Synthetically acetylated alginate is a superior in vitro biofilm model for antibiotic testing showing reduced tobramycin affinity

This study develops and validates synthetically acetylated alginate hydrogels as a reproducible in vitro model that more accurately mimics the physicochemical and mechanical properties of mucoid Pseudomonas aeruginosa biofilms relevant to cystic fibrosis infections. By comparing pristine seaweed-derived alginate, synthetically acetylated alginate, and native bacterial alginate, the authors demonstrate that acetylation significantly reduces hydrogel stiffness and alters antibiotic–matrix interactions in an ionic-strength-dependent manner. Micro-compression testing revealed that aminoglycoside antibiotics (tobramycin and gentamicin) dramatically stiffen alginate networks in Müller–Hinton broth, whereas this effect is suppressed for acetylated alginates under physiological saline conditions. Isothermal titration calorimetry and MRI diffusion mapping further showed that acetylation lowers tobramycin binding affinity and enables deeper antibiotic penetration into the gel matrix. Together, the results establish acetylated alginate as a mechanically and chemically faithful biofilm surrogate for antibiotic testing, overcoming the variability and experimental limitations of native biofilms.