Phage/nanoparticle cocktails for a biocompatible and environmentally friendly antibacterial therapy

Abstract

Antibiotic resistance continues to rise, necessitating alternative strategies. Bacteriophages have emerged as promising natural antibacterial agents, offering a targeted approach to combating bacterial infections. Combining bacteriophages with nanoparticles presents a novel approach that could enhance antibacterial potency while reducing the risk of resistance. While phage/antibiotic cocktails are widely explored to enhance antibacterial efficacy and prevent resistance, research on phage/nanoparticle combinations remains limited. We explore the synergy between green tea extract-capped silver nanoparticles (G-TeaNPs) and bacteriophages in combating pathogenic bacteria (methicillin-resistant Staphylococcus aureus, Salmonella enterica). G-TeaNPs show minimal antiphage activity, ensuring compatibility in phage-NP formulations. These combinations significantly reduce bacterial counts in a short time (only 3 h), e.g., S. aureus survival is around 30% after incubation with just 0.001 mg/mL of G-TeaNPs, while G-TeaNPs and phages alone result in around 80% and 70% survival, respectively. Cytotoxicity tests against eukaryotic 3T3 NIH fibroblast cells confirm biocompatibility at effective concentrations. Additionally, we examine G-TeaNPs' impact on the free-living protist Acanthamoeba castellanii. Both green tea extract and G-TeaNPs can reduce A. castellanii cell counts by 80%, but only at high concentrations. Microscopy revealed nanoparticle uptake by amoebae, causing intracellular accumulation and vacuolization, while green tea extract induced similar changes without uptake. Our findings highlight G-TeaNPs as safe, effective agents in phage/nanoparticle antibacterial formulations with dual antimicrobial and amoebicidal properties for therapeutic and environmental applications. KEYPOINTS: • Silver nanoparticles synthesized with tea extracts (G-TeaNPs) have a minimal effect on the tested viruses. • Combining G-TeaNP with bacteriophages offers new-generation antibacterial cocktails. • Green tea extracts and AgNPs present concentration-dependent anti-amoebic activity.

Keywords: Acanthamoeba castellanii; Antimicrobial agents; Antimicrobial combinations; Bacteriophages; Silver nanoparticles.

Fig. 1

a SEM image of G-TeaNPs (silver nanoparticles synthesized using green tea extract), inset: size distribution (diameter), line—kernel density. b Diffractogram of AgNPs capped with green tea extract. c FTIR spectra of G-TeaNPs and d green tea extract

Fig. 2

Antibacterial activity of phage/nanoparticle (phage/NP) cocktails and phage/antibiotic combinations against S. aureus (gram-positive) and S. enterica (gram-negative). a Bacterial survival after treatment with G-TeaNPs (0.1 mg/mL to 0.0001 mg/mL) combined with P22 (S. enterica) or vB_SauS_CS1 (S. aureus) bacteriophages (ROI = 1). b Effect of phage/NP cocktails ([G-TeaNPs] = 0.001 mg/mL) at different rates of infection (ROI = 1 to 100). c Comparison of bacterial survival following treatment with ampicillin (AMP) alone or in combination with bacteriophages at varying ROIs

Fig. 3

a Control cells of A. castellanii, non-exposed to G-TeaNPs. b A. castellanii exposed to G-TeaNPs, showing nanoparticle uptake and accumulation within intracellular vacuoles, compared to control cells. c Cells exposed to G-TeaNPs exhibit increased vacuolization and morphological changes: (1) trophozoites, (2) trophozoite vacuoles filled with AgNPs, and (3) cysts containing AgNPs. Similar vacuolization and size/shape reduction were observed in cells treated with green tea extract but without nanoparticle accumulation. These findings highlight the cellular uptake of nanoparticles and their potential influence on A. castellanii morphology. d Cytotoxic effects of citrate-capped C-AgNPs, G-TeaNPs, and green tea extract (GT) on A. castellanii over 7 days. G-TeaNPs inhibited amoebae growth only at high concentrations, while lower concentrations (i.e., 0.1 mg/mL as used in the other assays) were ineffective. GT alone was as effective as G-TeaNPs in reducing amoebae counts

Fig. 4

Optical microscopic images of 3 T3 NIH fibroblast cells after 24 h of incubation with different nanoparticle treatments: a untreated control cells. b Cells treated with 1% Triton X as a negative control. c Cells treated with only AlamarBlue (positive control). d Cells treated with control silver nanoparticles (C-AgNPs, 0.1 mg/mL), d) Cells treated with G-TeaNPs (0.1 mg/mL). e Viability of cells treated with AgNPs and G-TeaNPs after 24 h of incubation at 1 mg/mL and 0.1 mg/mL. f The survival of the fibroblasts after exposure to control AgNPs and G-TeaNPs at the concentrations of 1 mg/mL and 0.1 mg/mL. p < 0.001 by two-way ANOVA for both concentration and nanoparticle type, with significant interaction between factors