Evolution of antibiotic resistance in Pseudomonas aeruginosa using sequential regimen of antibiotic pair

Abstract

The WHO classifies MDR P . aeruginosa as a critical pathogen, underscoring the need for new treatments. In this study, diverse mutant lineages of P. aeruginosa AR06 were developed by exposing them to pairs of antibiotics from three different antibiotic groups. Notably, one mutant Mez(R)_Tob(R) surprisingly retained susceptibility despite sequential exposure to two different antibiotic stresses. Meanwhile, the mutant likely minimizes energy consumption for survival under stresses by deleting genes approximately 10% of its genes, especially those involved in efflux pump genes (e.g., MexV , MexW , MexJ , MexK , etc.) and their regulators (e.g., mexR , mexS , mexT ). These deletions, particularly efflux pump genes can lead to increased susceptibility to certain antibiotics due to the loss of efflux mechanisms, but the overall effect on resistance/susceptibility can be complex and context-dependent. Moreover, the energy minimization is further supplemented by porin impermeability (e.g., OprD, OprE, and OprF). This study provides first report on the mechanism underlying the persistence of susceptible phenotype in the mutant Mez(R)_Tob(R). However, the wild-type susceptible bacteria P. aeruginosa AR06 acquired high-level resistance when exposed to levofloxacin, with minimum inhibitory concentrations (MICs) exceeding 100 μg/mL. Subsequent treatment with tobramycin further increased resistance levels. In contrast, higher doses of mezlocillin were required to achieve similar resistance levels, indicating that β-lactams may be less prone to inducing resistance compared to fluoroquinolones and aminoglycosides. These findings emphasize the need for judicious antibiotic use and consideration of bacterial adaptive mechanisms to effectively manage and treat P . aeruginosa infections.