The in vitro Antimicrobial Properties of nano-Magnesium oxide (nMgO)

Introduction: Magnesium based nanoparticles are attractive for a number of reasons. We have previously documented the in vitro antimicrobial properties of this inexpensive bioabsorbable metal and unlike many of the other metal oxides; magnesium has very little inherent risk of toxicity. Huan et al. demonstrated that nMgO had an antibacterial effect alone and after incorporation in interior wall paint. In this study they also determined that the antibacterial efficacy increased with decreasing particle size. Lin et al. evaluated the antimicrobial activity of MgO nanoparticles (nMgO) on γ-Al2O3. They found that nMgO particles ranging in size from 4-10.8-nm loaded at 20% resulted in a bactericidal efficiency of approximately 99%. While the γ-Al2O3 appears to have provided an adequate support mechanism, it is unclear if such a material could be used in a implant setting. The goal of this study was to evaluate the in vitro antimicrobial properties of nano-magnesium oxide (nMgO). Our hypotheses were: (1) the addition of nMgO to a bacterial growth media will result in an increase in pH; (2) both nMgO particles and supernatant will result in a decrease in colony forming units (CFU)/ml of bacteria recovered; (3) the nMgO supernatant will result in a decrease in biofilm production. Materials and Methods: To determine the reaction properties of nMgO, nano-zinc oxide (nZnO) and magnesium metal mesh (Mg-mesh) 200-mg of each were placed in 8.0-ml of tryptic soy broth (TSB). The pH of the broth was serially measured over a 72-hour period. For the bacterial experiments 25-mg/ml of nMgO, nZnO and Mg-mesh were used as the source of nanoparticle. Enrofloxacin was used as a control for a bactericidal agent. All bacteria were cultured with no additive to serve as a negative control. The bacterial inoculum consisted of one of the following organisms: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Pseudomonas aeruginosa (MORF – isolated from an infected tibial implant), Staphylococcus aureus (ATCC 25923), Staphylococcus aureus (MORF 13G – isolated from an infected total hip arthroplasty), Staphylococcus epidermidis (RP62A). Pure cultures of all organisms were aerobically cultured for 24-hours at 37C on bovine blood agar plates. A supernatant was generated for each material by adding the material to TSB such that there was 25-mg/ml of nMgO, nZnO or Mg-mesh. This was incubated for 24-hours in a 37C waterbath with agitation at 200 rpm. After the incubation each suspension was filtered using a 0.22-μm syringe filter to generate a supernatant. Culture vials were prepared such that each contained 2-ml of sterile TSB broth and nMgO, nZnO, Mgmesh or antibiotic or a supernatant. Each was inoculated with ~10 of each organism. To assess biofilm prevention a 96-well plate method was used. 5% sucrose was added to the supernatant, antibiotic suspension and TSB. 180μl of the solution was added to each well and they were inoculated with ~10 CFU and incubated in a dry incubator at 37C for 24 and 48hours. Microtiter dilutions were performed to determine the CFU/ml in each sample. At 24 hours, the 96 well plates were stained for biofilm production. The OD570 was then and biofilm formation was categorized as highly positive (OD570 > 1), low-grade positive (0.1 < OD570 < 1) or negative (OD570 <0.1). Dilutions with up to 30 colonies present were used to calculate the median CFU/ml. Summary statistics were calculated and are presented as the median CFU/ml. The error bars in the figures represent the 25 and 75 percentile. Results: Hypothesis 1: The addition of both nMgO and Mg-mesh resulted in a rapid increase in pH. After 1-hour it was 10.33 and 10.46 for the nMgO and Mg-mesh respectively. In the nZnO the pH reached a maximum of 8.36 after 72-hours. Hypothesis 2: The experimental conditions resulted in an expected growth pattern of all six bacterial organisms as indicated by the CFU/ml recovered from the control vials. There were no CFU recovered from either the nMgO or Mg-mesh vials after 24-hours of incubation. Pseudomonas was recovered from the enrofloxacin vials, although the amount recovered was 10 times less than the CFU/ml in the control vials. CFU were recovered from the nZnO vials for each bacterium, with the exception of Staphylococcus epidermidis where no CFU were recovered.(Figure 1) Similar results were noted for the incubation with the supernatants. Hypothesis 3: The Staphylococcus epidermidis (RP62A) was the only bacterial inoculum that produced a highly positive biofilm in the control wells. Staphylococcus aureus ATCC produced a low-grade positive at 24-hours.(Table 1) Evaluating these bacteria only, the nMgO, nZnO, Mg-mesh and Enrofloxacin resulted in a negative biofilm at 24-hours. At 48-hours, only the nMgO, nZnO and Enrofloxacin resulted in negative biofilm results.