Adverse Impact of Gamma-Polyglutamic Acid on the Antimicrobial Efficacy of Cefiderocol and Nanosilver Against Gram-Negative Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii

<b>Background/Objectives:</b> Wound infections caused by multidrug-resistant Gram-negative bacteria, such as <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, and <i>Acinetobacter baumannii</i>, pose a major clinical challenge. This study evaluated the interactions between gamma-polyglutamic acid (γ-PGA), cefiderocol, and silver nanoparticles (AgNPs) within multilayer wound dressing configurations. The primary goal was to clarify the dual role of γ-PGA as a healing promoter and a potential protector of bacterial cells against antimicrobial agents. <b>Methods:</b> Multilayer dressing models were assembled in 96-well plates to simulate vertical stratification of antimicrobial layers4. Bacterial viability was assessed through relative OD₆₀₀ measurements following incubation with varying concentrations and spatial arrangements of cefiderocol, AgNPs, and γ-PGA. Data were analyzed using generalized linear modeling (GLM) with a gamma distribution and random forest regression to determine the relative importance of each factor in modulating bacterial survival. <b>Results:</b> γ-PGA concentration emerged as the dominant factor influencing bacterial viability, accounting for nearly 100% of variable importance in random forest analysis. Despite high antimicrobial pressure from cefiderocol and AgNPs, bacterial viability stabilized at approximately 40% in the presence of γ-PGA. The vertical positioning of γ-PGA significantly impacted survival; direct physical contact between the polymer and bacteria, particularly at high concentrations, enhanced bacterial persistence in <i>P. aeruginosa</i> and <i>E. coli</i>. Cefiderocol showed strain-specific potency, while AgNPs provided consistent growth inhibition. <b>Conclusions:</b> γ-PGA plays a paradoxical role in wound care by providing moisture retention while simultaneously acting as a cytoprotective agent that reduces antimicrobial efficacy, likely by facilitating biofilm formation. These findings underscore the necessity of optimizing the spatial layering and concentration of biopolymers in advanced dressings. Strategic design is crucial to balance regenerative benefits with maximal antimicrobial control to improve clinical outcomes in chronic wound management.