Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of ≥32 versus ≤8 μg/ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa , leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% ± 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% ± 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 ± 2.3 versus 24.2 ± 6.2 gold particles/bacterium, P ≤ 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells.

The aim of this study was to enhance the antimicrobial efficacy of a liposomal gentamicin formulation with gallium metal (Lipo-Ga-GEN) against clinical isolates of Pseudomonas aeruginosa. Sputum isolates of P. aeruginosa from cystic fibrosis patients were used to determine the MIC and MBC of Lipo-Ga-GEN. P. aeruginosa biofilms were formed and used to compare the minimum biofilm eradication concentration of the conventional drugs with that of Lipo-Ga-GEN. Quorum sensing (QS) molecule reduction of P. aeruginosa was determined by monitoring N-acyl homoserine lactone production using Agrobacterium tumefaciens reporter strain (A136). Viability of the cultured human lung epithelial cells (A549) was determined by Trypan Blue assay in order to assess Ga toxicity. MIC and MBC values indicated that gentamicin was more effective against a highly resistant strain of P. aeruginosa (PA-48913) when delivered as a Lipo-Ga-GEN formulation (256 mg/L free gentamicin versus 2 mg/L Lipo-Ga-GEN). Lipo-Ga-GEN was the only formulation that completely eradicated biofilms and blocked QS molecules at a very low concentration (0.94 mg/L gentamicin). The decrease in cell viability was less in A549 cells exposed to Lipo-Ga, suggesting that encapsulated Ga is safer. The results clearly indicate that the Lipo-Ga-GEN formulation is more effective than gentamicin alone in eradicating antibiotic-resistant P. aeruginosa isolates growing in a planktonic or biofilm community.

Lung injury in bacterial infection is a multifactorial phenomenon that involves bacterial metabolites and host factors. Primary isolates of type II pneumocytes and established cultures of Madin-Darby canine kidney (MDCK) cells were used to study effects of Pseudomonas aeruginosa exoproducts on epithelial paracellular permeability. The results indicate that elastase (PE) and exotoxin A (Exo A) have different, but complementary, actions that diminish epithelial barrier function. We measured transepithelial electrical resistance (TER) and permeability coefficient for mannitol (Pm) across cell monolayers plated on tissue culture membranes. Application of 100 ng/ml of Exo A to the basal side decreased TER from 1,405 +/- 106 to 462 +/- 50 ohm (omega) and increased Pm for mannitol 6-fold in 16 h (P < 0.05). Application of Exo A to the apical side did not affect either TER or Pm. In contrast, PE (6.5 U/ml) applied either apically or basolaterally reduced TER to 353 +/- 66 omega and increased Pm by 10-fold within 90 min (P < 0.05). The increase in permeability correlated with the number of bacteria that traversed the epithelial monolayers. Fluorescent staining and western immunoblot analysis of toxin-treated cells showed that two tight junctional proteins, ZO-1 and ZO-2, were depleted in monolayers treated with enzymatically active PE. The junctional proteins decreased in cells treated overnight with Exo A but were not depleted. Neither agent diminished cell viability as measured by trypan blue staining or release of radioactivity from 51 Cr-labeled cells. Elastase from P. aeruginosa thus seems to increase alveolar epithelial permeability by damaging tight junction-associated proteins. Exo A, through its effect on protein synthesis, may render the cells unable to restore the junctional proteins and thus the functional junctions.

Objectives: Chronic pulmonary infection by Pseudomonas aeruginosa in cystic fibrosis patients is virtually impossible to eradicate by means of existing free antibiotics. We sought to assess the antibacterial activities of liposomal gentamicin against clinical isolates of P. aeruginosa. Methods: Gentamicin was encapsulated into liposomes with different lipid compositions (1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol in the molar ratio of 2:1 by sonication. The in vitro stability of liposome-encapsulated gentamicin was studied over a 48 h period at 4 and 37°C in PBS and at 37°C in pooled plasma. The MICs of free and liposomal gentamicin for clinical isolates of P. aeruginosa were assessed by broth dilution. Results: The encapsulation efficiency of all liposomal preparations was 4%–5.18% of the initial amount of the drug in solution. The liposomes retained 60%–70% of the encapsulated gentamicin for 48 h when they were incubated in normal human pooled plasma or PBS at 4 or 37°C. The MICs of liposomal gentamicin for all clinical isolates of P. aeruginosa were lower than the MICs of free gentamicin. Importantly, liposomal gentamicin altered the susceptibilities of these clinical isolates from gentamicin resistant to either intermediate or susceptible. Conclusions: Taken together, these data indicate that liposomal gentamicin formulations could be more effective than the free drug in controlling pulmonary infections due to P. aeruginosa.