pH-Dependent Evolution of Delafloxacin and Ciprofloxacin Resistance in Pseudomonas aeruginosa from cystic fibrosis (CF) and non-CF-patients

ABSTRACT Delafloxacin (DLX) is a novel fluoroquinolone with enhanced antibacterial activity in acidic environments, a property that may be advantageous for treating Pseudomonas aeruginosa infections in cystic fibrosis (CF), where airway surface liquid pH is typically reduced (pH 5.5–6.7). However, the propensity for resistance development and the underlying mechanisms in P. aeruginosa remain incompletely defined. We conducted serial passage experiments on six clinical P. aeruginosa isolates (three CF-and three non-CF-derived) exposed to sub-inhibitory concentrations of DLX or ciprofloxacin (CIP) at pH 6.0 and 7.3 over nine days. Susceptibility was assessed by broth microdilution (BMD), and resistance mechanisms were characterized by whole-genome sequencing (WGS), efflux pump expression analysis (qRT-PCR), and functional validation using CRISPR/Cas9-mediated genome editing and complementation assays. DLX minimal inhibitory concentrations (MICs) rose only 10.1-to 28.5-fold over 9 days, compared with 77.6-to 97.8-fold for CIP, indicating a substantially higher genetic barrier to resistance. This barrier was most pronounced under acidic conditions: only 38.9% of DLX-passaged samples crossed the resistance breakpoint, versus 94.4% at neutral pH, whereas CIP resistance reached 100% regardless of pH. Cross-resistance was asymmetric: exposure to DLX consistently selected for CIP cross-resistance (97.2% of samples), whereas exposure to CIP induced DLX cross-resistance efficiently at neutral pH but only partially under acidic conditions. A previously undescribed gyrA mutation (p.Ala51Val) conferred a 4-fold increase in DLX MIC when introduced by CRISPR/Cas9, and upregulation of the MexEF-OprN efflux pump, reversible by mexS complementation, emerged as a prominent resistance mechanism. Overall, DLX exhibited a markedly higher genetic barrier to resistance than CIP in P. aeruginosa , particularly under the acidic conditions characteristic of the CF airway. However, its use may co-select for CIP cross-resistance through efflux upregulation, underscoring the need for careful stewardship in CF.