ANALYSES OF parC AND gyrA MUTATIONS IN CIPROFLOXACIN-RESISTANT AND SUSCEPTIBLE pseudomonas aeruginosa ISOLATED FROM SOIL BY PCR-RFLP AND SSCP METHOD

Abstract

The aims of this study were to assess the prevalence of gyrA and parC mutations in ciprofloxacin-resistant and susceptible Pseudomonas aeruginosa isolated from soil and to evaluate the suitability of the restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) techniques as screening methods for molecular characterizations of ciprofloxacin resistance. From the examined 21 P. aeruginosa isolates 9 strains were resistant to ciprofloxacin. These 21 P. aeruginosa isolates and one control strain were analyzed for alterations in the ciprofloxacin resistance determining region of gyrA and parC by RFLP and SSCP analyses. The PCR reaction confirmed the presence of the gyrA and parC genes in all of the strains. PCR-RFLP analyses with SacII for gyrA and HinfI for parC were performed as a screening method. We found that 18 and 17 out of 22 isolates have SacII and HinfI restriction site and 4 and 5 strains did not have the site recognized by these enzymes, respectively. Seven for gyrA and fourteen for parC PCR products were electrophoresed for SSCP. By SSCP analysis, 21 (in parC) and 15 (in gyrA) different band patterns were detected, and each pattern corresponded to a distinct mutation. As a result, the RFLP and SSCP methods are suitable for a molecular screening of ciprofloxacin resistant and susceptible P. aeruginosa isolates. SSCP analysis can also provide advantage for the detection of novel and multiple mutations. In addition, we can say that environmental monitoring followed by clinical surveillance can be successful in uncovering previously unrecognized cases of infection.