Effect of biofilm physical characteristics on their susceptibility to antibiotics: impacts of low-frequency ultrasound

Biofilms are highly resistant to antimicrobials, often causing chronic infections. Combining antimicrobials with low-frequency ultrasound (LFU) enhances antimicrobial efficiency, but little is known about the underlying mechanisms. Biofilm physical characteristics, which depend on factors such as growth conditions and age, can have significant effects on inactivation efficiency. In this study, we investigated the susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, with and without LFU treatment. The biofilms were grown under low and high fluid shear to provide different characteristics. Low-shear biofilms exhibited greater thickness, roughness, and porosity and lower density, compared to high-shear biofilms. The biofilm matrix of the high-shear biofilms had a three times higher protein-to-polysaccharide ratio, suggesting greater biofilm stiffness. This was supported by microrheology measurements of biofilm creep compliance. For the low-shear biofilms without LFU, the viability of the biofilms in their inner regions was largely unaffected by the antibiotic after a 2-hour treatment. However, when tobramycin was combined with LFU, the inactivation for the entire biofilm increased to 80% after 2 h. For the high-shear biofilms without LFU, higher LFU intensities were needed to achieve similar inactivation results. Microrheology measurements revealed that changes in biofilm inactivation profiles were closely related to changes in biofilm mechanical properties. Modeling suggests that LFU changes antibiotic diffusivity within the biofilm, probably due to a "decohesion" effect. Overall, this research suggests that biofilm physical characteristics (e.g., compliance, morphology) are linked to antimicrobial efficiency. LFU weakens the biofilm while increasing its diffusivity for antibiotics.