Amino acid Substitution Mutations Analysis of gyrA and parC Genes in Clonal Lineage of Klebsiella pneumoniae conferring High-Level Quinolone Resistance

Background: Emergence Klebsiella pneumoniae resistant to quinolone antibiotics due to mutations in gyrA and parC genes created problem for treatment of patients in different hospitals in Iran. The objective of this study was to determine the amino acid substitutions of GyrA and ParC proteins in certain clonal lineages of the K. pneumoniae conferring high level quinolone resistance. Methods: One hundred and eleven isolates of K. pneumoniae were recovered from clinical specimens in a teaching hospital in Kerman, Iran. The antibiotic susceptibility and MIC of quinolones were determined according to CLSI guidelines. Clonal lineages of the isolates were determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR amplification using ERIC specific primer sequences. Amino acid mutation profile of gyrA and parC amplicons of six high quinolone resistant isolates was also investigated by DNA sequencing. Results: Twenty two isolates were resistant to nalidixic acid (MIC ۲۵۶µg/ml), ciprofloxacin (MIC ۳۲µg/ml), levofloxacin (MIC ۳۲µg/ml), and ofloxacin (MIC ۳۲ µg/ml). Typing by ERIC-PCR identified ۴ clusters and six singleton, the largest one was belonged to cluster-۳ obtained from urine samples. Sequencing of gyrA gene showed three amino acid substitutions (Ser۸۳→Ile Lys۱۵۴→Arg Ser۱۷۱→Ala) in the strains ۱۸, ۲۰, ۳۳, two mutations (Lysine۱۵۴→Arg Ser۱۷۱→Ala) in the strains ۲۷, ۶۵ and six substitutions in the strain ۶۶, of which, three (Ser۸۳→Phe Asp۸۷→Ala Val۱۹۰→Gly) were unique for this strain. Sequencing of parC gene revealed double substitutions (Ser۱۲۹→Ala Ala۱۴۱→Val) in the strains ۱۸, ۲۷, ۶۶ and three aminoacid changes (Ser۸۰→Ile Ser۱۲۹→Ala Ala۱۴۱→Val) in the strains ۲۰ and ۳۳ respectively. Alignment and phylogenetic tree analysis of the gyrA sequence from highly quinolone resistant isolate ۶۶ with homologs sequences obtained from the NCBI database confirmed ۹۹.۸% similarity to gyrA gene of the K. pneumoniae ha۱۰ (GenBank: JX۱۲۳۰۱۷.۱). Conclusion: The results of present study suggest that acquisition of mutations in certain positions of gyrA and parC genes confer high level resistance to quinolones.

Keywords:

Klebsiella pneumoniae , quinolones resistance , ERIC-PCR , sequencing , GyrA and ParC mutations.