Phage-antibiotic combinations against multidrug-resistant <i>Pseudomonas aeruginosa</i> in <i>in vitro</i> static and dynamic biofilm models

Abstract

Biofilm-producing <i>Pseudomonas aeruginosa</i> infections pose a severe threat to public health and are responsible for high morbidity and mortality. Phage-antibiotic combinations (PACs) are a promising strategy for combatting multidrug-resistant (MDR), extensively drug-resistant (XDR), and difficult-to-treat <i>P. aeruginosa</i> infections. Ten MDR/XDR <i>P. aeruginosa</i> strains and five <i>P</i>. <i>aeruginosa</i>-specific phages were genetically characterized and evaluated based upon their antibiotic susceptibilities and phage sensitivities. Two selected strains, AR351 (XDR) and I0003-1 (MDR), were treated singly and in combination with either a broad-spectrum or narrow-spectrum phage, phage EM-T3762627-2_AH (EM), or 14207, respectively, and bactericidal antibiotics of five classes in biofilm time-kill analyses. Synergy and/or bactericidal activity was demonstrated with all PACs against one or both drug-resistant <i>P. aeruginosa</i> strains (average reduction: -Δ3.32 log₁₀ CFU/cm²). Slightly improved ciprofloxacin susceptibility was observed in both strains after exposure to phages (EM and 14207) in combination with ciprofloxacin and colistin. Based on phage cocktail optimization with four phages (EM, 14207, E20050-C (EC), and 109), we identified several effective phage-antibiotic cocktails for further analysis in a 4-day pharmacokinetic/pharmacodynamic <i>in vitro</i> biofilm model. Three-phage cocktail, EM + EC + 109, in combination with ciprofloxacin demonstrated the greatest biofilm reduction against AR351 (-Δ4.70 log₁₀ CFU/cm² from baseline). Of remarkable interest, the addition of phage 109 prevented phage resistance development to EM and EC in the biofilm model. PACs can demonstrate synergy and offer enhanced eradication of biofilm against drug-resistant <i>P. aeruginosa</i> while preventing the emergence of resistance.