Harnessing Ultrasound-Stimulated Phase Change Contrast Agents to Improve Antibiotic Efficacy Against Methicillin-ResistantStaphylococcus aureusBiofilms

Abstract

17Bacterial biofilms, often associated with chronic infections, respond poorly to antibiotic 18 therapy and frequently require surgical intervention. Biofilms harbor persister cells, metabolically 19 indolent cells, which are tolerant to most conventional antibiotics. In addition, the biofilm matrix 20 can act as a physical barrier, impeding diffusion of antibiotics. Novel therapeutic approaches 21 frequently improve biofilm killing, but usually fail to achieve eradication. Failure to eradicate the 22 biofilm leads to chronic and relapsing infection, associated with major financial healthcare costs 23 and significant morbidity and mortality. We address this problem with a two-pronged strategy 24 using 1) antibiotics that target persister cells and 2) ultrasound-stimulated phase-change 25 contrast agents (US-PCCA) , which improve antibiotic penetration. 26We previously demonstrated that rhamnolipids, produced by Pseudomonas aeruginosa, 27 could induce aminoglycoside uptake in gram-positive organisms, leading to persister cell death. 28We have also shown that US-PCCA can transiently disrupt biological barriers to improve 29 penetration of therapeutic macromolecules. We hypothesized that combining antibiotics which 30 target persister cells with US-PCCA to improve drug penetration could eradicate methicillin 31 resistant S. aureus (MRSA) biofilms. Aminoglycosides alone or in combination with US-PCCA 32 displayed limited efficacy against MRSA biofilms. In contrast, the anti-persister combination of 33 rhamnolipids and aminoglycosides combined with US-PCCA dramatically reduced biofilm 34 viability, frequently culminating in complete eradication of the biofilm. These data demonstrate 35 that biofilm eradication can be achieved using a combined approach of improving drug 36 penetration of therapeutics that target persister cells.37 38 42 osteomyelitis, endocarditis and pneumonia 2,3. In addition to the high degree of mortality, chronic 43 and relapsing S. aureus infections are common and associated with significant morbidity. This is 44 2 due to frequent treatment failure of S. aureus infections. This is best illustrated by SSTIs, with 45 some studies suggesting treatment failure rates as high as 45% and a recurrence rate of 70% 4. 46Importantly the failure of antibiotic therapy cannot be adequately explained by antibiotic 47 resistance 1. Failure to clear the infection leads to a need for prolonged antibiotic therapies, 48 increased morbidity and mortality, increased likelihood of antibiotic resistance development as 49 well as an enormous financial healthcare burden. 50 S. aureus forms biofilms, bacterial cells embedded in a self-produced extracellular 51 matrix, which act as a protective barrier from the host immune response and other 52 environmental assaults. Biofilms expand up to 1200μm in thickness when attached to indwelling 53 devices such as catheters 5. Non-surface attached biofilms, in chronic wounds and chronic lung 54 infections, harbor smaller non-surface attached cell aggregates ranging from 2-200μm in 5...

Making waves: how ultrasound-targeted drug delivery is changing pharmaceutical approaches

“…32,33 Similarly, a limited number of studies have demonstrated increased antibiotic efficacy against infections using therapeutic ultrasound. [34][35][36][37] Increased cytotoxicity is likely due to increased cell uptake of drug in response to ultrasound-induced increases in cell membrane permeability. 33,34,38 However, augmenting drug delivery via ultrasound insonation is typically more efficacious when combined with microbubble-based ultrasound contrast agents (UCA) (Fig.…”

“…Other phase-change contrast agents (typically liquid PFC droplet stabilized by a phospholipid shell) with 100-400 nm diameters have also been used to enhance antibiotic efficacy against biofilms by up to 94%. 37 Rapoport et al describe development of ultrasound-activated, paclitaxel-carrying, PEG-PLLA nanoemulsions that convert into microbubbles upon insonation (1 MHz, 3.4 W cm À2 ) for cancer treatment. This ultrasound-triggered nanotherapy causes tumor regression by an order of magnitude in ovarian, breast, and orthotopic pancreatic cancer mouse models.…”

Recalcitrant Staphylococcus aureus Infections: Obstacles and Solutions

“…A disadvantage of microbubbles is their short half-life following injection and their relatively large size (1 to 4 mm in diameter) (106), which may hamper penetration into biofilms. Maintaining microbubbles in the more stable liquid phase (nanodroplets) is hypothesized to improve penetration into biofilms due to their smaller size (0.1 to 0.4 mm in diameter) (107). Upon ultrasound activation, nanodroplets expand into microbubbles, creating shear stress from inside the biofilm ( 108).…”

“…Upon ultrasound activation, nanodroplets expand into microbubbles, creating shear stress from inside the biofilm ( 108). This experimental treatment strategy has successfully improved antibiotic efficacy against S. aureus biofilms in vitro (107,108), improved transdermal drug delivery (109), and transiently permeabilized the blood brain barrier (110); in theory, this method could be expanded to improve drug penetration to any niche that is vascularized. In addition, it has the potential for rapid clinical translation, as ultrasound is already used routinely in wound debridement, some nanodroplet formulations are variants of FDA-approved ultrasound contrast agents that are already in clinical practice (111), and the low-pressure ultrasound settings required can be achieved with existing ultrasound hardware at pressures below the FDA set limits for diagnostic imaging (107).…”