Evaluating Antibiotic Activity of AuNPs

The procedure for determining antibiotic activity followed a previous publication [27 (link)]. Escherichia coli ATCC 25922 (ATCC, Manassas, VA, USA) was sub-cultured in Tryptic soy broth (Thermo Scientific, Waltham, MA, USA) at 37 °C for 12 h. E. coli approximately at 2.4 × 10⁶ CFU, as determined by a spectrophotometer (ELx808 absorbance reader; BioTek, Shoreline, WA) at optical density 600 nm at 0.003 (OD 600 nm at 0.003), were diluted in Tryptic soy broth (Thermo Scientific) and added with AuNP in different concentrations: 12.5, 25 and 50 ppm, respectively. Then the solutions were incubated at 37 °C for 4 h. After that, the supernatant in serial dilution was plated in Tryptic soy agar (Thermo Scientific), kept at 37 °C overnight before bacterial colony enumeration. E. coli in Tryptic soy broth alone or in 100 μg/ml of gentamicin were used as the positive and negative control group, respectively.