Ceftolozane/tazobactam plus tobramycin against free-floating and biofilm bacteria of hypermutable Pseudomonas aeruginosa epidemic strains: Resistance mechanisms and synergistic activity

•Hypermutable biofilm-forming Pseudomonas aeruginosa in lungs of cystic fibrosis patients is a major treatment challenge.•Simulated exposures to ceftolozane/tazobactam (C/T) or tobramycin monotherapies were unable to suppress resistant subpopulations.•C/T resistance development was associated with AmpC overexpression and structural modification and novel CpxR mutations.•Combinations showed synergy and completely suppressed C/T and tobramycin less-susceptible free-floating and biofilm subpopulations.•A mechanism-based model well described the effects and will assist in future studies on this promising combination. Acute exacerbations of biofilm-associated Pseudomonas aeruginosa infections in cystic fibrosis (CF) have limited treatment options. Ceftolozane/tazobactam (alone and with a second antibiotic) has not yet been investigated against hypermutable clinical P. aeruginosa isolates in biofilm growth. This study aimed to evaluate, using an in vitro dynamic biofilm model, ceftolozane/tazobactam alone and in combination with tobramycin at simulated representative lung fluid pharmacokinetics against free-floating (planktonic) and biofilm states of two hypermutable P. aeruginosa epidemic strains (LES-1 and CC274) from adolescents with CF. Regimens were intravenous ceftolozane/tazobactam 4.5 g/day continuous infusion, inhaled tobramycin 300 mg 12-hourly, intravenous tobramycin 10 mg/kg 24-hourly, and both ceftolozane/tazobactam–tobramycin combinations. The isolates were susceptible to both antibiotics. Total and less-susceptible free-floating and biofilm bacteria were quantified over 120–168 h. Ceftolozane/tazobactam resistance mechanisms were investigated by whole-genome sequencing. Mechanism-based modelling of bacterial viable counts was performed. Monotherapies of ceftolozane/tazobactam and tobramycin did not sufficiently suppress emergence of less-susceptible subpopulations, although inhaled tobramycin was more effective than intravenous tobramycin. Ceftolozane/tazobactam resistance development was associated with classical (AmpC overexpression plus structural modification) and novel (CpxR mutations) mechanisms depending on the strain. Against both isolates, combination regimens demonstrated synergy and completely suppressed the emergence of ceftolozane/tazobactam and tobramycin less-susceptible free-floating and biofilm bacterial subpopulations. Mechanism-based modelling incorporating subpopulation and mechanistic synergy well described the antibacterial e