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Antibiotic resistance is emerging as a significant global health crisis, necessitating the urgent development of novel antibiotics or alternative therapies. Although it is recognized that bacteria require multiple mutations to develop resistance levels exceeding the mutant prevention concentration, the specific mutation combinations conferring high resistance have been largely undefined. Here, we investigated the multi-step evolution of fluoroquinolone resistance in through experimental evolution and whole-genome sequencing coupled with proteomic approaches. We discovered that in low-dose and high-dose experimental evolution scenarios, combinations of mutations in the negative regulators of efflux pumps () and DNA gyrases () contributed to the high-level resistance and some of these combinations were also prevalent in clinical isolates of . Notably, the selected mutation, which resulted in the overexpression of the MexCD-OprJ efflux pump, also exhibited collateral sensitivity to aminoglycosides and enhanced antibiotic tolerance. It was further revealed that the efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) could effectively prevent evolution to high-level resistance for both laboratory and clinical strains. Our work highlights the critical role of efflux pump repressor-related mutations in the evolution of high-level antibiotic resistance and demonstrates the potential of targeting these mutations to impede the evolution toward high-level resistance.IMPORTANCEIn this study, we examined the stepwise evolution of fluoroquinolone resistance in using experimental evolution, whole-genome sequencing, and proteomic analyses. Our findings revealed that under both low-dose and high-dose conditions, mutations in efflux pump regulators (/) and DNA gyrase genes (/) synergistically contributed to high-level resistance. These mutation combinations were not only observed in experimental settings but also detected in clinical isolates of . This work underscores the pivotal role of efflux pump repressor-related mutations in the progression to high-level antibiotic resistance. It also highlights the promise of targeting efflux pumps as a strategy to prevent the multi-step evolution of resistance in .

抗生素耐药性正成为一场重大的全球健康危机，这使得迫切需要开发新型抗生素或替代疗法。尽管人们认识到细菌需要多个突变才能产生超过突变预防浓度的耐药水平，但赋予高耐药性的具体突变组合在很大程度上仍不明确。在此，我们通过实验进化、全基因组测序以及蛋白质组学方法，研究了铜绿假单胞菌中氟喹诺酮耐药性的多步进化过程。我们发现，在低剂量和高剂量实验进化情况下，外排泵（MexCD-OprJ）和DNA促旋酶（gyrA）负调控因子中的突变组合导致了高水平耐药性，其中一些组合在铜绿假单胞菌临床分离株中也很常见。值得注意的是，所选的gyrA突变导致MexCD-OprJ外排泵过度表达，同时对氨基糖苷类药物表现出协同敏感性并增强了抗生素耐受性。进一步研究表明，外排泵抑制剂苯丙氨酸-精氨酸β-萘酰胺（PAβN）可以有效阻止实验室菌株和临床铜绿假单胞菌菌株进化为高水平耐药。我们的工作突出了外排泵阻遏物相关突变在高水平抗生素耐药性进化中的关键作用，并证明了针对这些突变来阻碍向高水平耐药性进化的潜力。

重要性

在本研究中，我们使用实验进化、全基因组测序和蛋白质组学分析方法，研究了铜绿假单胞菌中氟喹诺酮耐药性的逐步进化过程。我们的研究结果表明，在低剂量和高剂量条件下，外排泵调节因子（MexCD-OprJ）和DNA促旋酶基因（gyrA）中的突变协同导致了高水平耐药性。这些突变组合不仅在实验环境中观察到，在铜绿假单胞菌临床分离株中也被检测到。这项工作强调了外排泵阻遏物相关突变在进展为高水平抗生素耐药性过程中的关键作用。它还突出了以外排泵为靶点作为一种策略来防止铜绿假单胞菌耐药性多步进化的前景。