the likelihood of occurrence of XDR-TB, at present, in Kuwait.

Project description:BackgroundThe detection of mutations in the gyrA and gyrB genes in the Mycobacterium tuberculosis genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance.MethodsIn order to characterize the diversity and frequency of gyrA and gyrB mutations and to describe the global distribution of these mutations, we conducted a systematic review, from May 1996 to April 2013, of all published studies evaluating Mycobacterium tuberculosis mutations associated with resistance to fluoroquinolones. The overall goal of the study was to determine the potential utility and reliability of these mutations as diagnostic markers to detect phenotypic fluoroquinolone resistance in Mycobacterium tuberculosis and to describe their geographic distribution.ResultsForty-six studies, covering four continents and 18 countries, provided mutation data for 3,846 unique clinical isolates with phenotypic resistance profiles to fluoroquinolones. The gyrA mutations occurring most frequently in fluoroquinolone-resistant isolates, ranged from 21-32% for D94G and 13-20% for A90V, by drug. Eighty seven percent of all strains that were phenotypically resistant to moxifloxacin and 83% of ofloxacin resistant isolates contained mutations in gyrA. Additionally we found that 83% and 80% of moxifloxacin and ofloxacin resistant strains respectively, were observed to have mutations in the gyrA codons interrogated by the existing MTBDRsl line probe assay. In China and Russia, 83% and 84% of fluoroquinolone resistant strains respectively, were observed to have gyrA mutations in the gene regions covered by the MTBDRsl assay.ConclusionsMolecular diagnostics, specifically the Genotype MTBDRsl assay, focusing on codons 88-94 should have moderate to high sensitivity in most countries. While we did observe geographic differences in the frequencies of single gyrA mutations across countries, molecular diagnostics based on detection of all gyrA mutations demonstrated to confer resistance should have broad and global utility.

Project description:BackgroundFluoroquinolone resistance is a serious threat in the battle against the treatment of multi drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB). Fluoroquinolone resistant isolates from India had shown to have evolved several mutants in the quinolone resistance determining region (QRDR) of DNA gyrase A subunit (GyrA), the target of fluoroquinolone. In view of high prevalence of mutations in the 'hot spot' region, a study on combinatorial drug design was carried out to identify better analogues for the treatment of MDR-TB. The gyrA subunit 'hot spot' region of codons 90, 94 and 95 were modeled into their corresponding protein folds and used as receptors for the docking studies. Further, invitro tests were carried using the parent compounds, namely gatifloxacin and moxifloxacin and correlated with the obtained docking scores.ResultsMolecular docking and in vitro studies correlated well in demonstrating the enhanced activity of moxifloxacin, when compared to gatifloxacin, on ofloxacin sensitive and resistant strains comprising of clinical isolates of MDR-TB. The evolved lead structures targeting against mutant QRDR receptors were guanosine and cholesteryl esters of gatifloxacin and moxifloxacin. They showed consistently high binding affinity values of -10.3 and -10.1 kcal/mol respectively with the target receptors. Of these, the guanosine ester showed highest binding affinity score and its log P value lied within the Lipinski's range indicating that it could have better absorptivity when it is orally administered thereby having an enhanced activity against MTB.ConclusionsThe docking results showed that the addition of the cholesteryl and guanosine esters to the 'DNA gyrase binding' region of gatifloxacin and moxifloxacin enhanced the binding affinity of these parent molecules with the mutant DNA gyrase receptors. Viewing the positive correlation for the docking and in vitro results with the parent compounds, these lead structures could be further evaluated for their in vitro and in vivo activity against MDR-TB.

Dataset Information

Association of gyrA/B mutations and resistance levels to fluoroquinolones in clinical isolates of Mycobacterium tuberculosis.

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Association of gyrA/B mutations and resistance levels to fluoroquinolones in clinical isolates of Mycobacterium tuberculosis.

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