Potential novel therapeutic strategies in cystic fibrosis: antimicrobial and anti-biofilm activity of natural and designed α-helical peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia

Abstract

Background: Treatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains.

Results: Three α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin.

Conclusions: The activity shown by α-helical peptides against planktonic and biofilm cells makes them promising "lead compounds" for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease.

Figure 1

Time-killing kinetic of AMPs against CF strains. BMAP-27 (■), BMAP-28 (▴), P19(9/B) (×), and Tobramycin () were tested at MIC value against representative P. aeruginosa (Pa6, Pa15, and Pa22), S. maltophilia (Sm138, Sm143, and Sm192), and S. aureus (Sa4, Sa10, and Sa13) CF strains. Controls (♦) were not exposed to drugs. Values are the mean of two independent experiments performed in triplicate. The dotted line indicates a 3-log reduction in viability.

Figure 2

Effect of AMPs at sub-inhibitory concentrations against biofilm formation by CF strains. BMAP-27 (white bars), BMAP-28 (light gray bars), P19(9/B) (dark gray bars), and Tobramycin (black bars) were tested at 1/2x, 1/4x, and 1/8xMIC against biofilm formation by P. aeruginosa (n = 24, 24, 25, and 17, for BMAP-27, BMAP-28, P19(9/B) and Tobramycin, respectively), S. maltophilia (n = 14, 14, 27, and 5, for BMAP-27, BMAP-28, P19(9/B) and Tobramycin, respectively), and S. aureus (n = 11, 11, 8, and 3, for BMAP-27, BMAP-28, P19(9/B) and Tobramycin, respectively) CF strains. Prevention of biofilm formation was plotted as percentage of strains whose ability in forming biofilm was significantly decreased (of at least 25%) compared to controls (not exposed), as analyzed by a crystal violet staining assay.* p < 0.05; ** p < 0.0001, Fisher’s exact test.

Figure 3

Activity of AMPs at bactericidal concentrations against preformedP. aeruginosabiofilms. BMAP-27, BMAP-28, P19(9/B), and Tobramycin were tested at 1x (white bars), 5x (gray bars), and 10xMIC (black bars) against preformed biofilm by 6 P. aeruginosa CF strains. Results are expressed as percentage of biofilm’ viability compared to control (not exposed, 100% viability). ** p < 0.0001, Fisher’s exact test.