Antimicrobial susceptibility tests using broth dilution

The minimum inhibitory concentration (MIC) of HEScL and AgNPs-HEScL with bacterial and yeast strains was determined by broth microdilution, observing the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2008, 2012a,d, 2018), with some adaptations (Silva et al., 2018; Boriollo et al., 2020; de Carvalho Bernardo et al., 2021). These tests were performed in triplicate test systems using microdilution plates with multiple wells (96-well cell culture microplates, Corning Inc., NY, United States) containing 100 μl/well of sterile culture medium supplemented with HEScL or AgNPs-HEScL, including the microbial inoculum. The concentrations of HEScL used in the test ranged from 166,000 μg/ml to 324.2 μg/ml of culture medium, whereas those of AgNPs-HEScL varied between 8,000 μg/ml and 15.6 μg/ml of culture medium.

The recently grown microorganisms were adjusted to a transmittance of 79.5%–83.2% using a spectrophotometer (Thermo Scientific Multiskan GO UV/Vis, Microplate and Cuvette Spectrophotometer, Waltham, MA, United States) at wavelengths of 625 nm and 530 nm for bacteria and yeasts, respectively (T = 79.5–83.2% → A_{625 nm/530 nm} = 2 – log₁₀%T → A_{625 nm/530 nm} = 0.100–0.080). The culture media employed were as follows:

Afterwards, the microdilution plates were incubated at 35 or 37°C for 24–48 h under specific environmental conditions for each microorganism. These tests were performed in triplicate test systems and the interpretation of breakpoints was based on the guidelines of the Clinical and Laboratory Standards Institute. In addition, colorimetric assays based on resazurin biotransformation systems by bacteria (Resazurin sodium salt, Sigma-Aldrich Cat. # R7017, Merck KGaA, Darmstadt, Germany; Martin et al., 2003) and yeast TTZ (2, 3,5-Triphenyltetrazolium chloride, Sigma-Aldrich Cat. # T8877, Merck KGaA, Darmstadt, Germany; Silva et al., 2018; de Carvalho Bernardo et al., 2021) were performed in order to determine the minimum inhibitory concentrations of HEScL and AgNPs-HEScL.