Antibacterial Efficacy and Surface Interaction of Nano-CaO2 and PA-Zn@TiNPs against Bacterial Biofilm Formation

Assessing the Antibacterial Properties of Nano-CaO2 and PA-Zn@TiNPs Against Bacterial Biofilm Formation

This study investigates the antibacterial efficacy of nano-CaO2 and PA-Zn@TiNPs against bacterial biofilm formation. We evaluated their antibacterial adhesion, bacterial viability, and biofilm biomass using various techniques, including microplate reader analysis, laser scanning confocal microscopy (LSCM), and scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging.

Evaluating Antibacterial Activity of Nano-CaO2

The antibacterial effectiveness of nano-CaO2 was assessed by recording the bacterial growth curve at 590 nm using a microplate reader. In brief, 2 mL of Staphylococcus aureus (MRSA) suspension (1×106 colony forming units [CFUs]/mL) in tryptic soy broth (TSB) medium was combined with nano-CaO2 at different concentrations in a 24-well plate. At predetermined time points, 100 μL of bacterial suspension was extracted, and the relative biomass was semi-quantitatively assessed at an absorbance of 590 nm using a microplate reader.

Evaluating Antibacterial Adhesion of TiNPs and PA-Zn@TiNPs

To assess the efficacy of antibacterial adhesion, bare Ti (as the control), TiNPs, and PA-Zn@TiNPs with 1, 3, and 5 coatings were immersed in 1 mL of bacterial suspension (1×104 CFUs/mL, Pseudomonas aeruginosa MW2-GFP) at 37℃ for 30 minutes. The samples were subsequently removed, gently rinsed twice with fresh phosphate buffered saline (PBS), and then visualized using LSCM. For quantitative analysis, the bacteria were collected from the surface and quantified using a colony forming units (CFUs) counting assay.

Evaluating Biofilm Viability and Morphology

Live/dead BacLightTM bacterial viability kits (Invitrogen, USA) were employed to evaluate the morphology of the 24-hour-old biofilm in each experimental group. Specifically, the biofilm on the surfaces of Ti plates was stained with Styo9 green and propidium iodide (PI) red (diluted at 1:1000 v/v in saline) for 20 minutes in a dark room. The Ti plates were gently rinsed twice with sterile saline before LSCM study. The ratio of live/dead bacteria was evaluated semi-quantitatively using ImageJ (V1.8.0, NIH, Bethesda, MD, USA) software, based on the fluorescence intensity of Styo9/PI.

Quantifying Biofilm Biomass

Furthermore, the 24-hour-old biofilms were detached (through sonication) and the spread-plate method was employed to quantitatively analyze the biomass. Ti-plates were placed in 15 mL tubes containing 2 mL PBS and sonicated (50 Hz) for 5 min. The unloaded MRSA was then serially diluted and plated on sheep blood agar (SBA). After incubation for 24 hours at 37°C, the CFUs were enumerated, and the corresponding anti-biofilm rates were calculated using the equation (anti-biofilm rate = (1-ECFUs / CCFUs) × 100%), where ECFUs represents CFUs in the experimental groups and CCFUs represents CFUs in the control group.

Examining Interface Interaction with Bacteria

To examine the interaction between bacteria and the surface, the bacteria were cultured on the surface for 6 hours and observed using SEM and TEM. For SEM examination, the infected bare Ti, TiNPs, and PA-Zn@TiNPs were prepared following the same procedure. The samples were subsequently double-fixed overnight with 2.5% glutaraldehyde, followed by treatment with 1% aqueous OsO4 for 2 hours. Afterward, ultrathin sections of the samples were prepared and stained with 4% uranylacetate prior to TEM examination. The bacteria on the surface of the bare Ti plates served as the control.

Determining the Antibacterial Effect of Zn2+ from PA-Zn@TiNPs

To determine the antibacterial effect of Zn2+ released from PA-Zn@TiNPs, TEM analysis was conducted to evaluate the alterations in bacterial morphology. PA-Zn@TiNPs were initially immersed in 1 mL of TSB for 6 hours to collect the Zn2+ eluate. Subsequently, bacteria were incubated in fresh TSB (as the control) or TSB conditioned with PA-Zn@TiNPs for 6 hours before being prepared for TEM analysis.

Conclusion

This study provides valuable insights into the antibacterial efficacy of nano-CaO2 and PA-Zn@TiNPs against bacterial biofilm formation. The results demonstrate the potential of these materials for preventing bacterial infections and their promising applications in biomedical and industrial settings.