Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that sonobactericide can lead to bacterial dispersion or sonoporation (i.e., cell membrane permeabilization); however, real-time observations distinguishing individual bacteria during and directly after insonification are missing. Therefore, in this study, we investigated, in real-time and at high-resolution, the effects of ultrasound-induced microbubble oscillation on Staphylococcus aureus biofilms, without or with an antibiotic (oxacillin, 1 µg/mL). Biofilms were exposed to ultrasound (2 MHz, 100–400 kPa, 100–1000 cycles, every second for 30 s) during time-lapse confocal microscopy recordings of 10 min. Bacterial responses were quantified using post hoc image analysis with particle counting. Bacterial dispersion was observed as the dominant effect over sonoporation, resulting from oscillating microbubbles. Increasing pressure and cycles both led to significantly more dispersion, with the highest pressure leading to the most biofilm removal (up to 83.7%). Antibiotic presence led to more variable treatment responses, yet did not significantly impact the therapeutic efficacy of sonobactericide, suggesting synergism is not an immediate effect. These findings elucidate the direct effects induced by sonobactericide to best utilize its potential as a biofilm treatment strategy.
Bibliographical noteFunding Information:
This research was funded by the European Research Council, under the European Union’s Horizon 2020 research and innovation program, grant number 805308, as well as the Thoraxcenter of the Erasmus MC University Medical Center Rotterdam. The authors would like to thank Robert Beurskens from the Department of Biomedical Engineering and Andi R. Sultan and Corné H. Klaassen from the Department of Medical Microbiology and Infectious Diseases, as well as all at the Erasmus MC University Medical Center Rotterdam, for their skillful assistance. The authors also thank the members of the Therapeutic Ultrasound Contrast Agent group (Biomedical Engineering Dept.) and S. aureus working group (Medical Microbiology and Infectious Diseases Dept.) for their useful discussions.
Funding: This research was funded by the European Research Council, under the European Union’s Horizon 2020 research and innovation program, grant number 805308, as well as the Thoraxcenter of the Erasmus MC University Medical Center Rotterdam.
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