The single most common microbe causing cardiovascular infections is Staphylococcus aureus (S. aureus). S. aureus produces coagulase that converts fibrinogen to fibrin, which is incorporated into biofilms. This process aids in adherence to intravascular structures, defense against the host immune system, and resistance to antimicrobial treatment. Despite its significance, fibrin formation in S. aureus biofilms remains poorly understood. Therefore, this study aimed to elucidate the early development of cardiovascular biofilms. Clinically isolated coagulase-positive S. aureus and coagulase-negative Staphylococcus lugdunensis (S. lugdunensis) from patients with cardiovascular infections, and a coagulase mutant S. aureus Δcoa, were grown in tryptic soy broth (TSB), Iscove's Modified Dulbecco's Medium (IMDM), and pooled human plasma, with or without porcine heart valves. Bacterial growth, metabolic activity, and bacterial fibrinogen utilization were measured over 24 h at 37 °C. Time-lapse confocal microscopy was used to visualize and track biofilm development. S. aureus exhibited more growth in TSB and human plasma than S. lugdunensis and S. aureus Δcoa, but showed similar growth in IMDM after 24 h. Peak metabolic activity for all isolates was highest in TSB and lowest in human plasma. The presence of porcine valves caused strain-dependent alterations in time to peak metabolic activity. Confocal imaging revealed fibrin-based biofilm development exclusively in the coagulase-producing S. aureus strains. Between 2 and 6 h of biofilm development, 74.9 % (p = 0.034) of the fibrinogen from the medium was converted to fibrin. Variations in fibrin network porosity and density were observed among different coagulase-producing S. aureus strains. Fibrin formation is mediated by S. aureus coagulase and first strands occurred within 3 h for clinical strains after exposure to human plasma. This study stresses the importance of experimental design given the bacterial changes due to different media and substrates and provides insights into the early pathogenesis of S. aureus cardiovascular biofilms.

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.

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.