Dataset Information

Analysis of DNA gyrA Gene Mutation in Clinical and Environmental Ciprofloxacin-Resistant Isolates of Non-Tuberculous Mycobacteria Using Molecular Methods.

ABSTRACT: BACKGROUND:During the past several years, nontuberculous mycobacteria (NTM) have been reported as some of the most important agents of infection in immunocompromised patients. OBJECTIVES:The aim of this study was to evaluate the ciprofloxacin susceptibility of clinical and environmental NTM species isolated from Isfahan province, Iran, using the agar dilution method, and to perform an analysis of gyrA gene-related ciprofloxacin resistance. MATERIALS AND METHODS:A total of 41 clinical and environmental isolates of NTM were identified by conventional and multiplex PCR techniques. The isolates were separated out of water, blood, abscess, and bronchial samples. The susceptibility of the isolates to 1 µg/mL, 2 µg/mL and 4 µg/mL of ciprofloxacin concentrations was determined by the agar dilution method according to CLSI guidelines. A 120-bp area of the gyrA gene was amplified, and PCR-SSCP templates were defined using polyacrylamide gel electrophoresis. The 120-bp of gyrA amplicons with different PCR-SSCP patterns were sequenced. RESULTS:The frequency of the identified isolates was as follows: Mycobacterium fortuitum, 27 cases; M. gordonae, 10 cases; M. smegmatis, one case; M. conceptionense, one case; and M. abscessus, two cases. All isolates except for M. abscessus were sensitive to all three concentrations of ciprofloxacin. The PCR-SSCP pattern of the gyrA gene of resistant M. abscessus isolates showed four different bands. The gyrA sequencing of resistant M. abscessus isolates showed 12 alterations in nucleotides compared to the M. abscessus ATCC 19977 resistant strain; however, the amino acid sequences were similar. CONCLUSIONS:This study demonstrated the specificity and sensitivity of the PCR-SSCP method for finding mutations in the gyrA gene. Due to the sensitivity of most isolates to ciprofloxacin, this antibiotic should be considered an appropriate drug for the treatment of related diseases.

SUBMITTER: Nasr Esfahani B

PROVIDER: S-EPMC4870840 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Analysis of DNA gyrA Gene Mutation in Clinical and Environmental Ciprofloxacin-Resistant Isolates of Non-Tuberculous Mycobacteria Using Molecular Methods.

Nasr Esfahani Bahram B Zarkesh Esfahani Fatemeh Sadat FS Bahador Nima N Moghim Sharareh S Radaei Tooba T Rezaei Yazdi Hadi H Ghasemian Safaei Hajiyeh H Fazeli Hossein H

Jundishapur journal of microbiology 20160311 3

<h4>Background</h4>During the past several years, nontuberculous mycobacteria (NTM) have been reported as some of the most important agents of infection in immunocompromised patients.<h4>Objectives</h4>The aim of this study was to evaluate the ciprofloxacin susceptibility of clinical and environmental NTM species isolated from Isfahan province, Iran, using the agar dilution method, and to perform an analysis of gyrA gene-related ciprofloxacin resistance.<h4>Materials and methods</h4>A total of 4 ...[more]

PMID: 27217921

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Project description:BACKGROUND:Phenotypic fluoroquinolone resistance was first reported in Western Kenya in 2009 and later in Coastal Kenya and Nairobi. Until recently gonococcal fluoroquinolone resistance mechanisms in Kenya had not been elucidated. The aim of this paper is to analyze mutations in both gyrA and parC responsible for elevated fluoroquinolone Minimum Inhibitory Concentrations (MICs) in Neisseria gonorrhoeae (GC) isolated from heterosexual individuals from different locations in Kenya between 2013 and 2017. METHODS:Antimicrobial Susceptibility Tests were done on 84 GC in an ongoing Sexually Transmitted Infections (STI) surveillance program. Of the 84 isolates, 22 resistant to two or more classes of antimicrobials were chosen for analysis. Antimicrobial susceptibility tests were done using E-test (BioMerieux) and the results were interpreted with reference to European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. The isolates were sub-cultured, and whole genomes were sequenced using Illumina platform. Reads were assembled de novo using Velvet, and mutations in the GC Quinolone Resistant Determining Regions identified using Bioedit sequence alignment editor. Single Nucleotide Polymorphism based phylogeny was inferred using RaxML. RESULTS:Double GyrA amino acid substitutions; S91F and D95G/D95A were identified in 20 isolates. Of these 20 isolates, 14 had an additional E91G ParC substitution and significantly higher ciprofloxacin MICs (p?=?0.0044*). On the contrary, norfloxacin MICs of isolates expressing both GyrA and ParC QRDR amino acid changes were not significantly high (p?=?0.82) compared to MICs of isolates expressing GyrA substitutions alone. No single GyrA substitution was found in the analyzed isolates, and no isolate contained a ParC substitution without the simultaneous presence of double GyrA substitutions. Maximum likelihood tree clustered the 22 isolates into 6 distinct clades. CONCLUSION:Simultaneous presence of amino acid substitutions in ParC and GyrA has been reported to increase gonococcal fluoroquinolone resistance from different regions in the world. Our findings indicate that GyrA S91F, D95G/D95A and ParC E91G amino acid substitutions mediate high fluoroquinolone resistance in the analyzed Kenyan GC.

Dataset Information

Analysis of DNA gyrA Gene Mutation in Clinical and Environmental Ciprofloxacin-Resistant Isolates of Non-Tuberculous Mycobacteria Using Molecular Methods.

Publications

Analysis of DNA gyrA Gene Mutation in Clinical and Environmental Ciprofloxacin-Resistant Isolates of Non-Tuberculous Mycobacteria Using Molecular Methods.

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