Bianca Natasha Oliveira de Moraes¹, Susana de Souza Barreto¹, Gabriel de Barros Correa¹, Claudia Trigo Pedroso Moraes¹, Walidr Pereira Elias¹, Angela Silva Barbosa¹, Marcia Regina Franzolin¹, Lilia Coronato Courrol²

Introduction: The indiscriminate use of antibiotics has led to an increase in multidrug-resistant strains, which can cause severe infections and even lead to death. As a result, new studies on antimicrobial therapies have been emerging over the years. Metallic nanoparticles have proven to be a promising alternative treatment due to their antimicrobial capacity. Their mechanisms of action involve the release of metal ions, reactive oxygen species (ROS), cell lysis, and immunostimulatory effects. Upon contact with human cells, these nanoparticles can interact with cellular membranes and immune system components, being capable of modulating inflammatory responses. Objective: This study aimed to evaluate the antimicrobial activity of silver, copper, and silver-copper nanoparticles against Gram-positive and Gram-negative bacteria, as well as yeast. Additionally, we assessed their effect on molecules of the complement system. Materials and Methods: Silver and copper nanoparticles were synthesized using banana peel (BAN Ag, Cu, and AgCu) and pineapple peel (PIN Ag, Cu, and AgCu) extracts. They were tested against: Staphylococcus aureus ATCC 25923, Methicillin Resistant S. aureus, Escherichia coli ATCC 25922, E. coli EAEC 042, Klebsiella pneumoniae, Pseudomonas aeruginosa ATCC 27853, Stenotrophomonas maltophilia, and the yeast Candida albicans ATCC 10231. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using broth microdilution and incubation at 37°C for 20 hours, with turbidity measured at 595 nm. The inhibitory activity on biofilm formation in microplates was assessed using nanoparticles diluted 1:80 and 1:160 against EAEC 042, MRSA, and P. aeruginosa ATCC 27853. Cultures were incubated at 37°C for 24 hours and stained with 0.5% crystal violet, with quantification at 595 nm. A proteolytic assay employing immunoblotting was also performed to evaluate the interaction of silver-copper nanoparticles with the EAEC 042 derived Pet (Plasmid-encoded toxin) and the complement system molecule C9, as Pet is known to degrade this molecule. Results and Discussion: AgCuNPs diluted 1:20 showed excellent antimicrobial activity (94–100%) against Gram-positive and Gram-negative bacteria, and yeast. The MIC for PIN AgCuNPs against Gram-positive strains ranged from 1/5 to 1/20, with greater activity against Gram-negative strains (1/10 to 1/160). For BAN AgCuNPs, MIC values ranged from 1/10 to 1/20 for Gram-positive isolates and 1/10 to 1/80 for Gram-negative ones. For C. albicans, both PIN and BAN AgCuNPs showed antimicrobial effects at dilutions between 1/10 and 1/20. MBC values ranged from <1/5 to 1/80. The inhibition of biofilm formation was dose-dependent for BAN NPs, ranging from 28.25 to 50.9% for MRSA; 44.1 to 85.0% for EAEC 042; and 17.7 to 63.3% for P. aeruginosa. However, BAN NPs alone showed lower efficacy: 12.52 to 39.5% inhibition for MRSA; 0 to 1.77% for EAEC 042; and 26.62 to 59.2% for P. aeruginosa. The assay with Pet protein showed partial inhibition of its catalytic activity on C9. Conclusion: The nanoparticles did not degrade the C9 molecule in the absence of Pet and also stimulated the production of C9 polymers. Silver and copper nanoparticles biosynthesized from banana and pineapple peel extracts show promise as potential antimicrobial agents.

Agradecimentos: CAPES; FAPESP; Fundação Butantan