3.11. Antimicrobial Activity Assays

Inhibitory concentration assays were conducted using a standard broth microdilution method, according to Clinical and Laboratory Standards Institute (CLSI) protocol [47,51]. Briefly, two-fold serial dilutions were prepared with sterile deionized water to achieve concentrations ranging from 5 to 1250 μg/mL in a volume of 20 μL, after which 80 μL of BHI broth and 100 μL of bacterial culture containing 2 × 10⁴ CFU/mL were added. Thus, each well-contained peptide or MA-peptide monomer concentrations ranging from 0.5 to 125 μg/mL and a final bacterial concentration of 1 × 10⁴ CFU/mL. The well that exclusively contained S. mutans served as the negative control for bacterial growth/negative control for antibacterial activity. The positive control for bacterial growth/positive control for antibacterial activity was identical to the negative control, except that the 20 μL H₂O was replaced with 20 μL of 63 μg/mL CHX solution. The blank well contained 20 μL H₂O and 180 μL medium without cells. The 96-well plate was incubated in the presence of 5% CO₂ at 37 °C overnight. The solution of each well was transferred to a new plate. The bacterial cell viability was assessed by fluorescence intensity using an established AlamarBlue assay [52,53]. The minimum inhibitory concentration (MIC) value was defined as the lowest peptide or MA-peptide monomer concentration corresponding to the zero-value measured via AlamarBlue assay. Experiments were repeated three times per specimen treatment and concentration.

In-solution antimicrobial activity of peptide conjugated resin discs was assessed. The round, disc polymer specimens were soaked in water for five days to remove unreacted components [54]. The polymer specimens were subsequently assessed for in-solution antimicrobial activity. Briefly, the soaked disc samples were blotted to remove excess water and one disc was transferred to the microwell in a 96-well plate. Following, 20 μL H₂O, 80 μL of BHI broth, and 100 μL of BHI broth containing 2 × 10⁴ CFU/mL S. mutans cells were added to each well to a final bacterial concentration of 1 × 10⁴ CFU/mL. After overnight incubation in the presence of 5% CO₂ at 37 °C, the solution of each well was transferred to a new plate, and AlamarBlue dye was added, following the manufacturer‘s protocol. Samples were incubated for 2 h before fluorescence intensity was measured at Ex565/Em595. The well that contained S. mutans served as the negative control. Polymer-only demonstrates the antibacterial activity of the system without MA-peptide monomer in the formulation. The blank well contained 20 μL H₂O and 180 μL medium without cells. Experiments were repeated a minimum of three times per specimen treatment.