Neoteric Silver-Ceria as a Rapid, Residual Antibacterial Agent for Abiotic Surfaces

<b>Introduction:</b> High rates of mortality and morbidity due to hospital acquired infectious (HAI) disease remains a serious problem in the healthcare sector. The application of nanotechnology as a novel antimicrobial agent has proven effective and is of high interest. Among them, cerium- and cerium oxide-based (ceria) nanoparticles (CNPs) have received much attention owing to their unique surface kinetics <i>via</i> auto-catalytic cycling between two oxidative states (Ce⁴⁺→Ce³⁺→Ce⁴⁺). CNPs have been shown to be less toxic to mammalian cell lines but effective in eradicating bacteria through the formation of reactive oxygen species (ROS). Nano-based silver is also known for its efficient antimicrobial action. The aim of this study was to investigate the antibacterial activity of silver-doped ceria nanoparticles (AgCNPs) on three bacterial genera both in a liquid and dry form and its potential role as a novel, self-disinfecting and residual coating for high-touch abiotic surfaces. We hypothesize that through heightened generation of ROS, AgCNPs interact with the bacterial membrane leading to oxidative stress and bacterial death.<br/><b>Materials and Method: </b>AgCNP was synthesized using a base-mediated forced hydrolysis method and material characterization performed. The minimum inhibitory concentration (MIC) of AgCNPs and CNPs was determined for <i>Pseudomonas aeruginosa</i> (PA), <i>Staphylococcus aureus</i> (SA), and methicillin resistant <i>Staphylococcus aureus</i> (MRSA). Experiments were conducted with AgCNP and CNP at concentrations of 100 µg/mL (PA), 300 µg/mL (SA and MRSA), AgNO₃ (20 µg/mL) and gentamicin (20 µg/mL). Cell viability, a time-scale assay using TEM, intracellular ROS, DNA, and membrane damage were analysed. The ability of each agent to generate superoxide (O₂^●-) and hydroxyl (OH^●) radicals, RNS, and H₂O₂ was determined. To test each agent’s residual antibacterial activity, solid surface (individual and multiple species) re-challenge tests were carried out with four cumulative doses (~10⁸ cells/mL) of bacteria. Biofilm from each bacterial species was allowed to form followed by treatment with each agent for three days at intervals of 24 h. Biofilm thickness and bacterial viability was assessed. All experiments were carried out in triplicate and a Mann-Whitney U test was used for statistical analysis where <i>p</i> values &lt; 0.05 were considered significant.<br/><b>Results:</b> AgCNP consisted of particles composed of 53.7% Ce³⁺:Ce⁴⁺(%Ce³⁺) and 14.6% [Ag]/[Ag+Ce]. Treatment with AgCNP resulted in significant PA cell death (100%) after 60 mins (<i>p</i> &lt; 0.0001) and SA and MRSA death after 180 mins (both <i>p</i> &lt; 0.0001). AgCNP significantly induced ROS generation when given to all three bacteria (PA (~20-fold), SA (~7.4fold) and MRSA (~21fold)) when compared to control (all <i>p</i> &lt; 0.0001). TEM analysis showed AgCNPs localized onto the bacterial membrane resulting in a significant and fatal increase in cell size (PA (<i>p</i>&lt;0.01), SA and MRSA (<i>p</i>&lt;0.05)). Results also revealed a significant increase in DNA damage following AgCNP treatment in all groups when compared to control (all <i>p</i> &lt; 0.0001). Further, AgCNP induced a significantly increased amount of O₂^●-, OH^●, H₂O₂ and RNS compared to all other agents. An AgCNP-coated glass slide surface re-challenge test showed 100% inhibition of PA, SA and MRSA both individually and as a multispecies inoculation. Finally, AgCNPs were able to disrupt pre-formed biofilm in all three species and significantly eradicated shielded bacteria within (all <i>p</i> &lt; 0.0001). AgCNPs outperformed CNPs, AgNO₃ and gentamicin.<br/><b>Discussion and Significance: </b>This study showed that AgCNPs, whether in a liquid or dry form, were able to eradicate <i>S. aureus</i>, MRSA<i>,</i> and <i>P. aeruginosa</i>. AgCNPs displayed synergistic and augmented antibacterial activity when compared to Ag or CNPs alone. We have demonstrated that AgCNP coatings may offer a new type of continuous disinfectant that leaves behind a continually disinfecting film with excellent antibacterial efficacy.