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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:BackgroundNon-tuberculous mycobacteria (NTM) treatment constitutes a macrolide-based antibiotic regimen in combination with aminoglycosides for Rapid-Growing Mycobacteria (RGM), and rifampicin for Slow-Growing Mycobacteria (SGM). Mutations in the anti-NTM drug target regions promote NTM evolution to mutant strains that are insusceptible to NTM drugs leading to treatment failure. We, therefore, described the mutation patterns of anti-NTM drug target genes including rrl, rrs, and rpoB in NTM isolates from Kenya. Methods: We carried out a cross-sectional study that included 122 NTM obtained from the sputum of symptomatic tuberculosis-negative patients in Kenya. All 122 NTM underwent targeted sequencing of the rrl gene. The 54 RGM were also sequenced for rrs, and the 68 SGM were sequenced for rpoB genes using ABI 3730XL analyzer. The obtained sequences were aligned to their wild-type reference sequences for each gene using Geneious then mutations were identified. Pearson chi-square at a 95% confidence interval tested the association of NTM to mutation patterns for each gene.ResultsNTM harboring mutations associated with resistance to at least one of the antibiotics used in the macrolide-based therapy were 23% (28/122). Of these NTM, 10.4% (12/122) had mutations in the rrl gene with 58.3% (7/12) comprising RGM and 41.7% (5/12) being SGM. Mutation at position 2058 (A2058G, A2058C, A2058T) of the rrl gene was seen for 83.3% (10/12) of NTM, while 16.6% (2/12) harbored a A2059G mutation. Of the 54 RGM included for rrs characterization, 11.1% (6/54) exhibited mutations at position 1408(A1408G), while 14.7% (10/68) of the SGM had mutations in the rpoB gene at positions S531W, S531L, S531Y, F506L, E509H with M.gastri having multiple mutations at positions D516V, H526D and, S531F.ConclusionWe demonstrated a significant level of mutations associated with drug resistance for macrolides, aminoglycosides, and rifampicin in NTM isolated from symptomatic TB negative patients in Kenya.

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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