Activity of AMP2041 against human and animal multidrug resistant Pseudomonas aeruginosa clinical isolates.
BACKGROUND
Antimicrobial resistance is a growing threat to public health. Pseudomonas aeruginosa is a relevant pathogen causing human and animal infections, frequently displaying high levels of resistance to commonly used antimicrobials. The increasing difficulty to develop new effective antibiotics have discouraged investment in this area and only a few new antibiotics are currently under development. An approach to overcome antibiotic resistance could be based on antimicrobial peptides since they offer advantages over currently used microbicides.


METHODS
The antimicrobial activity of the synthetic peptide AMP2041 was evaluated against 49 P. aeruginosa clinical strains with high levels of antimicrobial resistance, isolated from humans (n = 19) and animals (n = 30). In vitro activity was evaluated by a microdilution assay for lethal dose 90% (LD90), while the activity over time was performed by time-kill assay with 12.5 µg/ml of AMP2014. Evidences for a direct membrane damage were investigated on P. aeruginosa ATCC 27853 reference strain, on animal isolate PA-VET 38 and on human isolate PA-H 24 by propidium iodide and on P. aeruginosa ATCC 27853 by scanning electron microscopy.


RESULTS
AMP2041 showed a dose-dependent activity, with a mean (SEM) LD90of 1.69 and 3.3 µg/ml for animal and human strains, respectively. AMP2041 showed microbicidal activity on P. aeruginosa isolates from a patient with cystic fibrosis (CF) and resistance increased from first infection isolate (LD90= 0.3 μg/ml) to the mucoid phenotype (LD90= 10.4 μg/ml). The time-kill assay showed a time-dependent bactericidal effect of AMP2041 and LD90was reached within 20 min for all the strains. The stain-dead assay showed an increasing of membrane permeabilization and SEM analysis revealed holes, dents and bursts throughout bacterial cell wall after 30 min of incubation with AMP2041.


CONCLUSIONS
The obtained results assessed for the first time the good antimicrobial activity of AMP2041 on P. aeruginosa strains of human origin, including those deriving from a CF patient. We confirmed the excellent antimicrobial activity of AMP2041 on P. aeruginosa strains derived from dog otitis. We also assessed that AMP2041 antimicrobial activity is linked to changes of the P. aeruginosa cell wall morphology and to the increasing of membrane permeability.
Department of Veterinary Science, University of Parma, Via del Taglio 10, 43126, Parma, Italy.
Clotilde Silvia
CS
Cabassi
Department of Veterinary Science, University of Parma, Via del Taglio 10, 43126, Parma, Italy.
Andrea
A
Sala
Department of Veterinary Science, University of Parma, Via del Taglio 10, 43126, Parma, Italy.
Davide
D
Santospirito
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 7/9, 25124, Brescia, Italy.
Giovanni Loris
GL
Alborali
Arcispedale S. Maria Nuova, Viale Risorgimento 80, 42123, Reggio Emilia, Italy.
Edoardo
E
Carretto
Clinica Veterinaria Malpensa, Via Marconi 27, 21017, Samarate, VA, Italy.
Giovanni
G
Ghibaudo
Department of Veterinary Science, University of Parma, Via del Taglio 10, 43126, Parma, Italy. simone.taddei@unipr.it.
Simone
S
Taddei
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002473:Cell Wall / Q000187:drug effects / Q000648:ultrastructure; D004285:Dogs; D024901:Drug Resistance, Multiple, Bacterial; D006801:Humans; D050296:Microbial Viability / Q000187:drug effects; D008855:Microscopy, Electron, Scanning; D010455:Peptides / Q000494:pharmacology*; D011552:Pseudomonas Infections / Q000382:microbiology* / Q000662:veterinary*; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000302:isolation & purification
D000900:Anti-Bacterial Agents; D010455:Peptides
Antimicrobial peptide; Bacterial membrane damage; Cinical isolates; Multidrug resistance; Pseudomonas aeruginosa