Infection-dependent antibiotic response phenotypes emerge during chronic airway adaptation of Pseudomonas aeruginosa

Objectives Chronic Pseudomonas aeruginosa airway infections can persist for years despite intensive antibiotic therapy guided by standard antimicrobial susceptibility testing. While this technique captures the emergence of resistance, it does not reproduce many features of the infection environment, where adaptation acquired during chronic infection may alter bacterial responses to antibiotics in ways that are not fully reflected by conventional susceptibility testing. Here we investigated antibiotic responses of longitudinal clinical isolates under standard laboratory conditions and in an airway epithelial infection model. Methods Six longitudinal pairs of P. aeruginosa isolates from people with cystic fibrosis were analysed. Changes in bacterial growth following exposure to tobramycin, ceftazidime and ciprofloxacin were quantified under standard laboratory conditions and compared with changes in bacterial burden after treatment measured in differentiated airway epithelial cultures maintained under air-liquid interface (ALI) conditions that reproduce key features of the airway infection environment. Representative phenotypes were visualized by confocal microscopy and interpreted in the context of available whole-genome sequencing data. Results Major resistance phenotypes identified by conventional susceptibility testing were largely reproduced in the ALI model. However, concordance between phenotypes observed under standard laboratory and ALI conditions differed between antibiotics. Several isolates classified as susceptible according to conventional susceptibility testing displayed substantial survival in the ALI model, and these discordant phenotypes were observed predominantly among those isolates recovered after prolonged airway persistence. Genomic analyses identified mutations associated with both classical resistance mechanisms and chronic adaptation. Conclusions Phenotypes observed under conventional laboratory conditions do not uniformly predict those observed in the ALI airway conditions. Discordant phenotypes were most apparent among chronically adapted isolates and varied between antibiotics, suggesting that adaptation acquired during chronic infection can alter antibiotic responses in ways that are not fully reflected by conventional susceptibility testing.