PCR-RFLP-Based Detection of Mutations in the Chromosomal Fluoroquinolone Targets g yr A and p ar C Genes of Acinetobacter b aumanii Clinical Isolates from a Tertiary Hospital in Cairo, Egypt

Abstract

Background and Aim: Acinetobacter baumannii is one of the most antimicrobial resistant nosocomial pathogens encountered clinically worldwide. The emergence of fluoroquinolone resistance among A. baumannii isolates is currently of concern. This study aimed to investigate the antimicrobial susceptibility patterns among 49 clinical isolates of A baumannii collected from a tertiary care hospital in Egypt. These isolates were analysed for the mechanism of fluoroquinolone resistance based on the presence of mutations in the quinolone resistance-determining regions (QRDRs) of the chromosomal quinolone resistance determinants gyrA and parC genes. Methods: A. baumannii isolates were identified using conventional biochemical testing, VITEK 2 automated system and polymerase chain reaction (PCR) assay targeting the intrinsic blaOXA-51-like gene of A. baumannii species. Antimicrobial susceptibility to different antimicrobial agents was tested using agar disk diffusion method and the minimum inhibitory concentration (MIC) of ciprofloxacin was determined using E-test (bioMerieux, France) on Mueller-Hinton agar medium following the Clinical and Laboratory Standards Institute guidelines. The QRDRs of the gyrA and parC genes in A. baumannii isolates were amplified by PCR using specific primers. Mutations in theses QRDRs were detected by HinfI restriction fragment length polymorphism (RFLP) of PCR products and sequencing. Results: The blaOXA-51-like gene was detected in all isolates confirming identification as A. baumannii. Antimicrobial susceptibility study showed that all isolates (100 %) were MDR. They were 100 % resistant to the tested fluoroquinolones, ciprofloxacin and levofloxacin. The MIC of ciprofloxacin ranged from 4 to ≥ 32 μg/mL. All A. baumannii isolates (100 %) were found to harbour gyrA and parC genes. HinfI restriction analysis showed a detectable mutation in QRDRs at position Ser-83 of gyrA gene and Ser-80 of parC gene. There was single mutation in either gyrA or parC in 11 isolates showed ciprofloxacin MIC of < 32 μg/mL, while 38 isolates with MIC of ≥ 32 μg/mL had double mutations in QRDRs of both genes. Conclusions: Resistance of A. baumannii isolates to fluoroquinolones in Egypt is alarming as all MDR A. baumannii isolates in the current study were mostly highly resistant to ciprofloxacin with MIC ≥ 32 μg/mL, limiting the remaining therapeutic options and a public health policy on appropriate prescribing and thus the rational use of antimicrobial agents is required. Double mutation with substitutions at positions Ser-83 and Ser-80 of gyrA and parC genes, respectively, could lead to high‐level ciprofloxacin resistant phenotype than a single mutation in one of them. Further extensive studies including a larger number of isolates from different geographic areas in Egypt and investigating other fluoroquinolone resistance mechanisms are warranted.