The evolution of antimicrobial resistance (AMR) in biofilms has been repeatedly studied by experimental evolution in vitro, but rarely in vivo. The complex microenvironment at the infection site imposes selective pressures on the bacterial biofilms, potentially influencing the development of AMR. We report here the development of AMR in an in vivo mouse model of Pseudomonas aeruginosa biofilm lung infection. P. aeruginosa embedded in seaweed alginate beads underwent four successive lung infection passages with or without ciprofloxacin exposure (CIP). The development of CIP resistance was assessed at each passage by population analysis of the bacterial populations recovered from the lungs of CIP-treated and control mice, with subsequent whole genome sequencing of selected isolates. As inflammation plays a crucial role in shaping the microenvironment at the infection site, its impact was explored through the measurement of cytokine levels in the lung homogenate. A rapid development of AMR was observed starting from the second passage in the CIP-treated mice. Genetic analysis revealed mutations in nfxB, efflux pumps (mexZ), and two-component systems (parS) contribution to CIP resistance. The control group isolates exhibited mutations in the dipA gene, likely associated with biofilm dispersion. In the initial two passages, the CIP-treated group exhibited an elevated inflammatory response compared to the control group. This increase may potentially contribute to the release of mutagenic reactive oxygen species and the development of AMR. In conclusion, this study illustrates the complex relationship between infection, antibiotic treatment, and immune response.

中文翻译：

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铜绿假单胞菌肺部感染生物膜模型中抗菌药物耐药性的体内进化

生物膜中抗菌素耐药性（AMR）的进化已经通过体外实验进化进行了反复研究，但很少在体内进行。感染部位复杂的微环境对细菌生物膜施加选择性压力，可能影响抗菌素耐药性的发展。我们在此报告了铜绿假单胞菌生物膜肺部感染体内小鼠模型中 AMR 的发展。嵌入海藻藻酸盐珠中的铜绿假单胞菌在有或没有环丙沙星暴露（CIP）的情况下经历了四次连续的肺部感染传代。通过对从 CIP 处理小鼠和对照小鼠肺部回收的细菌种群进行种群分析，以及随后对选定分离株进行全基因组测序，评估每次传代时 CIP 抗性的发展。由于炎症在塑造感染部位的微环境中起着至关重要的作用，因此通过测量肺匀浆中的细胞因子水平来探讨其影响。在 CIP 处理的小鼠中，从第二代开始观察到 AMR 的快速发展。遗传分析揭示了 nfxB、外排泵 (mexZ) 和双组分系统 (parS) 的突变对 CIP 耐药性的贡献。对照组分离株表现出 dipA 基因突变，可能与生物膜分散有关。在最初的两代中，与对照组相比，CIP 治疗组表现出升高的炎症反应。这种增加可能有助于诱变活性氧的释放和抗菌素耐药性的发展。总之，这项研究阐明了感染、抗生素治疗和免疫反应之间的复杂关系。