Determination of Minimum Inhibitory Concentrations

The minimum inhibitory concentrations (MICs) values of Ag-NPs alone, neomycin, and its combination with Ag-NPs against two selected P. aeruginosa isolates (P₈ and P₁₄) were calculated using resazurin-based microtiter dilution assay (Wiegand et al., 2008; Abdelaziz et al., 2014).

Firstly, the stock Ag-NPs colloidal suspension was prepared by the chemical reduction of silver nitrate solution in Deionized Distilled Water (DDW) as previously described by Khalil et al. (2017). The concentration of the prepared stock Ag-NPs colloidal suspension was determined by Equation 2 as described by Liu et al. (2007).

Where;

C: molar concentration of nanoparticle solution,

n_t: total number of silver atoms added as AgNO₃= 1 M,

n: number of atoms per nanoparticle (calculated by Equation 3),

v: volume of the reaction solution in liter,

n_a: Avogadro’s number (=6.023 × 10²³).

Where;

N: number of atoms per nanoparticles,

π = 3.14,

ρ: density of face-centered, cubic (fcc) silver (=10.5 g/cm³),

D: average diameter of nanoparticles (measured by TEM),

M: atomic mass of silver (=107.868 g),

n_a: Avogadro’s number (=6.023 × 10²³).

The previously prepared stock colloidal solution of Ag-NPs (1.7 μg/ml) was diluted with sterile dist. H₂O to obtain the working solutions, each having a concentration two times the appropriate final concentrations obtained by diluting the samples with MHB media in the microtiter plates. The 96-well microtiter plates were loaded with various concentrations Ag-NPs (100 μl each) and bacterial inoculum (100 μl, 1 × 10⁵ CFU/ml). Plates were then incubated at 37°C for 18 h (Chan and Mashitah, 2013). After incubation, 20 μl of membrane-filtered resazurin dye (0.1% w/v in dist. H₂O) was applied to each well, and the plates were incubated in an incubator shaker (37°C, 1 h). The transformation of purple resazurin into a fluorescent pink to red showed actively metabolizing cells, while the presence of dark blue suggested full suppression of bacterial growth in microtiter plate wells. The MICs were detected as the lowest concentrations of Ag-NPs in the wells remained blue. All experiments were conducted in triplicates (Lipuma et al., 2009; Singh et al., 2013; Abdelaziz et al., 2014).

Neomycin (aminoglycosides) was chosen for the following in vivo study, as neomycin is commonly used in the topical treatment of many skin infections, including burns. The MIC value of the antibiotic was determined either individually or combined with Ag-NPs against P. aeruginosa isolates (P₈ and P₁₄). Antibiotic dry powder (10.000 μg/ml) was mixed in DDW to prepare a stock of the antibiotics, which was subsequently sterilized using bacterial filters (pore size, 0.22 μm). To prepare twofold serial dilutions of antibiotics, the stock concentrations were either diluted with sterile dist. H₂O for detecting MIC of free drug or diluted with Ag-NPs suspension (at sub-inhibiting concentration) for the combination. Every working solution had a concentration two times the final concentrations required, which was achieved by diluting the working samples with MHB media. The 96-well microtiter plates were loaded with 100 μl of each antibiotic concentration, either alone or in combination with Ag-NPs, and inoculated with 100 μl of bacterial culture (10⁵ CFU/ml). The plates were incubated at 37°C for 18 h. MICs were observed by broth microdilution technique using resazurin dye as stated above.