Fluoroquinolone resistance in Pseudomonas aeruginosa typically arises through site-specific mutations and overexpression of efflux pumps. In this study, we investigated the dynamics of different resistance mechanisms in P. aeruginosa populations that have evolved under fluoroquinolone pressure, as well as the interactions between these mechanisms in evolutionary trajectories. Bacteria of strain ATCC27853 were selected under different concentrations of ciprofloxacin and levofloxacin for six parallel lineages, followed by amplification of four target genes in the quinolone-resistance determining region (QRDR) and Sanger sequencing to identify the mutations. The expression of four efflux pump proteins was evaluated by real-time polymerase chain reaction using the relative quantitation method, with the ATCC27853 strain used as a control. We found that ciprofloxacin killed P. aeruginosa sooner than did levofloxacin. Further, we identified five different mutations in three subunits of QRDRs, with gyrA as the main mutated gene associated with conferring fluoroquinolone resistance. Additionally, we found a larger number of mutations appearing at 2 mg/L and 4 mg/L of ciprofloxacin and levofloxacin, respectively. Moreover, we identified the main efflux pump being expressed as MexCD-OprJ, with initial overexpression observed at 0.25 mg/L and 0.5 mg/L of ciprofloxacin and levofloxacin, respectively. These results demonstrated gyrA83 mutation and MexCD-OprJ overexpression as the primary mechanism conferring ciprofloxacin and levofloxacin resistance in P. aeruginosa. In addition, we also show that ciprofloxacin exhibited a stronger ability to kill the bacteria while potentially rendering it more susceptible to resistance.

中文翻译：

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铜绿假单胞菌对氟喹诺酮类药物的体外耐药性的发展。

铜绿假单胞菌中的氟喹诺酮耐药性通常是由于位点特异性突变和外排泵的过表达引起的。在这项研究中，我们调查了在氟喹诺酮压力下进化的铜绿假单胞菌种群中不同抗性机制的动力学，以及这些机制在进化轨迹中的相互作用。在不同浓度的环丙沙星和左氧氟沙星中选择菌株ATCC27853的细菌用于六个平行谱系，然后在喹诺酮抗性确定区域（QRDR）中扩增四个靶基因，并进行Sanger测序以鉴定突变。使用相对定量方法，通过实时聚合酶链反应，以ATCC27853菌株为对照，评估了四个外排泵蛋白的表达。我们发现环丙沙星比左氧氟沙星更早杀死铜绿假单胞菌。此外，我们确定了QRDRs的三个亚基中的五个不同突变，其中gyrA是与赋予氟喹诺酮耐药性相关的主要突变基因。此外，我们发现在环丙沙星和左氧氟沙星分别以2 mg / L和4 mg / L出现大量突变。此外，我们确定了主要外排泵表示为MexCD-OprJ，在环丙沙星和左氧氟沙星分别观察到初始过表达分别为0.25 mg / L和0.5 mg / L。这些结果证明，gyrA83突变和MexCD-OprJ过表达是赋予铜绿假单胞菌环丙沙星和左氧氟沙星抗性的主要机制。此外，