The Antimicrobial Activity of the AGXX® Surface Coating Requires a Small Particle Size to Efficiently Kill Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) isolates are often resistant to multiple antibiotics and pose a major health burden due to limited treatment options. The novel AGXX® surface coating exerts strong antimicrobial activity and successfully kills multi-resistant pathogens, including MRSA. The mode of action of AGXX® particles involves the generation of reactive oxygen species (ROS), which induce an oxidative and metal stress response, increased protein thiol-oxidations, protein aggregations, and an oxidized bacillithiol (BSH) redox state in S. aureus. In this work, we report that the AGXX® particle size determines the effective dose and time-course of S. aureus USA300JE2 killing. We found that the two charges AGXX®373 and AGXX®383 differ strongly in their effective concentrations and times required for microbial killing. While 20-40 μg/ml AGXX®373 of the smaller particle size of 1.5-2.5 μm resulted in >99.9% killing after 2 h, much higher amounts of 60-80 μg/ml AGXX®383 of the larger particle size of >3.2 μm led to a >99% killing of S. aureus USA300JE2 within 3 h. Smaller AGXX® particles have a higher surface/volume ratio and therefore higher antimicrobial activity to kill at lower concentrations in a shorter time period compared to the larger particles. Thus, in future preparations of AGXX® particles, the size of the particles should be kept at a minimum for maximal antimicrobial activity.

Keywords: AGXX®; Staphylococcus aureus; antimicrobial activity; contact killing; metal particles.

Figure 1

The proposed mode of action of AGXX® and the responses in Staphylococcus aureus. AGXX® is composed of silver (Ag⁺) and ruthenium (Ru^x), which are connected by two redox cycles and form an electric field, leading to electron transfer to molecular oxygen (O₂), with subsequent reactive oxygen species (ROS) generation, such as superoxide anion O₂•⁻, hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH•). First, Ag⁰ is oxidized to Ag⁺, which is back-reduced to Ag⁰ possibly by electrons from cellular donors, such as the thioredoxin (Trx)/thioredoxin reductase (TrxR) system. Second, Ru^x+1 is reduced by electrons from Ag⁰, leading to formation of Ru^x, which is re-oxidized to Ru^x+1. ROS are generated in the AGXX® redox cycles upon oxidation of Ag⁰ and Ru^x. Furthermore, Ag⁺ ions and O₂•⁻ have been described to damage FeS clusters and release Fe²⁺, which potentiates OH• generation via the Fenton chemistry. In S. aureus, AGXX® induces antioxidant enzymes, such as catalases, peroxiredoxins, and superoxide dismutases for detoxification of ROS (Loi et al., 2018). AGXX® leads to thiol-oxidation of the HypR repressor, resulting in upregulation of the HypR-controlled flavin disulfide reductase MerA, which probably detoxifies OH•. In addition, AGXX® exposure resulted in increased protein S-bacillithiolation of GapDH and an oxidative shift of the BSH redox potential, supporting an impaired thiol-redox homeostasis. GapDH-SSB can be de-bacillithiolated in the Brx/BSH/YpdA redox pathway to regenerate GapDH. Consequently, BSH and MerA were shown to protect S. aureus against AGXX® toxicity (Loi et al., 2018). In addition, AGXX® causes oxidative protein unfolding and protein aggregates, which are degraded by the Clp protease complex. The figure is modified from (Guridi et al., 2015) including previous results from (Loi et al., 2018).

Figure 2

The charges AGXX®373 (A) and AGXX®383 (B) cause different antimicrobial effects in S. aureus USA300JE2. Survival assays of S. aureus USA300JE2 cells were performed at an OD₅₀₀ of 0.5 after exposure to 10–40 μg/ml AGXX®373 (A) or 40–100 μg/ml AGXX® 383 (B). Serial dilutions of the cultures were spotted after 1–6 h of AGXX® exposure onto Luria Bertani (LB) agar plates to observe colony forming units (CFUs) after 24 h incubation. The presented results are representatives of three biological replicates. The results indicate that AGXX®373 particles have a stronger killing effect compared to AGXX®383 particles in S. aureus.

Figure 3

Survival assays indicate 2-3-fold increased killing efficiencies of smaller AGXX®373 versus larger AGXX®383 particles in S. aureus. S. aureus USA300JE2 was exposed to 10–40 μg/ml AGXX®373 particles (A,B) and 50–100 μg/ml AGXX®383 particles (C,D) at an OD₅₀₀ of 0.5. After 1–24 h of AGXX® treatment, CFUs were determined from serial dilutions plated overnight on LB agar plates. The survival ratios of the CFUs after AGXX® stress were calculated relative to the untreated control (co), which was set to 100%. Mean values of 3–5 biological replicates are presented, and error bars represent the SD. For statistical analyses of the killing effect, the 100% survival of the untreated control (co) was compared to 10, 20, 30, and 40 μg/ml AGXX®373 (A) or 50, 60, 80, and 100 μg/ml AGXX®383 (C), respectively, and the calculations were performed using a Student’s unpaired two-tailed t-test by the graph prism software. Symbols are ns > 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001.