Cytation 3

Assays were carried out under CLSI M100-S23 guidelines⁴⁰ . A working culture was created by standardizing liquid culture using a BioTek Cytation3 and inoculating into CAMHB to a concentration of 5.0 × 10⁵ CFU/mL. Working culture was added to extracts and controls in 96-well microtiter plates (Grenier-Bio 655-185) such that each well contained a total volume of 0.2 mL. Vehicle controls and antibiotic controls (ampicillin, kanamycin, and vancomycin for Staphylococcus spp. assays, gentamicin, tetracycline, and meropenem for other species, 0.5 to 64 µg/mL) were included for each strain. Extracts and vehicle were tested at a concentration range of 2.0 to 256 µg/mL, using 2-fold serial dilution. Plates were incubated at 37 °C, with S. aureus, S. epidermidis, and P. aeruginosa for 18 hours and A. baumannii and K. pneumoniae for 22 hours. Optical density (OD₆₀₀) was measured using a BioTek Cytation3 plate reader at initial and final time points, to account for extract colour. The IC₅₀ for growth was defined as the lowest concentration at which an extract displayed ≥50% inhibition and MIC (IC₉₀) at ≥90% inhibition.
Extracts active against multidrug-resistant A. baumannii (OIFC143) and K. pneumoniae (NR-15410) were tested for growth inhibition of S. epidermidis and additional strains of A. baumannii and K. pneumoniae.

RT-QuIC assay was performed using standard protocols from published reports23 (link)30 (link)51 (link) with slight modifications. Unless otherwise specified, the reaction mixtures consisted of final concentrations of 350 mM NaCl, 0.1 mM EDTA, 10 μM ThT, 0.1 mg/ml rPrP and 0.0025% SDS in 1X PBS. First, 5-μL samples from either brain homogenates or slice culture homogenates were diluted in a total reaction mixture of 100 μL. Plates subjected to RT-QuIC assay were sealed with Nalgene Nunc plate sealer. Plates were incubated at 42 °C in either a CLARIOstar (BMG) or Cytation3 (BioTek) plate reader with alternating 1-min shake (double orbital) and rest cycles. All samples were run at least in triplicates, and samples were judged to be positive as reported previously when samples were run in quadruplicates30 (link)50 (link). Whenever triplicates were run, we averaged their fluorescence readings, and additionally we selected 10xSD (standard deviation of negative controls) as the criteria for determining the threshold. Bottom plate recordings of ThT fluorescence (450 ± 15 nm excitation and 480 ± 10 nm emission) were taken every 30 min and data analysis was performed as reported previously16 (link) using MARS version 5.2.R8 or Gen 5 version 2.07.17 software when CLARIOstar (BMG) or Cytation3 (BioTek) were used, respectively, and the data were exported to Microsoft Excel for graphing.

Biofilm inhibition of S. aureus was performed as described previously^{41 (link)}. Briefly, supplemented TSB with 3% NaCl, 0.5% dextrose, and 2% human plasma was used in 96-well microtiter plates (Falcon 35–1172). Working cultures of UAMS-1 (wt) and UAMS-929 (isogenic ΔsarA mutant of UAMS-1) were standardized to a concentration of 5 × 10⁵ CFU/mL and the final well volume was 0.2 mL. Extracts were assessed at sub-IC₅₀ concentrations for growth, ranging from 2.0 to 256 µg/mL. The vehicle and positive control, 220D-F2, were assessed from 2.0 to 256 µg/mL. All experiments were incubated statically at 37 °C for 22 hours. Optical density (OD₆₀₀) was measured using a BioTek Cytation3 plate reader at initial and final time points, to account for extract colour. Biofilms were rinsed twice with 1X PBS, fixed with 100% EtOH, and stained with crystal violet. The dry stain was eluted with ethanol, diluted in PBS, and quantified at 595 nm using a BioTek Cytation 3 plate reader. The MBIC₅₀ (minimum biofilm inhibitory concentration) was defined as the lowest concentration at which an extract displayed ≥50% inhibition and MBIC₉₀ at ≥90% inhibition.