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

Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

Staphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.</p

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

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

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

Native valve, prosthetic valve, and cardiac device-related infective endocarditis: A review and update on current innovative diagnostic and therapeutic strategies

Infective endocarditis (IE) is a life-threatening microbial infection of native and prosthetic heart valves, endocardial surface, and/or indwelling cardiac device. Prevalence of IE is increasing and mortality has not significantly improved despite technological advances. This review provides an updated overview using recent literature on the clinical presentation, diagnosis, imaging, causative pathogens, treatment, and outcomes in native valve, prosthetic valve, and cardiac device-related IE. In addition, the experimental approaches used in IE research to improve the understanding of disease mechanisms and the current diagnostic pipelines are discussed, as well as potential innovative diagnostic and therapeutic strategies. This will ultimately help towards deriving better diagnostic tools and treatments to improve IE patient outcomes