In vitro Antibacterial and Antibiofilm Activity of Biogenic Silver Nanoparticles Synthesized using Cell-free Supernatant from Probiotic Enterococcus durans

Abstract

Antimicrobial-resistant bacterial infections remain a significant public health concern, creating a need for new strategies to address this global problem. This study aimed to evaluate the antimicrobial activity of biogenic silver nanoparticles (Bio- AgNPs) synthesized using cell-free supernatant from the previously characterized probiotic Enterococcus durans strain against resistant bacterial strains. The antibacterial activity of Bio-AgNPs against vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) was evaluated based on bacterial growth (minimum inhibitory concentration [MIC] using resazurin staining), viability (minimum bactericidal concentration [MBC] using CFU/mL), and time-kill assays (CFU/mL). Antibiofilm activity was evaluated using crystal violet staining to assess biofilm biomass and the XTT assay to assess metabolic activity. The viability of sessile biofilmforming cells was determined by CFU/mL. Bacterial membrane permeability was assessed using propidium iodide (PI) staining. Bio-AgNPs exhibited inhibitory activity, with MIC values of 1.5 μg/mL for VRE E. faecium and 3.1 μg/mL for MRSA S. aureus. The MBC of Bio-AgNPs was twice the MIC for both strains. Bio-AgNPs demonstrated bactericidal activity, with a significant reduction in the number of surviving cells at concentrations equal to or greater than 0.15 μg/mL for E. faecium and 0.7 μg/mL for S. aureus. The time-kill assay showed a marked reduction in viable cells following exposure to Bio-AgNPs at 1× MIC after 12 h. Reductions of up to 53% in biofilm biomass and 92% in metabolic activity were observed. Bio-AgNPs also significantly reduced the number of viable sessile cells within biofilms. Bio-AgNPs synthesized using cell-free supernatant from E. durans exhibited bactericidal activity against the MRSA and VRE strains evaluated, with low MIC and MBC values and a concentration-dependent effect on bacterial growth and survival. Fluorescence microscopy with PI suggested compromised bacterial membrane integrity following Bio- AgNP treatment, indicating that membrane disruption may contribute to their antibacterial activity. Bio-AgNPs also reduced biofilm biomass, metabolic activity, and the number of viable sessile cells. These findings support further investigation of Bio-AgNPs as biogenic antimicrobial candidates. These findings demonstrate that Bio-AgNPs synthesized using cell-free supernatant from E. durans exhibited bactericidal and antibiofilm activity against the resistant strains evaluated in vitro, supporting their potential as biogenic antimicrobial candidates for further investigation.