Introduction: The rise in antibiotic resistance has become a daunting concern in the last decade, including strains like methicillin-resistant Staphylococcus aureus (MRSA), causing significant health problems and extensive medical bills. It is important, then, to explore novel, non-drug antibiotics to reduce these life-threatening infections. Selenium nanoparticles (SeNP) have been shown to exhibit specific toxicity to bacteria while remaining non-toxic to healthy mammalian cells.
Selenium is a trace element micronutrient that is associated with antioxidant and metabolic mechanisms in the body. Selenium nanoparticles (SeNP) have effectively reduced both gram-positive (S. aureus) and gram-negative (E. coli and P. aeruginosa) bacteria, while having a safe toxicity profile to healthy fibroblasts and osteoblasts. While the mechanism of antibacterial action is unknown, we hypothesize here that SeNP exerts its antibacterial action by depleting glutathione (GSH), an essential antioxidant that is needed to neutralize reactive oxygen species (ROS) in bacteria. Results showed that SeNP can increase the lag time and significantly decrease the growth rate of S. aureus, while re-introduction of the antioxidant protein glutathione can reverse these effects.
Materials and Methods: SeNP synthesis was performed by the mixture of GSH, Na2SeO3 with a small amount of bovine serum albumin (BSA) in DiH2O. Particles were precipitated into solution upon the addition of NaOH and rinsed by centrifugation and resuspension in PBS. S. aureus (ATCC 12600) was cultured in tryptic soy broth at 37ºC with 5% CO2. Kinetic growth curves were measured using a SpectraMax® Paradigm® with optical densities (OD) of the cultures measured every 2 minutes. Bacteria were treated with SeNP at a range of concentrations, and to explore the mechanism of action, two additional conditions were considered: 1) “rescuing” the bacteria with small amounts of antioxidants and 2) adding small concentrations of ROS to accelerate the killing process. Changes in growth rate (μmax) and the duration of the lag phase were calculated via the Gompertz model for statistical analysis.
Human dermal fibroblasts (Lonza) were also used to determine mammalian cell response to these same conditions and to determine the “therapeutic window” in which bacteria are killed yet mammalian cells remain healthy. All experiments were performed in triplicate and repeated at least three times.
Results and Discussion: The resulting SeNPs were monodispersed (~110–130 nm) and had a negative zeta potential of -22.6 mV. Over several days in storage, no significant agglomeration or changes in NP size were observed. Bacteria treatment with SeNP only showed an increase in the lag phase with a slight decrease in growth rate with increasing concentrations of SeNP. The addition of GSH, as a rescue agent, removed the change in lag phase but still showed a decrease in growth rate with increased SeNP. The addition of 0.01% H2O2 increased the changes observed with SeNP alone, supporting the hypothesis that selenium kills bacteria by reacting with ROS.
Figure 1. Kinetic growth curves of S. aureus treated with SeNP compared to non-treated (black) bacteria over 24 hours. Lag time increases and exponential growth decreases with increasing concentrations of SeNP. N = 3.
Conclusions: Selenium nanoparticles are easily-prepared, monodispersed particles that can be developed as efficient antimicrobial agents with inherent activity. SeNPs can reduce bacterial growth and adhesion without harming healthy mammalian tissue cells, and can be used for numerous antimicrobial applications in wound healing and medical implant devices.
Acknowledgements: The authors would like to thank Northeastern University for funding.
Northeastern University