Strain-specific proteome responses of Pseudomonas aeruginosa to biofilm-associated growth and to calcium

Abstract

Liposomal amikacin (Arikace™) is a liposome-encapsulated form of amikacin that is formulated to treat chronic P. aeruginosa infections in cystic fibrosis patients. These liposomes carry a zwitterionic surface charge and are composed of lipids found naturally within the lung. A key aspect of the activity of the formulation is the ability to penetrate to the sites of Pseudomonas biofilm-like growth in the lung. Experiments were designed to investigate the penetration of liposomes into P. aeruginosa biofilms and in vitro activity. Methods and Results: Model liposomes of the same size and lipid composition as liposomal amikacin (Arikace™) were prepared with membrane-associated or encapsulated fluorescent labels, a hydrophobic carbocyanine dye and calcein, respectively. A mucoid strain of Pseudomonas aeruginosa (PA3064) was used to establish biofilms in rectangular optical grade glass flow cells. Biofilms were observed after four days of growth by confocal laser scanning microscopy using a focal plane set to view within the biofilm cluster or outside as a control. Time dependent accumulation of fluorescent liposomes within the biofilms was measured by the spatial distribution of fluorescence intensity in regions within or outside of the biofilm. Images indicated significant penetration of liposomes into the interior of biofilms under these conditions. The rate of penetration was considerably slower than typical rates for small molecules, consistent with the size of the liposomes. Liposome concentrations were higher near the periphery than the interior. However, even the interior concentration was at least as high as the concentration of liposomes in the fluid outside of the biofilm, suggesting some binding or trapping of the liposomes within the biofilm. Penetration of liposomes was observed under flow or static conditions. In a “washout” experiment, where medium is passed through the biofilms previously treated with liposomes, a significant portion of the liposomes remained associated with the biofilms for an extended period of time. The penetration of liposomes was reflected in the observation of killing of bacteria in colonies in the interior of agar beads. Exposure of these cultures to liposomal amikacin resulted in a large reduction of viable bacteria throughout the beads as monitored by a fluorescent DNA content assay. Similar colony forming unit reductions in animal models (to be shown in other poster presentations) suggest that these principles also operate in vivo. Conclusions: Liposomes similar to liposomal amikacin (Arikace™) readily penetrate into biofilms of Pseudomonas aeruginosa and may even have enhanced binding to biofilms. This binding along with localized release can explain the substantial efficacy observed in animal models.