Receptor-Targeted Sequential Phage Dosing against Pseudomonas aeruginosa PAO1: An In Vitro Proof-of-Concept Study

Abstract Purpose Antibiotic resistance has intensified the need for alternative therapies, such as bacteriophage administration. However, the optimal delivery strategy—specifically simultaneous versus sequential dosing—remains poorly defined for ensuring sustainable efficacy. This single-strain, in vitro proof-of-concept study evaluated receptor-targeted sequential dosing using phages Dobby, targeting lipopolysaccharide, and MPK7, targeting type IV pili, against P. aeruginosa PAO1 . Methods Time-kill assays were conducted at various multiplicities of infection (MOI: 0.01, 1, and 100) to compare simultaneous and sequential dosing regimens. Resistance profiling was performed to assess the fitness costs of resulting isolates, while atomic force microscopy infrared spectroscopy (AFM-IR) was utilized to identify chemical signatures and underlying resistance mechanisms in phage-resistant mutants. Results Sequential treatment, particularly initiating therapy with the LPS-specific phage Dobby followed by MPK7, was more effective than simultaneous dosing, achieving bacterial reductions exceeding 3-log10 within 48 h. Notably, low MOI (0.01) sequential treatments maintained sustained population suppression, whereas high MOI (100) treatments triggered rapid bacterial rebound. Resistance profiling confirmed that Dobby-resistant isolates exhibited lower fitness costs, rendering them more susceptible to subsequent MPK7 treatment. AFM-IR revealed unique chemical alterations in resistant mutants, specifically linked to LPS truncation and TFP dysfunction. Conclusions In the Dobby–MPK7–PAO1 model, receptor-specific sequential dosing produced more sustained bacterial suppression than simultaneous treatment. This proof-of-concept supports further evaluation of temporal phage dosing across diverse P. aeruginosa strains and phage combinations.