Comparison of the bactericidal efficacy of silver nanoparticles with that of conventional chemical disinfectants -

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Comparison of the bactericidal efficacy of silver nanoparticles with that of conventional chemical disinfectants

Introduction

The creation of metal nanoparticles presents quantum-level benefits, including surface-enhanced Raman spectroscopy and metal-enhanced fluorescence. In the realm of biological applications, silver (Ag) and gold (Au) have garnered considerable interest owing to their distinctive electrical and optical characteristics. Notably, the noble metal Ag is gaining heightened focus for its extensively documented biomedical applications. Nevertheless, alternative chemical disinfection approaches have persisted as the primary choice, primarily because of challenges associated with generating stable and efficient nanoparticles in the domain of medical hygiene. Concerning the commonalities and distinctions in the bactericidal mechanisms of silver and various chemical disinfectants, it has been suggested that the inhibition of microorganisms occurs through the interaction with cysteine residues located in crucial protein regions, leading to their deactivation. The current study seeks to establish standardized operating procedures as a reference for evaluating model microbes against silver nanoparticles produced through environmentally friendly colloidal chemistry.

Methodology

Several formulations were experimented with to optimize the ultimate composition of Ag-NPs for enhanced bactericidal efficiency. Silver nitrate (AgNO3, 0.0071 moles) was dissolved in 30.00 mL of Milli-Q water. A 3.00% solution of Arabic gum (AG) was introduced to prevent particle agglomeration. Various reducing agents, including ascorbic acid (C6H8O6), sodium citrate (C6H5ONa3), sodium borohydride (NaBH4), and dimethylamine borane (DMAB, C2H10NB), were employed for the complete chemical reduction of silver cations (Ag+). Subsequently, the mixture was stirred for 30 minutes at 60 °C to obtain a stable colloidal suspension, utilized as a disinfectant. The bactericidal effectiveness of the produced Ag-NPs relies on factors such as size, structure, and synthesis conditions. However, the variation in the minimal bacterial concentration (MBC) measurements did not exceed a factor of four across different Ag reductants.

The bactericidal activity assays were performed using Escherichia coli (E. coli, K12 strain) as the model organism. Before disinfectant treatment, around 1.5 × 10^7 colony-forming units (cfu) of E. coli were cultured in a total volume of 3 ml of disinfectants at different concentrations.  Following the treatment, 50 μl of the sample (approximately 2.5 × 10^5 cfu) was introduced into 3 ml of nutrient broth and then incubated for 16 hours. A parallel procedure was conducted with Staphylococcus aureus (S. aureus) to evaluate the impact of Ag-NPs on Gram-positive bacteria. High-resolution transmission electron microscopy (HR-TEM) and electron energy loss spectroscopy (EELS) were additionally utilized to ascertain the elemental composition distribution, facilitating a deeper comprehension of the mechanism involved in E. coli inactivation.

Results: While Ag-NPs [1:1 citrate] did not exhibit complete bactericidal activity during a 10-minute treatment, they demonstrated a minimal bactericidal concentration (MBC) of 40 ppm after 2 hours. As the treatment duration extended to 6 hours, the Ag-NPs displayed an MBC of 0.6 ppm. Notably, Ag-NPs demonstrated a prolonged and persistent impact on E. coli inactivation with a high efficiency rate of 100%. This suggests that Ag nanoparticles present promising prospects for applications in water treatment or air quality control. TEM morphological analyses reveal several heterogeneous regions characterized by variations in darkness and lightness. These distinctions imply that the dark regions may be linked to the condensation of nucleic acids towards the center of the cell, while the light regions could signify the detachment of the plasma membrane from the cell wall.  The higher the quantity of interacting sites/particles, the stronger the bactericidal impact, thereby indicating increased potency, as assessed by minimal bactericidal concentrations (MBCs).

Conclusion: Silver nanoparticles were produced through a straightforward synthesis method using four different reducing agents, with sodium citrate and DMAB chosen for highlighting their efficacy in E. coli inactivation. Despite variations in antibacterial activity among different nanoparticle formulations, those with a 1:1 silver-to-citrate ratio or a 1:1 or 1:2 silver-to-DMAB ratio proved most efficient, as indicated by reported minimal bactericidal concentrations (MBCs). While the 1:2 ratio (silver:DMAB) demonstrated the highest antimicrobial potency or the lowest minimal bacteria concentration, the 1:1 ratio (silver:citrate) had the advantage of environmental friendliness. The procedures employed resulted in monodisperse and stable colloidal suspensions of nanoparticles.

Comparison with two chemical disinfectants affirmed that Ag-NPs exhibited persistent and effective bactericidal activity even at the lowest concentration tested (0.6 ppm for 6 hours), while hypochlorite disinfectant acted rapidly but required higher concentrations (16 ppm for 0.2 hours). Ag-NPs displayed greater persistence, whereas hypochlorite was active within minutes but lacked long-term efficacy (<8 hours).

Our observations on Ag-NP action supported the hypothesis that efficacy depends on the ratio of E. coli to nanoparticles rather than the concentration of Ag-NPs. Higher ratios resulted in a faster bactericidal effect. Gram-positive bacteria S. aureus exhibited higher resistance to Ag-NPs, with an MBC of 0.6 ppm for E. coli at 6 hours versus 2.5 ppm for S. aureus at 8 hours.

Based on our findings and a literature review, we concur that the mechanism of E. coli inactivation using Ag-NPs may involve multiple processes, including indirect generation of reactive oxygen species (ROS), direct interaction of silver with cell wall proteins and lipids, compromising their function, and potential interaction with DNA once the wall and plasma membrane are compromised, although our results did not detect lingering nanoparticles in the cytoplasm.

Reference:

Chamakura, K., Perez-Ballestero, R., Luo, Z., Bashir, S. and Liu, J., 2011. Comparison of bactericidal activities of silver nanoparticles with common chemical disinfectants. Colloids and Surfaces B: biointerfaces, 84(1), pp.88-96.