Quantifying cavitation behavior of activated nanodroplets during antibiotic treatment of methicillin-resistant Staphylococcus aureus biofilms in murine wounds

Chronic wounds are frequently infected with bacterial biofilms that hinder antibiotic efficacy and promote relapse through persister cells. Ultrasound and phase-change contrast agents (PCCAs) have shown promise in enhancing antibiotic delivery and biofilm disruption. However, cavitation feedback during in vivo biofilm treatment has yet to be investigated. In a methicillin-resistant Staphylococcus aureus (MRSA) murine wound model (n = 5), wounds were treated twice daily with gentamicin (0.1%) and palmitoleic acid (3%) from days 2–4 post-infection. One daily treatment was followed by OFP PCCA and ultrasound (1.1 MHz, 9.09% duty cycle, 700 or 1700 kPa). Passive cavitation signals were recorded every 15 s during five 1-min treatments. Total net cavitation dose was quantified from the third to fifth harmonic and bacterial burden was assessed on day 5. Nonlinear regression analysis found strong negative correlation between cavitation dose and bacterial burden (R2 = 0.88), indicating that higher cavitation exposure was associated with greater bacterial reduction. Cavitation was sustained during treatment with 1700 kPa and significantly greater than at 700 kPa (p = 0.0145), where signal dropped towards baseline. Increasing wound coverage (∼40% in these treatments) may further improve burden reduction. This is the first in vivo demonstration linking higher cavitation dose to bacterial reduction in biofilms, supporting cavitation monitoring as a potential feedback tool for optimizing ultrasound-enhanced antimicrobial therapies.