Pseudomonas aeruginosa frequently causes antibiotic-recalcitrant pneumonia, but the mechanisms driving its adaptation during human infections remain unclear. To reveal the selective pressures and adaptation strategies at the mucosal surface, here we investigated P. aeruginosa growth and antibiotic tolerance in tissue-engineered airways by transposon insertion sequencing (Tn-seq). Metabolic modelling based on Tn-seq data revealed the nutritional requirements for P. aeruginosa growth, highlighting reliance on glucose and lactate and varying requirements for amino acid biosynthesis. Tn-seq also revealed selection against biofilm formation during mucosal growth in the absence of antibiotics. Live imaging in engineered organoids showed that biofilm-dwelling cells remained sessile while colonizing the mucosal surface, limiting nutrient foraging and reduced growth. Conversely, biofilm formation increased antibiotic tolerance at the mucosal surface. Moreover, mutants with exacerbated biofilm phenotypes protected less tolerant but more cytotoxic strains, contributing to phenotypic heterogeneity. P. aeruginosa must therefore navigate conflicting physical and biological selective pressures to establish chronic infections.

铜绿假单胞菌经常引起抗生素顽固性肺炎，但在人类感染期间驱动其适应的机制仍不清楚。为了揭示粘膜表面的选择压力和适应策略，我们在这里通过转座子插入测序 （Tn-seq） 研究了组织工程气道中铜绿假单胞菌的生长和抗生素耐受性。基于 Tn-seq 数据的代谢模型揭示了铜绿假单胞菌生长的营养需求，突出了对葡萄糖和乳酸的依赖以及对氨基酸生物合成的不同需求。Tn-seq 还揭示了在没有抗生素的情况下，粘膜生长过程中对生物膜形成的选择。工程类器官的实时成像显示，生物膜驻留细胞在粘膜表面定植时保持无柄，限制了营养物质的觅食并减少了生长。相反，生物膜的形成增加了粘膜表面的抗生素耐受性。此外，生物膜表型加剧的突变体保护的耐受性较低但细胞毒性较大的菌株，导致表型异质性。因此，铜绿假单胞菌必须驾驭相互冲突的物理和生物选择压力才能建立慢性感染。