The Semi-Synthetic Peptide Lin-SB056-1 in Combination with EDTA Exerts Strong Antimicrobial and Antibiofilm Activity against Pseudomonas aeruginosa in Conditions Mimicking Cystic Fibrosis Sputum

Abstract

Pseudomonas aeruginosa is a major cause of chronic lung infections in cystic fibrosis (CF) patients. The ability of the bacterium to form biofilms and the presence of a thick and stagnant mucus in the airways of CF patients largely contribute to antibiotic therapy failure and demand for new antimicrobial agents able to act in the CF environment. The present study investigated the anti-P. aeruginosa activity of lin-SB056-1, a recently described semi-synthetic antimicrobial peptide, used alone and in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA). Bactericidal assays were carried out in standard culture conditions and in an artificial sputum medium (ASM) closely resembling the CF environment. Peptide’s structure and interaction with large unilamellar vesicles in media with different ionic strengths were also investigated through infrared spectroscopy. Lin-SB056-1 demonstrated fast and strong bactericidal activity against both mucoid and non-mucoid strains of P. aeruginosa in planktonic form and, in combination with EDTA, caused significant reduction of the biomass of P. aeruginosa mature biofilms. In ASM, the peptide/EDTA combination exerted a strong bactericidal effect and inhibited the formation of biofilm-like structures of P. aeruginosa. Overall, the results obtained highlight the potential of the lin-SB056-1/EDTA combination for the treatment of P. aeruginosa lung infections in CF patients.

Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential

“…Differently from artificial sputum medium, patients' sputum contains host/bacterial components such as cell-derived factors, normal flora, inflammatory mediators, proteases, or peptidases that may exert an additional inhibitory effect on the peptide's activity. Nevertheless, herein, we showed that the combination of lin-SB056-1 and EDTA at sub-active and non-cytotoxic concentrations [16,27] determined a significant reduction in P. aeruginosa load in sputa of PCD chronically infected patients, despite certain differences in the level of reduction among different sputum samples being observed. Such differences are not surprising considering that inter-patient variables (e.g., clinical stage and severity of lung infection, bacterial load, sputum sample composition, and consistency) were not standardized in our experiments, in the attempt to mimic conditions found during the actual antimicrobial therapy.…”

“…EDTA is prescribed in a number of clinical conditions demonstrating high tolerability (up to 2 g once a week, intravenously injected) and efficacy [15]. Recently, we demonstrated that the optimized semi-synthetic antimicrobial peptide lin-SB056-1 in combination with EDTA is able to exert a synergistic bactericidal effect against P. aeruginosa in an artificial sputum medium resembling CF sputum [16]. Similarities and differences between CF and PDC sputum have been reported.…”

“…Similar to CF patients, eradication of chronic P. aeruginosa infection in PCD lungs is hardly obtained, and the reduction of bacterial density during chronic colonization or exacerbations is often the aim of the antimicrobial therapy [3]. In previous studies, we have shown that the combination of lin-SB056-1/EDTA possesses antimicrobial activity against P. aeruginosa in artificial sputum medium and prevents P. aeruginosa biofilm formation in an in vivo-like three-dimensional (3D) lung epithelial cell model [16,27]. Despite resembling the airway mucus, artificial sputum media normally behave like Newtonian fluids lacking many of the intramolecular interactions and covalent cross-links that give respiratory secretions their viscoelastic characteristic [28].…”

“…Due to the chelation of divalent cations from their binding sites in lipopolysaccharide (LPS), EDTA may destabilize the bacterial outer membrane, thus increasing the permeability to lin-SB056-1 molecules and their interaction with the bacterial membranes. In addition, at least part of the synergistic effect observed in sputum could be ascribed to the ability of EDTA to reduce sputum viscosity, thus favoring peptide diffusion [16]. Finally, EDTA could also neutralize the inhibitory effect of sputum on the peptide's activity, sequestering cations that may interfere with the electrostatic interactions of the peptide with bacterial surface [9,10].…”

Antibacterial, Antibiofilm, and Antiadhesive Properties of Different Quaternized Chitosan Derivatives

“…Overall, these findings suggest that the high lipophilic outer membrane of Gram-negative bacteria may function as an efficient barrier against hydrophilic macromolecules such as Ch-derivatives. Combining Ch-derivatives with agents (such as EDTA) able to destabilize the outer membrane of Gram-negative bacteria as well as to sequester positively charged medium components could help in increasing their activity against this group of bacteria [35]. However, a contrasting study, aiming at correlating the antibacterial activity of Ch and the surface characteristic of bacterial cell wall, found that the negative charge on the cell surface of Gram-negative bacteria (P. aeruginosa, Salmonella typhimurium and E. coli) was higher than that of Gram-positive one (Staphylococcus aureus and Streptococcus faecalis) resulting in more Ch adsorbed and higher inhibitory effect against the Gram-negative bacteria [36].…”