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

Delafloxacin (DLX) is a novel fluoroquinolone with increased antibacterial potency in acidic environments, potentially beneficial for treating Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients. However, the development of resistance and its underlying mechanisms in P. aeruginosa remain unclear. To investigate this, researchers performed serial passage experiments on six clinical P. aeruginosa isolates (three derived from CF patients and three from non-CF patients) exposed to sub-inhibitory concentrations of DLX or ciprofloxacin (CIP) at pH 6.0 and 7.3 over a period of nine days.

Susceptibility to the antibiotics was evaluated using broth microdilution, while resistance mechanisms were characterized through whole-genome sequencing, efflux pump expression analysis, and functional validation via CRISPR/Cas9-mediated genome editing and complementation assays. The minimal inhibitory concentrations (MICs) of DLX only increased 10.1- to 28.5-fold over nine days, while those of CIP increased 77.6- to 97.8-fold, suggesting a significantly higher genetic resistance barrier to CIP.

This barrier was most evident under acidic conditions, with only 38.9% of DLX-passaged samples reaching the resistance threshold compared to 94.4% at neutral pH, while CIP resistance was achieved at 100% regardless of pH. There was also an observed asymmetric cross-resistance pattern, as exposure to DLX consistently selected for resistance to CIP in 97.2% of samples, but exposure to CIP only induced DLX cross-resistance efficiently at neutral pH but partially under acidic conditions.

A novel gyrA mutation (p.Ala51Val) was identified, which conferred a 4-fold increase in DLX MIC when introduced via CRISPR/Cas9. Furthermore, upregulation of the MexEF-OprN efflux pump, which could be reversed by mexS complementation, emerged as a significant resistance mechanism. In summary, the study found that DLX demonstrated a considerably higher genetic resistance barrier to resistance compared to CIP in P. aeruginosa, especially under the acidic conditions prevalent in the CF airway.

However, the use of DLX may inadvertently select for CIP cross-resistance through efflux pump upregulation, highlighting the necessity for cautious usage in CF patients.