Disrupting Biofilms by Combining Ultrasound-Stimulated Microbubbles, Anti-Biofilm Agents and Antibiotics.

OBJECTIVE: Chronic infections are often sustained by biofilms, which exhibit high tolerance to antimicrobials and immune clearance. Ultrasound (US)-stimulated microbubbles (MBs) have shown promise for biofilm disruption, yet their integration with chemical anti-biofilm agents and antibiotics has not been systematically explored. This study reports, for the first time, the combined application of MBs with tetrasodium ethylenediaminetetraacetic acid (T-EDTA), nitric oxide (NO)-releasing NONOates (PAPA NONOate and spermine NONOate) and ciprofloxacin against Pseudomonas aeruginosa PAO1 biofilms. METHODS: P. aeruginosa PAO1 biofilms were grown in Ibidi channel slides in LB broth for 24 or 48 h. Biofilms were then treated with lipid-shell MBs in combination with anti-biofilm agents (4% w/v T-EDTA or 250 µM NONOates) and 0.25 µg/mL ciprofloxacin. Biofilms were exposed to US at 1.1 MHz and 1.0 MPa peak-negative pressure. Biofilm surface area was determined by fluorescence microscopy. RESULTS: MBs demonstrated stable physicochemical properties and cavitation profiles, with 1 MPa peak-negative pressure producing the greatest biofilm dispersal. Among all regimens, T-EDTA combined with ciprofloxacin and US-stimulated MBs achieved the most consistent and substantial biofilm removal, highlighting multimodal efficacy combining physical disruption, matrix modification and antibiotic action. NONOates showed limited benefit when paired with US-stimulated MBs, suggesting cavitation does not enhance NO-mediated dispersal. Biofilm maturity (24 vs. 48 h) significantly influenced treatment outcomes, with early stage biofilms responding more readily to cavitation-driven disruption. CONCLUSION: These findings establish a novel multimodal strategy for anti-biofilm therapy and underscore the importance of considering biofilm developmental stage in ultrasound-enhanced treatment design.