Bactericidal and anti-biofilm effects of uncharged and cationic ultrasound-responsive nitric oxide microbubbles on Pseudomonas aeruginosa biofilms

Abstract

Bacterial biofilms are a major and ongoing concern for public health, featuring both inherited genetic resistance traits and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing need for novel methods of drug delivery, to increase the efficacy of antimicrobial agents. This research evaluated the anti-biofilm and bactericidal effects of ultrasound responsive gas-microbubbles (MBs) of either air or nitric oxide, using an in vitro Pseudomonas aeruginosa biofilm model grown in artificial wound medium. The four lipid-based MB formulations evaluated were room-air MBs (RAMBs) and nitric oxide MBs (NOMBs) with no electrical charge, as well as cationic (+) RAMBs+ and NOMBs+. Two principal treatment conditions were used: i) ultrasound stimulated MBs only, and ii) ultrasound stimulated MBs with a sub-inhibitory concentration (4 µg/mL) of the antibiotic gentamicin. The total treatment time was divided into a 60 second passive MB interaction period prior to 40 second ultrasound exposure; each MB formulation was tested in triplicate. Ultrasound stimulated RAMBs and NOMBs without antibiotic achieved reductions in biofilm biomass of 93.3% and 94.0%, respectively. Their bactericidal efficacy however was limited, with a reduction in culturable cells of 26.9% and 65.3%, respectively. NOMBs with sub-inhibitory antibiotic produced the most significant reduction in biofilm biomass, corresponding to a 99.9% (SD ± 5.21%); and a 99.9% (SD ± 0.07%) (3-log) reduction in culturable bacterial cells. Cationic MBs were initially manufactured to promote binding of MBs to negatively charged biofilms, but these formulations also demonstrated intrinsic bactericidal properties. In the absence of antibiotic, the bactericidal efficacy of RAMB+ and NOMB+ was greater that of uncharged counterparts, reducing culturable cells by 84.7% and 86.1% respectively; increasing to 99.8% when combined with antibiotic. This study thus demonstrates the anti-biofilm and bactericidal utility of ultrasound stimulated MBs, and specifically is the first to demonstrate the efficacy of a NOMB for the dispersal and potentiation of antibiotics against bacterial biofilms in vitro. Importantly the biofilm system and complex growth-medium were selected to recapitulate key morphological features of in vivo biofilms. The results us offer new insight for the development of new clinical treatments, for example, in chronic wounds.

Research hotspot and trend of chronic wounds: A bibliometric analysis from 2013 to 2022

“…Many researchers have recently focused on developing safe and effective ways to better destroy biofilms to treat chronic wound infections. For instance, negative pressure wound therapy (NPWT), ultrasound, antibacterial nanoparticles, phage therapy, quorum sensing inhibitor antimicrobial peptides (AMP) and maggot debridement therapy 101–110 . Most chronic wound biofilms (Over 90%) are composed of various microorganisms 111 .…”

“…For instance, negative pressure wound therapy (NPWT), ultrasound, antibacterial nanoparticles, phage therapy, quorum sensing inhibitor antimicrobial peptides (AMP) and maggot debridement therapy. [101][102][103][104][105][106][107][108][109][110] Most chronic wound biofilms (Over 90%) are composed of various microorganisms. 111 However, many studies have used a single bacterial biofilm.…”

Cationic Microbubbles for Non-Selective Binding of Cavitation Nuclei to Bacterial Biofilms

“…Cationic MBs (+) were produced by adding the cationic phospholipid 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC) (Avanti Polar Lipids, Alabaster, AL, USA) dissolved in chloroform to the DSPC and PEG40s mixture in quantities determined by the desired molar ratio, final lipid concentration, and sample volume as described in other research [ 36 , 37 , 40 , 41 ] (see Table 1 ).…”

“…The use of a non-selectively targeted MB has demonstrated improved treatment efficacy by reducing the diffusion distance between this highly reactive gas and the biofilm. While utility and success of this cationic MB formulation and therapeutic approach have been demonstrated in other work [ 36 ], the present study aims to outline the formulation and testing underpinning its successful implementation. Herein, we hypothesised that cationic microbubbles could therefore be utilised as a non-selective means of targeting bacterial biofilms to achieve greater local concentration of MBs associated with and proximal to the biofilm and in turn increase the efficacy of ultrasound-mediated antibiofilm therapy.…”

Sonosensitive Cavitation Nuclei—A Customisable Platform Technology for Enhanced Therapeutic Delivery

“…CN have been shown to be effective in treating a wide variety of infections, including in vitro monospecies biofilms of both Gram-positive [213,214] and Gram-negative [215,216] bacterial species as well as more complex models, such as an infected clot model [217], a bladder organoid model [218], and an in vivo infected catheter model [219]. In all cases, the benefit of applying CN to a biofilm is twofold; first, cavitation can disrupt the structural integrity of the biofilm matrix via production of craters [220] and micropores [221], and second, it can enhance the delivery of antibiotic drugs, including gentamicin [216,218,220], vancomycin [213,221], oxacillin [212], and streptomycin [220].…”