Higazy, D. | Search bioRxiv

bioRxiv2026

Evolutionary Trajectories of Ciprofloxacin Resistance in P. aeruginosa Lung Biofilms: Mutation Dynamics, Metabolomic Shifts, and Collateral Sensitivity

The evolution of antimicrobial resistance (AMR) in chronic biofilms is often viewed as a unidirectional path toward higher fitness, yet the metabolic constraints governing these trajectories remain poorly understood. We performed a four-passage evolution experiment using a murine lung biofilm model to assess the impact of prolonged ciprofloxacin (CIP) exposure on resistance and host response. This approach integrated population-level adaptive dynamics, whole-genome sequencing (WGS), and NMR-based metabolomics, alongside histopathology and cytokine analysis. Prolonged CIP treatment accelerated resistance, with isolates reaching MICs of 8-12 mg/L (a 32- to 48-fold increase) by the fourth passage. WGS revealed distinct evolutionary trajectories: control isolates accumulated metabolic and regulatory mutations without susceptibility changes, while CIP-treated isolates exhibited a stepwise progression from metabolic adaptation to high-level resistance, marked by early nfxB and late gyrA mutations. Metabolomic profiling revealed progressive divergence, with PCA identifying the nfxB genotype as the primary driver of variation (49.1% of variance). This resistant metabolic state was characterized by the depletion of central carbon metabolites, including glucose and tyrosine, alongside the accumulation of essential amino acids. Importantly, these changes were accompanied by a distinct trade-off; high-level CIP resistance triggered collateral sensitivity to tobramycin and aztreonam. While CIP treatment ultimately reduced neutrophilic inflammation (p = 0.011) and mucin production (p = 0.0496), early-passage lungs exhibited transient elevations in pro-inflammatory cytokines (CXCL2, MMP2, TNF-). In conclusion, the adaptive trajectory to CIP resistance involves metabolic rewiring and collateral sensitivity, offering a framework to exploit the evolutionary costs of resistance in chronic biofilm infections.

microbiology↗

bioRxiv2024

Diversification of Pseudomonas aeruginosa biofilm populations under repeated phage exposures decreases the efficacy of the treatment

Phage therapy has been proposed as a therapeutic alternative to antibiotics for treatment of chronic, biofilm-related P. aeruginosa infections. To get a deeper insight into the complex biofilm-phage interactions, we investigated in the present study the effect of three successive exposures to lytic phages of biofilms formed by the reference strains PAO1 and PA14 as well as of two sequential clinical P. aeruginosa isolates from the sputum of a patient with cystic fibrosis (CF). The Calgary device was employed as biofilm model and the efficacy of phage treatment was evaluated by measurements of the biomass stained with crystal violet (CV) and of the cell density of the biofilm bacterial population (CFU/ml) after each of the three phage exposures. The genetic alterations of P. aeruginosa isolates from biofilms exposed to phages were investigated by whole genome sequencing. We show here that the anti-biofilm efficacy of the phage treatment decreased rapidly with repeated applications of lytic phages on P. aeruginosa strains with different genetic background. Although we observed the maintenance of a small subpopulation of sensitive cells after repeated phage treatments, a fast recruitment of mechanisms involved in the persistence of biofilms to the phage attack occurred, mainly by mutations causing alterations of the phage receptors. However, mutations causing phage tolerant phenotypes such as alginate-hyperproducing mutants were also observed. In conclusion, a decreased anti-biofilm effect occurred after repeated exposure to lytic phages of P. aeruginosa biofilms due to recruitment of different resistance and tolerance mechanisms.

microbiology↗