Ireddy, N. | Search bioRxiv

bioRxiv2026

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 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-derived 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, compared with 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.

microbiology↗

bioRxiv2025

Multi-omics profiling of cross-resistance between ceftazidime-avibactam and meropenem identifies common and strain-specific mechanisms in Pseudomonas aeruginosa clinical isolates

Pseudomonas aeruginosa is a highly versatile and resilient pathogen that can infect different tissues and rapidly develop resistance to multiple drugs. Ceftazidime-avibactam (CZA) is an antibiotic often used to treat multidrug resistant infections, however, the knowledge on the CZA resistance mechanisms in P. aeruginosa is limited. Here we performed laboratory evolution of eight clinical isolates of P. aeruginosa exposed to either CZA or meropenem (MEM) in sub-inhibitory concentrations, and used multi-omics profiling to investigate emerging resistance mechanisms. The majority of strains exposed to MEM developed high resistance (83%, 20/24 strains from eight clinical isolates), with only 17% (4/24) acquiring cross-resistance to CZA. The rate of resistance evolution to CZA was substantially lower (21%, 5/24), while 38% (9/24) acquired cross-resistance to MEM. Whole-genome sequencing revealed strain heterogeneity and different evolutionary paths, with three genes mutated in three or more strains: dacB in CZA-treated strains and oprD and ftsI in MEM-treated strains. Transcriptomic and proteomic analysis underlined heterogeneous strain response to antibiotic treatment with few commonly regulated genes and proteins. To identify genes potentially associated with antibiotic resistance, we built a machine learning model that could separate CZA- and MEM-resistant from sensitive strains based on gene expression and protein abundances. To test some of the identified associations, we performed CRISPR/Cas9 genome editing that demonstrated that mutations in dacB, ampD, and to a lesser extent in mexR directly affected CZA resistance. Overall, this study provides novel insights into the strain-specific molecular mechanisms regulating CZA resistance in Pseudomonas aeruginosa. ImportancePseudomonas aeruginosa is one of the most difficult-to-treat pathogens in the hospital, which often acquires resistance to multiple antibiotics. Ceftazidime-avibactam (CZA) is an essential antibiotic used to treat multidrug resistant infections, but its resistance mechanisms are not well understood. Here we investigated the evolution of resistance to CZA and meropenem (MEM) in eight clinical bacterial isolates from patients blood, urine, and sputum. While the rate of resistance evolution to MEM was higher than to CZA, MEM-resistant strains rarely acquired cross-resistance towards CZA. To identify changes at the genome, transcriptome and proteome levels during antibiotic exposure, we performed multi-omics profiling of the evolved strains, and confirmed the effect of several genes on antibiotic resistance with genetic engineering. Altogether, our study provides insights into the molecular response of P. aeruginosa to CZA and MEM and informs therapeutic interventions, suggesting that CZA could still be effective for patients infected with MEM-resistant pathogens.

microbiology↗