Bactericidal Effect of Ultrasound-Responsive Microbubbles and Sub-inhibitory Gentamicin against Pseudomonas aeruginosa Biofilms on Substrates With Differing Acoustic Impedance

The aim of this research was to explore the interaction between ultrasound-activated microbubbles (MBs) and Pseudomonas aeruginosa biofilms, specifically the effects of MB concentration, ultrasound exposure and substrate properties on bactericidal efficacy. Biofilms were grown using a Centre for Disease Control (CDC) bioreactor on polypropylene or stainless-steel coupons as acoustic analogues for soft and hard tissue, respectively. Biofilms were treated with different concentrations of phospholipid-shelled MBs (10⁷-10⁸ MB/mL), a sub-inhibitory concentration of gentamicin (4 µg/mL) and 1-MHz ultrasound with a continuous or pulsed (100-kHz pulse repetition frequency, 25% duty cycle, 0.5-MPa peak-to-peak pressure) wave. The effect of repeated ultrasound exposure with intervals of either 15- or 60-min was also investigated. With polypropylene coupons, the greatest bactericidal effect was achieved with 2 × 5 min of pulsed ultrasound separated by 60 min and a microbubble concentration of 5 × 10⁷ MBs/mL. A 0.76 log (83%) additional reduction in the number of bacteria was achieved compared with the use of an antibiotic alone. With stainless-steel coupons, a 67% (0.46 log) reduction was obtained under the same exposure conditions, possibly due to enhancement of a standing wave field which inhibited MB penetration in the biofilm. These findings demonstrate the importance of treatment parameter selection in antimicrobial applications of MBs and ultrasound in different tissue environments.