Use of static overpressure to assess the role of acoustic cavitation in ultrasound-mediated gene transfection

Ultrasound-mediated DNA delivery (UDD) is a technique that utilizes ultrasound to aid the transfer of genetic materials into bacterial cells of interest (either in their planktonic or biofilm modes), resulting in new functionalities being displayed by the target microorganisms in-situ. The physical mechanism of UDD is poorly understood, which hampers its widespread adoption and scaling. To investigate the role of cavitation in UDD, we utilized a static pressure vessel designed to suppress cavitation while leaving other acoustic effects unaltered. Pseudomonas putida UWC1, a soil bacterium that is not naturally competent, was suspended in water and exposed to 40 kHz burst-mode ultrasound (400 cycle burst, 10 Hz repetition frequency, 60 s total) at an ambient pressure of either 1 bar or 9.5 bars. Noise diagnostics yielded inertial cavitation levels and transformed cells were quantified using a doubly selective reporter plasmid DNA. Samples exposed at 9.5 bars overpressure exhibited a drastic reduction in both cavitation activity and the number of transformed cells compared to those exposed at 1 bar. (Cell viability was not affected by pressure change.) The fact that transformation rates are reduced significantly when cavitation is suppressed suggests that acoustic cavitation is the major contributing factor in UDD.