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

Abstract

Interactions between antibiotic compounds and bacterial biofilms can have detrimental impact on their efficacy during treatment of bacterial infections. Despite acetylated alginate being the main structural compound of P. aeruginosa’s biofilms, most in vitro models use pristine seaweed-derived alginate. Therefore, in this work synthetically acetylated seaweed-derived alginate is tested under various conditions and compared to pristine seaweed‑derived and native bacterial alginate. It is demonstrated that acetylated alginates are softer than non-acetylated alginates and interact vastly different with antibiotics in physiological saline solution than in standard bacteriological medium. All tested alginates show massively increased Young’s moduli after treatment with the aminoglycosides tobramycin and gentamicin in Müller‑Hinton broth, whereas in physiological solution this interaction is lowered and absent, respectively, for acetylated alginates. Magnetic resonance imaging experiments show that tobramycin reaches and affects the molecular network in the centre of a Ca‑alginate bead only if the alginate backbone is acetylated. Acetylated alginates have drastically reduced affinity towards tobramycin as measured by isothermal titration calorimetry. ΔHapp and KD are dependent on the ionic strength of the buffer and are increased for acetylated alginates compared to pristine seaweed-derived alginate. Throughout these results it is shown that synthetically acetylated seaweed-alginate is matching native bacterial alginate (physico-)chemical properties more closely than pristine seaweed-derived alginate.