Liposomal N-acetylcysteine Modulates the Pathogenesis of P. aeruginosa Isolated from the Lungs of Cystic Fibrosis Patient

Abstract

N-acetylcysteine (NAC) is a mucolytic agent with antimicrobial potential. We evaluated the antimicrobial activity of the free and liposomal NAC (F-NAC; L-NAC) against Pseudomonas aeruginosa. The minimum inhibitory concentrations (MIC), the minimum bactericidal concentrations (MBC) and the in vitro time kill studies of L-NAC were determined by broth-dilution method. Efficacy of the formulations on the production of N-acyl homoserine lactone molecules, virulence factors and motility were determined. Eradication of bacterial community within biofilms was assessed using the Calgary Biofilm Device. The L-NAC Cytotoxicity and anti-bacterial adhesion potential to human lung cells were examined using pulmonary A549 cell lines. The MIC of L-NAC was lower than the free drug (1250 mg/L and 5000 mg/L, respectively). MBC for L-NAC was 2500 mg/L compared to 5000 mg/L for F-NAC. L-NAC at 2500 mg/L killed bacteria in 2 h, whereas F-NAC exhibited the same effect at 5000 mg/L. Quorum sensing was significantly inhibited by L-NAC (P<0.001). At 1/8 MIC, L-NAC reduced the production of bacterial proteases significantly more than that of F-NAC at 1/4 MIC. L-NAC was also able to reduce the bacterial motility at eightfold lower concentration than F-NAC (P<0.001). As for biofilms, L-NAC provided 75% protection against biofilm formation, 90% reduction in the formed biofilms, and a 46% eradication effect on bacterial community within biofilms compared to treated biofilm with PBS (P<0.001). Finally, L-NAC at 2500 mg/L was safe to A549 cells, reduced bacterial adhesion by 15% compared to control (P<0.001). These data indicate that L-NAC formulation is more effective than F-NAC against P. aeruginosa and has the potential to improve therapeutic outcomes in CF patient. (VF) [17]. P. aeruginosa pathogenesis is closely associated with its motility such as swimming, swarming and twitching, which allows it to attach to different surfaces forming a biofilm [18]. P. aeruginosa grows into biofilms as a survival strategy. These biofilms are described as communities of microbial cells which are enclosed in polysaccharides matrix, proteins and nucleic acids [19]. P. aeruginosa forms three dimensional mushroom shaped colonies attached to the surface with the help of flagellar motility and type IV pili-mediated twitching motility [20]. Biofilms work as a shield to protect bacterial cells against the host immune system and antibiotics [21-24]. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibiotics are found to be 100 to 1000 fold higher for mature biofilms when compared to planktonic cells or young biofilms [25]. These communities are predominantly associated with persistence of infection as well as resistant to antibiotic; thereby making the biofilms of P. aeruginosa hard to control and eradicate [26]. P. aeruginosa releases several VF such as elastase, chitinase, lipase and protease [27-29]. These VF have a major effect on the alveolar epithelial permeability by damaging collagen, α-1-antitrypsin, elastin, im...

Antibacterial Efficacy of Liposomal Formulations Containing Tobramycin and N-Acetylcysteine against Tobramycin-Resistant Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii

“…As reported by Messiaen et al, 2013, the size of the prepared tobramycin liposomes were 426.3 (±26.4) and 228.5 (±34.9), making the sizes of the TL and TNL average [71]. However, the size of the TNL liposomes were closer to those described by Hasanin and others, which had a mean diameter of 200 nm [72,75]. The polydispersity index (PDI), also known as the heterogeneity index, is a description of the size distribution in the tested sample.…”

“…On other hand, the sizes of the liposome formulations were expected to range from 25 nm to 2.5 µm, based on which they were classified as small (≤100 nm), intermediate (100-250 nm), large (≥250 nm) or giant (>1 µm) [71,72] The average size of bacterial cells is approximately 1 µm, and for liposomal formulations to fuse properly with the bacterial membrane and release their contents, they must be smaller than the bacterial cells. The size of the nanoparticle is an important factor in drug delivery to eukaryotic cells.…”

Efficacy of neutral and negatively charged liposome-loaded gentamicin on planktonic bacteria and biofilm communities

“…The biofilm reduction assay was performed to determine whether the liposomal formulations at a concentration of 16× MIC could inhibit the formation of biofilm, as described previously. 44 For biofilm reduction, bacteria were allowed to attach to the surface of a 96-well polystyrene plate and allowed to form a biofilm for 18 h. The next day, unadhered bacteria were washed and the biofilm was incubated again for 18 h with treatments at 16× MIC. Biofilm formation was measured as above.…”