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

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.

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.

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.

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.