Sonobactericide

This thesis proposes sonobactericide, which is the use of ultrasound and exogenous cavitation nuclei, such as microbubbles, either alone or as a therapeutic complement for biofilm eradication or as a theragnostic for bacterial infections. Using infective endocarditis clinical isolates of _Staphylococcus aureus_, three different translational _in vitro_ biofilm models were developed and used in this thesis. Observed sonobactericide effects that can enhance therapy were biofilm disruption with bacterial dispersal, sonoporation (membrane permeabilization), fibrin strand manipulation (bending, recovery, and breaking), and synergistic responses when a thrombolytic and/or antibiotic were present. This thesis paves the way towards the clinical translation of sonobactericide as an effective treatment, diagnostic, or theranostic strategy employed in infectious disease care

An in vitro proof-of-principle study of sonobactericide

Infective endocarditis (IE) is associated with high morbidity and mortality rates. The predominant bacteria causing IE is Staphylococcus aureus (S. aureus), which can bind to existing thrombi on heart valves and generate vegetations (biofilms). In this in vitro flow study, we evaluated sonobactericide as a novel strategy to treat IE, using ultrasound and an ultrasound contrast agent with or without other therapeutics. We developed a model of IE biofilm using human whole-blood clots infected with patient-derived S. aureus (infected clots). Histology and live-cell imaging revealed a biofilm layer of fibrin-embedded living Staphylococci around a dense erythrocyte core. Infected clots were treated under flow for 30 minutes and degradation was assessed by time-lapse microscopy imaging. Treatments consisted of either continuous plasma flow alone or with different combinations of therapeutics: oxacillin (antibiotic), recombinant tissue plasminogen activator (rt-PA; thrombolytic), intermittent continuous-wave low-frequency ultrasound (120-kHz, 0.44 MPa peak-to-peak pressure), and an ultrasound contrast agent (Definity). Infected clots exposed to the combination of oxacillin, rt-PA, ultrasound, and Definity achieved 99.3 ± 1.7% loss, which was greater than the other treatment arms. Effluent size measurements suggested low likelihood of emboli formation. These results support the continued investigation of sonobactericide as a therapeutic strategy for IE

Sonobactericide: An Emerging Treatment Strategy for Bacterial Infections

Ultrasound has been developed as both a diagnostic tool and a potent promoter of beneficial bio-effects for the treatment of chronic bacterial infections. Bacterial infections, especially those involving biofilm on implants, indwelling catheters and heart valves, affect millions of people each year, and many deaths occur as a consequence. Exposure of microbubbles or droplets to ultrasound can directly affect bacteria and enhance the efficacy of antibiotics or other therapeutics

Opening of endothelial cell–cell contacts due to sonoporation

Ultrasound insonification of microbubbles can locally increase vascular permeability to enhance drug delivery. To control and optimize the therapeutic potential, we need to better understand the underlying biological mechanisms of the drug delivery pathways. The aim of this in vitro study was to elucidate the microbubble-endothelial cell interaction using the Brandaris 128 ultra-high-speed camera (up to 25 Mfps) coupled to a custom-built Nikon confocal microscope, to visualize both microbubble oscillation and the cellular response. Sonoporation and opening of cell-cell contacts by single αVβ3-targeted microbubbles (n = 152) was monitored up to 4 min after ultrasound insonification (2 MHz, 100–400 kPa, 10 cycles). Sonoporation occurred when microbubble excursion amplitudes exceeded 0.7 μm. Quantification of the influx of the fluorescent model drug propidium iodide upon sonoporation showed that the size of the created pore increased for larger microbubble excursion amplitudes. Microbubble-mediated opening of cell-cell contacts occurred as a cellular response upon sonoporation and did not correlate with the microbubble excursion amplitude itself. The initial integrity of the cell-cell contacts affected the susceptibly to drug delivery, since cell-cell contacts opened more often when cells were only partially attached to their neighbors (48%) than when fully attached (14%). The drug delivery outcomes were independent of nonlinear microbubble behavior, microbubble location, and cell size. In conclusion, by studying the microbubble–cell interaction at nanosecond and nanometer resolution the relationship between drug delivery pathways and their underlying mechanisms was further unraveled. These novel insights will aid the development of safe and efficient microbubble-mediated drug delivery

Combined Confocal Microscope and Brandaris 128 Ultra-High-Speed Camera

Controlling microbubble-mediated drug delivery requires the underlying biological and physical mechanisms to be unraveled. To image both microbubble oscillation upon ultrasound insonification and the resulting cellular response, we developed an optical imaging system that can achieve the necessary nanosecond temporal and nanometer spatial resolutions. We coupled the Brandaris 128 ultra-high-speed camera (up to 25 million frames per second) to a custom-built Nikon A1R+ confocal microscope. The unique capabilities of this combined system are demonstrated with three experiments showing microbubble oscillation leading to either endothelial drug delivery, bacterial biofilm disruption, or structural changes in the microbubble coating. In conclusion, using this state-of-the-art optical imaging system, microbubble-mediated drug delivery can be studied with high temporal resolution to resolve microbubble oscillation and high spatial resolution and detector sensitivity to discern cellular response. Combining these two imaging technologies will substantially advance our knowledge on microbubble behavior and its role in drug delivery

Vancomycin-decorated microbubbles as a theranostic agent for Staphylococcus aureus biofilms

Bacterial biofilms are a huge burden on our healthcare systems worldwide. The lack of specificity in diagnostic and treatment possibilities result in difficult-to-treat and persistent infections. The aim of this in vitro study was to investigate if microbubbles targeted specifically to bacteria in biofilms could be used both for diagnosis as well for sonobactericide treatment and demonstrate their theranostic potential for biofilm infection management. The antibiotic vancomycin was chemically coupled to the lipid shell of microbubbles and validated using mass spectrometry and high-axial resolution 4Pi confocal microscopy. Theranostic proof-of-principle was investigated by demonstrating the specific binding of vancomycin-decorated microbubbles (vMB) to statically and flow grown Staphylococcus aureus (S. aureus) biofilms under increasing shear stress flow conditions (0–12 dyn/cm2), as well as confirmation of microbubble oscillation and biofilm disruption upon ultrasound exposure (2 MHz, 250 kPa, and 5,000 or 10,000 cycles) during flow shear stress of 5 dyn/cm2 using time-lapse confocal microscopy combined with the Brandaris 128 ultra-high-speed camera. Vancomycin was successfully incorporated into the microbubble lipid shell. vMB bound significantly more often than control microbubbles to biofilms, also in the presence of free vancomycin (up to 1000 µg/mL) and remained bound under increasing shear stress flow conditions (up to 12 dyn/cm2). Upon ultrasound insonification biofilm area was reduced of up to 28%, as confirmed by confocal microscopy. Our results confirm the successful production of vMB and support their potential as a new theranostic tool for S. aureus biofilm infections by allowing for specific bacterial detection and biofilm disruption.</p