Impact of Low-Frequency Ultrasound on Physical Properties and Antibiotic Susceptibility of a Mucoid Biofilm

<i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>) is a pathogen commonly associated with lung infections in cystic fibrosis (CF) patients, often developing a mucoid phenotype that overproduces alginate, a major component of the biofilm's extracellular polymeric substances (EPS) matrix, increasing tolerance to antibiotics. Past studies have shown that low-frequency ultrasound (LFU) increases the antibiotic susceptibility of <i>P. aeruginosa</i> in biofilms, but its effects on mucoid strains are unknown. In this study, we assessed the combined application of LFU and antibiotics on <i>P. aeruginosa</i> FRD1, a mucoid strain, and compared it to nonmucoid <i>P. aeruginosa</i> PAO1 biofilms. The mucoid biofilm exhibited greater stiffness, thickness, and density, with polysaccharides (likely alginate) comprising over 70% of the EPS matrix. Although LFU application led to a 50% increase in biofilm creep compliance, no synergistic effect was observed with tobramycin. However, the same LFU treatment significantly improved ciprofloxacin susceptibility. We implemented a one-dimensional model that estimated a 25-fold increase in the diffusion coefficient for ciprofloxacin following LFU, though higher intensities were needed to induce comparable effects in diffusivity and mechanical properties compared to the nonmucoid biofilm. The model also suggested that tobramycin likely sorbed to alginate, hindering diffusion to the biofilm interior, thereby reducing its inactivation efficiency. Our findings suggest that LFU can impact antibiotic diffusion through the biofilm and mechanical properties but not necessarily overcome antibiotic interactions with the EPS matrix. Additionally, variations in EPS matrix composition may require different LFU intensities for effective outcomes. These results may have significant implications for potential clinical applications, especially for CF patients.