الفهرس 
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Abstract
Quinolone resistance is traditionally mediated by chromosomal mutations that alter the target enzymes (DNA gyrase and/or topoisomerase IV), until plasmid mediated quinolones resistance (PMQR) was described in a clinical isolate of Klebsiella pneumoniae in 1998.
Although the chromosomal mutations in quinolone resistance determining regions (QRDR) play an important role in conferring a high level of quinolone resistance, while the PMQR genes lead to increased minimal inhibitory concentrations (MICs) of quinolones in many cases but are still below susceptible MIC breakpoints, some of researchers believed that the acquisition of the hidden PMQR genes will accelerate the development of mutations in gyrA QRDR region that result in facilitating the emergence of high level resistance in the presence of quinolones at therapeutic levels and a decrease in therapeutic efficacy.
This study was conducted on 100 non duplicate isolates, 64 Enterobacteriaceae spp. (32 klebsiella and 32 E.coli) and 36 Pseudomonas, recovered from clinical specimens referred to Central Microbiology Laboratory, Ain shams university Hospital for routine culture and sensitivity, aiming to determine the occurrence of PMQR determinants by multiplex PCR and chromosomal mutations by PCR restriction fragment length polymorphism (PCR-RFLP). Also, identify different mechanisms of FQs resistance and determine FQs resistance pattern among the studied isolates.
All isolates were subjected to subculture on MacConky agar media to obtain fresh isolates followed by identification of the isolates using VITEK® 2 Compact system, then antibiotic susceptibility to FQs group by disc diffusion method according to CLSI, 2015 and VITEK® 2 Compact system. The detection of PMQR determinants such as (qnrB, qnrS, oqxAB, qepA and aac(6’)-Ib-cr) was performed by using multiplex PCR. While, the gyrA mutations for chromosomal quinolones resistances were detected by PCR-RFLP.
As regards PMQR results, 77% of FQs resistant isolates were positive to one or more plasmids, plasmids were significantly higher among Pseudomonas isolates followed by Klebsiella then E.coli isolates P (< 0.05). OqxAB was significantly higher PMQR gene recovered among Klebsiella isolates followed by Pseudomonas then E.coli P (<0.05). The most frequent plasmids among positive extended spectrum β-lactamases (ESBL) producing isolates and multidrug resistant (MDR) isolates were oqxAB followed by qnrS.
As regards gyrA gene mutations, 78% of FQs resistant isolates were positive for gyrA mutations, the frequency of mutations was higher in Pseudomonas isolates followed by E.coli then klebsiella isolates P (< 0.05). Asp-87 mutation was higher than Ser-83 mutation. And Asp-87 mutation was significantly higher in E.coli isolates followed by klebsiella then Pseudomonas isolates P (< 0.05).
The prevalence of gyrA gene mutations was statistically significant higher among FQs resistant isolates than FQs moderate sensitive clinical isolates P (< 0.05), this may be explained by the higher presence of gyrA double mutation in FQs resistance isolates.
Consequently, detection of PMQR genes in the current study might be potentially indicative of a rising rate of PMQR genes among clinical isolates. Multiplex PCR for monitoring of PMQR and PCR-RFLP for gyrA mutations detection in Enterobacteriaceae and Pseudomonas spp. played an important role in simple and rapid detection of different mechanisms of FQs resistance.
As regards limitations of this study, the other types of PMQR genes did not included in this work as every plasmid has different primer sequence that make PCR multiplex not reliable especially when they have the nearest base pair. And also, other Enterobacteriaceae spp. did not included in this current study as every organism has different primer sequence for detection gyrA mutations that make the PCR-RFLP more costly.