Combination therapy is a common antibiotic treatment strategy that aims at minimizing the risk of resistance evolution in several infectious diseases. Nonetheless, evidence supporting its efficacy against the nosocomial opportunistic pathogen Pseudomonas aeruginosa remains elusive. Identification of the possible evolutionary paths to resistance in multidrug environments can help to explain treatment outcome. For this purpose, we here performed whole-genome sequencing of 127 previously evolved populations of P. aeruginosa adapted to sub-lethal doses of distinct antibiotic combinations and corresponding single drug treatments, and experimentally characterized several of the identified variants. We found that alterations in the regulation of efflux pumps is the most favored mechanism of resistance, regardless of the environment. Unexpectedly, we repeatedly identified intergenic variants in the adapted populations, often with no additional mutations and usually associated with genes involved in efflux pump expression, possibly indicating a regulatory function of the intergenic regions. The experimental analysis of these variants demonstrated that the intergenic changes caused similar increases in resistance against single and multidrug treatments as those seen for efflux regulatory gene mutants. Surprisingly, we could find no substantial fitness costs for a majority of these variants, most likely enhancing their competitiveness towards sensitive cells, even in antibiotic-free environments. We conclude that the regulation of efflux is a central target of antibiotic-mediated selection in P. aeruginosa and that, importantly, changes in intergenic regions may represent a usually neglected alternative process underlying bacterial resistance evolution, which clearly deserves further attention in the future.

中文翻译：

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铜绿假单胞菌快速适应抗生素联合治疗的基因组基础。

联合治疗是一种常见的抗生素治疗策略，旨在将几种传染病中的耐药性进化风​​险降至最低。但是，仍然有证据支持其对抗医院内机会病原体铜绿假单胞菌的功效。确定在多药环境中耐药性的可能进化途径可以帮助解释治疗结果。为此，我们在此对127个先前进化的铜绿假单胞菌种群进行了全基因组测序适应亚致死剂量的不同抗生素组合和相应的单药治疗，并通过实验表征了几种已鉴定的变体。我们发现，不管环境如何，外排泵调节的改变是最受人们欢迎的阻力机制。出乎意料的是，我们在适应的人群中反复鉴定出基因间变异，通常没有其他突变，并且通常与外排泵表达相关的基因有关，这可能表明基因间区域的调控功能。这些变体的实验分析表明，基因间的变化导致对单药和多药治疗的耐药性增加，与外排调节基因突变体所见的类似。出奇，我们发现这些变体中的大多数都没有实质性的适用成本，即使在无抗生素的环境中，也很有可能增强其对敏感细胞的竞争力。我们得出的结论是，外排的调节是抗生素介导的选择的主要目标。铜绿假单胞菌，重要的是，基因间区域的变化可能代表了细菌抗性进化背后通常被忽略的替代过程，这显然在未来值得进一步关注。