Analysis of amino acid substitution mutations of gyrA and parC genes in the clonal lineage of Klebsiella pneumoniae conferring high-level quinolone resistance

Abstract

Introduction: The objective of this study was to determine the amino acid substitutions in gyrA and parC proteins in certain clonal lineages of K. pneumoniae conferring high-level quinolone resistance. Methods: One hundred and eleven K. pneumoniae isolates were recovered from clinical specimens in a teaching hospital in Kerman, Iran. The antibiotic susceptibility and MIC of quinolones were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Clonal lineages of the isolates were confirmed by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Amino acid mutation profiles of gyrA and parC amplicons of 6 high quinolone-resistant isolates were also determined by DNA sequencing. Results: Twenty-two isolates were resistant to nalidixic acid (MIC, 256 µg/ml), ciprofloxacin (MIC, 32 µg/ml), levofloxacin (MIC, 32 µg/ml), and ofloxacin (MIC, 32 µg/ml). ERIC-PCR typing identified 4 clusters and 6 singleton, the largest belonged to cluster 3. Sequencing of gyrA gene showed 3 amino acid substitutions ( Ser83→Ile, Lys154→Arg, and Ser171→Ala) in the strains 18, 20, 33; two substitutions (Lysine154→Arg; Ser171→Ala) in the strains 27 and 65 and 6 substitutions in the strain 66, of which, 3 (Ser83→Phe, Asp87→Ala, and Val190→Gly) were unique for this strain. Sequencing of parC gene revealed two substitutions (Ser129→Ala and Ala141→Val) in the strains 18, 27, 66, and 3 amino acid changes (Ser80→Ile, Ser129→Ala, and Ala141→Val) in the strains 20 and 33, respectively. Alignment and phylogenetic tree analysis of the gyrA from isolate 66 with homologous sequences obtained from the NCBI database, revealed 99.8% similarity to gyrA gene of K. pneumoniae ha10. Conclusion: The results suggest that acquisition of mutations in certain positions of gyrA and parC genes confers high-level resistance to quinolones. J Med Microbiol Infec Dis, 2014, 2 (3): 109-117.