Antimicrobial Susceptibility Testing

Minimum inhibitory concentrations (MICs) were determined in round-bottom 96-well microtiter trays (Sarstedt 82.1582.001), using the modified broth microdilution method recommended by the Clinical and Laboratory Standards Institute [CLSI], 2017. Testing concentrations were as follows: NCL195: 256–0.25 μg/mL; EDTA: 3,800–45 μg/mL; PMBN: 32–0.06 μg/mL; PMB: 32–0.06 μg/mL. Luria–Bertani (LB) broth (Oxoid, VIC, Australia) was used instead of cation-adjusted Mueller–Hinton broth as it was shown previously that robenidine can chelate calcium ions resulting in loss of activity. In addition, twofold serial dilutions of NCL195 were performed in 100% DMSO, with 1 μL added to each well, as NCL compounds have very low solubility in aqueous environments (Abraham et al., 2016). The MICs for ampicillin, gentamicin, and apramycin against each isolate were determined for each test to serve as an internal quality control. The MICs of isolates were determined by visual reading and using an EnSpire Multimode Plate Reader 2300 at A_{600 nm}. MIC₅₀, MIC₉₀, and MIC ranges for NCL195, EDTA, PMBN, PMB, or combinations were then determined (Venter, 2019).