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. 2016 Mar 11;9(3):e30018.
doi: 10.5812/jjm.30018. eCollection 2016 Mar.

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

Affiliations

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

Bahram Nasr Esfahani et al. Jundishapur J Microbiol. .

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.

Keywords: Ciprofloxacin; DNA Gyrase A; PCR-SSCP.

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Figures

Figure 1.
Figure 1.. PCR Amplification of 210 bps of gyrA Gene
Lane 1, molecular sized marker (100 bp DNA ladder); lane 2, gyrA PCR product from M. abscessus; lane 3, gyrA PCR product from M. gordonae; lanes 4 - 5, gyrA product from M. fortuitum ATCC49403 and M. fortuitum; lane 6, gyrA negative control.
Figure 2.
Figure 2.. PCR-SSCP of 210 bps of gyrA in 8 Isolates of NTM
Lanes 1 and 2, M. abscessus (resistance to ciprofloxacin); lane 3, M. fortuitum ATCC 49403; lanes 4 - 6, M. fortuitum; lanes 7 and 8, M. gordonae (sensitive to ciprofloxacin).
Figure 3.
Figure 3.. My Query: M. abscessus Resistant to Ciprofloxacin
M. abscessus, M. abscessus ATCC 19977. The amino acid sequence of M. abscessus resistant to ciprofloxacin was 100% similar to M. abscessus ATCC 19977 (HQ324097.1) and M. gordonae, but the nucleotide sequence was different at 12 points (with M. gordonae at 13 points).
Figure 4.
Figure 4.. My Query: M. fortuitum Sensitive to Ciprofloxacin
M. fortuitum, M. fortuitum ATCC 6841. The amino acid sequence of M. fortuitum sensitive to ciprofloxacin was 100% similar to M. fortuitum ATCC 6841(AJ564392.1).
Figure 5.
Figure 5.. My Query: M. gordonae Sensitive to Ciprofloxacin
The amino acid sequence of M. gordonae sensitive to ciprofloxacin was 100% similar to M. gordonae ATCC 14470 (AJ564389.1).

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