<p>Synergistic antibacterial effect of ultrasound microbubbles combined with chitosan-modified polymyxin B-loaded liposomes on biofilm-producing <em>Acinetobacter baumannii</em></p>

Abstract

Purpose: Resistant strains of Acinetobacter baumannii (AB) that can form biofilms are resistant to polymyxin. Therefore, effective and safe polymyxin preparations against biofilm-producing AB are urgently needed. This study aims to prepare chitosan-modified polymyxin B-loaded liposomes (CLPs) and ultrasound microbubbles (USMBs) and then explore the synergistic antibacterial effects of USMBs combined with CLPs in vitro. Methods: CLPs were prepared using a modified injection method, and microbubbles were prepared using a simple mechanical vibration method. Minimal biofilm inhibitory concentration (MBIC) of CLPs against resistant biofilm-producing AB was determined. Antibacterial activities of CLPs with or without USMBs were analyzed by crystal violet staining and resazurin assays to evaluate biofilm mass and viable counts, respectively. Then, the anti-biofilm effects of CLPs with or without USMBs on biofilm-producing AB were confirmed via scanning electron microscopy (SEM) analysis. Results: We prepared CLPs that were 225.17±17.85 nm in size and carried positive charges of 12.64±1.44 mV. These CLPs, with higher encapsulation efficiency and drug loading, could exhibit a sustained release effect. We prepared microbubbles that were 2.391±0.052 µm in size and carried negative charges of −4.32±0.43 mV. The MBICs of the CLPs on the biofilmproducing AB was 8±2 µg/mL, while that of polymyxin B was 32±2 µg/mL. USMBs in combination with 2 µg/mL of polymyxin B could completely eliminate the biofilm-producing AB and achieve the maximum antimicrobial effects (P.0.05 vs sterile blank control). SEM imaging revealed some scattered bacteria without a biofilm structure in the USMB combined with the CLP group, confirming that this combination has the greatest anti-biofilm effects. Conclusion: In this research, we successfully prepared USMBs and CLPs that have a more significant antibacterial effect on biofilm-forming AB than polymyxin B alone. Experiments in vitro indicate that the synergistic antibacterial effect of combining USMBs with CLPs containing as little as 2 µg/mL of polymyxin B is sufficient to almost eliminate drug-resistant biofilm-producing AB.

Sonobactericide: An Emerging Treatment Strategy for Bacterial Infections

“…A similar increase in bio-effects with increasing microbubble concentration has been reported for sonoporation of eukaryotic cells (Ward et al 2000) and bloodÀbrain barrier disruption (Song et al 2017). Increasing the antibiotic concentration also resulted in an increasing dose-Àeffect relationship for all treatments of Acinetobacter baumannii, including microbubbles combined with free polymyxin B and microbubbles combined with free chitosan-modified polymyxin B-loaded liposomes (Fu et al 2019). Horsley et al (2019) increased both antibiotic and microbubble concentrations, because gentamicin was encapsulated on liposomes bound to the microbubbles, which led to an enhanced reduction of bacterial load in infected urothelial cells.…”

“…Two articles compared two different types of bacteria (Zhu et al 2013;Li et al 2015), and another compared a bacterium and a fungus (Tandiono et al 2012). Only four studies used patient-derived clinical isolates originating from a central venous catheter (Hu et al 2018b), infective endocarditis blood culture (Lattwein et al 2018), pneumonia-induced sputum (Fu et al 2019) and urine from a patient with lower urinary tract symptoms (Horsley et al 2019). With the exception of Agarwal et al (2014), who used bacteria from a wastewater reclamation plant to investigate membrane biofouling removal, the other groups used lab-derived strains, which may limit clinical applicability.…”

“…Changes in biofilm metabolism, in response to different treatments, were measured by confocal imaging with 5-cyano-2,3-ditolyltetrazolium chloride dye (Hu et al, 2018b), a CO 2 -evolution monitoring system (Ronan et al 2016) or an absorbance-based resazurin assay (Guo et al 2017;Fu et al 2019). Although investigation of cellular metabolism provides an indication of the overall bio-effect, the growth rate, biomass, cell viability and persister development could not be specified.…”

“…Four groups produced custom microbubbles for coadministration with an antibiotic (Ikeda-Dantsuji et al 2011;Dong et al 2013Dong et al , 2017Dong et al , 2018, an antibiotic encapsulated in a liposome (Fu et al 2019) or stem cells (Yi et al 2016). Dong et al (2013Dong et al ( , 2017Dong et al ( , 2018 produced lipid-coated microbubbles with a gas core of C 3 F 8 and diameter of 4À6 mm.…”

“…Ikeda-Dantsuji et al (2011) produced microbubbles (»1 mm) encapsulating C 3 F 8 gas and coated with DSPC and DSPE-PEG(2000), at a molar ratio of 94:6. Microbubbles (mean diameter: 2.39 § 0.05 mm) with a coating of 1,2ÀdipalmitoylÀsnÀglyceroÀ3Àphosphocholine, DSPE and cholesterol, mass ratio of 10:4:1 and a C 3 F 8 gas core were produced by Fu et al (2019). A thorough characterization of the response of these microbubbles to ultrasound was not reported in these publications.…”