Abstract
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.
Highlights
80% of all bacterial infections are associated with biofilms [1], where bacteria encase themselves in a protective matrix hindering antibiotic effectiveness up to 1,000-fold compared to freefloating bacteria and facilitating development of antibiotic resistance [2, 3]
We investigated the capability of the vancomycin9 decorated microbubbles (vMB) to remain bound to bacterial biofilms under static and shear stress conditions and evaluated their theranostic potential using confocal microscopy combined with ultra-high-speed imaging using the Brandaris 128
The multiplicity of PEG units in the lipid and the presence of the chlorine isotopes in vancomycin led to intricated spectra where the average molecular weight (MW) determination of the conjugate was not possible
Summary
80% of all bacterial infections are associated with biofilms [1], where bacteria encase themselves in a protective matrix hindering antibiotic effectiveness up to 1,000-fold compared to freefloating (i.e., planktonic) bacteria and facilitating development of antibiotic resistance [2, 3]. This increased resistance is largely due to the reduced metabolic activity of biofilm-embedded bacteria and the limited penetration of antibiotics throughout the biofilm [4,5,6]. Delayed diagnosis of infective endocarditis is associated with increased mortality and early diagnosis of biofilm infection is critical [11, 15, 16]
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