Mahsa Nabilou1, Laleh Babaeekhou2, Maryam Ghane1. 1. Department of Biology, Islamshahr Branch, Islamic Azad University, Islamshahr, Iran. 2. Department of Biology, Islamshahr Branch, Islamic Azad University, Islamshahr, Iran. babaeekhou@iiau.ac.ir.
Abstract
BACKGROUND: Fluoroquinolones (FQs) including ciprofloxacin (CIP) are key antibiotics for the treatment of Pseudomonas aeruginosa infections, but resistance to FQs is developing as a result of chromosomal mutations or efflux pump effects. Plasmid-mediated quinolone resistance (PMQR) has been recently reported in the Enterobacteriaceae family. This study aimed to investigate the mechanisms of CIP insusceptibility in P. aeruginosa isolates from ICU patients and to characterize their genotypes. METHODS: A total of 40 ciprofloxacin unsusceptible (CIP-US) P. aeruginosa isolates from Tehran hospitals were recruited in this study. A broth microdilution assay was performed to find acquired resistance profiles of the isolates. All isolates were screened for target-site mutations (gyrA and parC), PMQR genes, and efflux pumps (mexB, D, Y, and E) expression. Clonality was determined by random amplified polymorphic DNA (RAPD)-PCR, and genotyping was performed on 5 selected isolates by analyzing 7 loci in the existing multilocus sequence typing scheme. RESULTS: Thirty-eight out of 40 CIP-US isolates (95%) were categorized as MDR. Seven (17.5%) had gyrA mutation in codons 83, and no mutation was detected in parC; 77.5% of the isolates were positive for PMQR genes. Among PMQR genes, qnrB (30%), qnrC (35%), and qnrD (30%) predominated, while qnrA, qnrS, and aac(6)-Ib genes were harbored by 20.5%, 12.5%, and 15% of the isolates respectively. Efflux pump protein expression was observed in 35% of the isolates. After RAPD-PCR, 19 different genotypes were yielded, and 5 of them were classified into sequence types (STs): 773, 1160, 2011, 2386, and 359. CONCLUSION: In this first-time study on P. aeruginosa CIP-US strains from Iranian ICU patients, three main CIP unsusceptibility mechanisms were investigated. A single mutation in one CIP target enzyme could explain high CIP resistance. qnr genes in the isolates can be considered as a CIP-unsusceptibility mechanism among studied isolates. Efflux pumps have more contribution in multidrug resistance than CIP susceptibility. CIP-US isolates of this study have not spread from distinct clonal strains and probably emerged from different sources. STs identified for the first time in this study in Iran should be considered as emerging MDR strains.
BACKGROUND: Fluoroquinolones (FQs) including ciprofloxacin (CIP) are key antibiotics for the treatment of Pseudomonas aeruginosa infections, but resistance to FQs is developing as a result of chromosomal mutations or efflux pump effects. Plasmid-mediated quinolone resistance (PMQR) has been recently reported in the Enterobacteriaceae family. This study aimed to investigate the mechanisms of CIP insusceptibility in P. aeruginosa isolates from ICU patients and to characterize their genotypes. METHODS: A total of 40 ciprofloxacin unsusceptible (CIP-US) P. aeruginosa isolates from Tehran hospitals were recruited in this study. A broth microdilution assay was performed to find acquired resistance profiles of the isolates. All isolates were screened for target-site mutations (gyrA and parC), PMQR genes, and efflux pumps (mexB, D, Y, and E) expression. Clonality was determined by random amplified polymorphic DNA (RAPD)-PCR, and genotyping was performed on 5 selected isolates by analyzing 7 loci in the existing multilocus sequence typing scheme. RESULTS: Thirty-eight out of 40 CIP-US isolates (95%) were categorized as MDR. Seven (17.5%) had gyrA mutation in codons 83, and no mutation was detected in parC; 77.5% of the isolates were positive for PMQR genes. Among PMQR genes, qnrB (30%), qnrC (35%), and qnrD (30%) predominated, while qnrA, qnrS, and aac(6)-Ib genes were harbored by 20.5%, 12.5%, and 15% of the isolates respectively. Efflux pump protein expression was observed in 35% of the isolates. After RAPD-PCR, 19 different genotypes were yielded, and 5 of them were classified into sequence types (STs): 773, 1160, 2011, 2386, and 359. CONCLUSION: In this first-time study on P. aeruginosa CIP-US strains from Iranian ICU patients, three main CIP unsusceptibility mechanisms were investigated. A single mutation in one CIP target enzyme could explain high CIP resistance. qnr genes in the isolates can be considered as a CIP-unsusceptibility mechanism among studied isolates. Efflux pumps have more contribution in multidrug resistance than CIP susceptibility. CIP-US isolates of this study have not spread from distinct clonal strains and probably emerged from different sources. STs identified for the first time in this study in Iran should be considered as emerging MDR strains.