Phage-based decontamination: Exploring resistance, disinfectant tolerance, and virulence trade-offs in Acinetobacter baumannii

Increasing multidrug resistance in Acinetobacter baumannii necessitates a better understanding of how phage resistance influences bacterial fitness, disinfectant tolerance, and virulence. In this study, we induced phage-resistant mutants from the reference strain ATCC 17978 using phages previously applied in clinical decontamination, and we evaluated their physiological adaptations. The wild-type strain initially showed high phage susceptibility but rapidly developed resistance within 24 h. Phage-resistant mutants exhibited changes in plaque morphology and developed at frequencies between 7.6 × 10⁻⁷ and 2.11 × 10⁻⁵. In many cases, these mutants required alternative phages for successful reinfection. Certain phage-resistant strains (e.g., 2R and RB71RR) showed initial fitness reductions (15-20 % fewer generations), which were restored to wild-type levels after 24 h. Whole-genome sequencing of JB68R revealed mutations in glycosyltransferases, IS3 transposases, and peptidoglycan DD-metalloendopeptidases, which correlated with increased ethanol sensitivity (50 %) and enhanced NaDCC tolerance-suggesting potential membrane remodeling as an adaptive mechanism. All phage-resistant mutants exhibited reduced virulence in a Galleria mellonella model, as indicated by lower mortality and melanization responses. To provide a comparative perspective, we also examined colistin-resistant strains generated from ATCC 17978, given the critical role of colistin as a last-line antibiotic. These strains displayed persistent growth impairment (22 % reduced generation number, 32 % lower growth rate; p < 0.01) alongside increased tolerance to hydrogen peroxide (3.3-fold) and benzalkonium chloride (2-fold), indicating a distinct set of resistance trade-offs. These findings underscore the ecological and physiological differences between phage and antibiotic resistance and highlight exploitable vulnerabilities, which could guide the development of more effective, integrated infection control strategies in healthcare settings.