Molecular Detection of gyrA Mutation in Clinical Strains of Klebsiella pneumoniae

Multi-drug resistant (MDR) Klebsiella pneumoniae strains cause the majority of community acquired and life-threatening infections. We aimed to detect the gyrA mutations in the clinical strains of nalidixic acid and ciprofloxacin resistant K. pneumoniae isolated from the patients with urinary tract infections.

Bacterial strains were isolated from the patients with urinary tract infections admitted to a major hospital in Tehran, Iran (2017-2018). Bacterial identification was done according to standard microbiological tests. Antimicrobial susceptibility testing for quinolones and fluoroquinolone antibiotics was done using both disc diffusion and minimal inhibitory concentrations (MICs) methods. PCR-RFLP was used to detect the probable mutation in the gyrA gene in nalidixic acid and ciprofloxacin resistant strains. Finally sequencing was performed to detect point mutations in isolated K. pneumoniae strains.

One hundred K. pneumonia isolates were recovered from the urine samples of the clinical cases. Antibiotic resistance testing showed that among all K. pneumoniae isolates, 26% and 19% of the strains were resistant to nalidixic acid and ciprofloxacin respectively. MIC value was ≥4 µg/ml for ciprofloxacin resistant isolates. The results of RFLP on gyrA PCR amplicons using HinfI restriction enzyme showed point mutation in this gene in 46% of nalidixic acid and ciprofloxacin resistant K. pneumonia. The data obtained from the sequencing confirmed the RFLP results and indicated the presence of point mutations in codons 83 and 87 in the gyrA gene which leads to the substitution of different amino acids in gyrA protein.

Our findings indicated a relative increased rate of resistance against quinolones and fluoroquinolone antibiotics that raised a concern about extensive dissemination of clinical strains of nalidixic acid and ciprofloxacin resistant K. pneumonia. Point mutation of gyrA gene was responsible for the resistance in our strains however to gain more insight into the molecular characterization of quinolone-resistant isolates, other possible mechanisms of the resistance should also be investigated.