title: 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.
  
  
  OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, IR Iran.
authors.forename: Bahram
authors.initials: B
authors.lastname: Nasr Esfahani
authors.affiliation: Department of Microbiology, Science and Research Branch, Islamic Azad University, Fars, IR Iran.
authors.forename: Fatemeh Sadat
authors.initials: FS
authors.lastname: Zarkesh Esfahani
authors.affiliation: Department of Microbiology, Science and Research Branch, Islamic Azad University, Fars, IR Iran.
authors.forename: Nima
authors.initials: N
authors.lastname: Bahador
authors.affiliation: Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, IR Iran.
authors.forename: Sharareh
authors.initials: S
authors.lastname: Moghim
authors.affiliation: Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, IR Iran.
authors.forename: Tooba
authors.initials: T
authors.lastname: Radaei
authors.affiliation: Department of Microbiology, Jahrom University of Medical Sciences, Jahrom, IR Iran.
authors.forename: Hadi
authors.initials: H
authors.lastname: Rezaei Yazdi
authors.affiliation: Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, IR Iran.
authors.forename: Hajiyeh
authors.initials: H
authors.lastname: Ghasemian Safaei
authors.affiliation: Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, IR Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Fazeli
keywords: Ciprofloxacin; DNA Gyrase A; PCR-SSCP