Nunc sealing tape

MIC was determined using the broth microdilution method in 7H9 as described previously with minor modifications (25 (link)). SPR719 and comparator antibiotics were serially diluted 2-fold in 100 μL volume of broth up to 10 concentration points in a 96-well plate format (flat bottom, Corning). Bacteria were grown to mid-log phase (OD₆₀₀ 0.4 to 0.6) and the cultures were diluted to OD₆₀₀ = 0.1. 100 μL of bacterial suspensions were dispensed into the wells, resulting in a final volume of 200 μL and a seeding density of OD₆₀₀ = 0.05. After reading at day 0, OD₆₀₀, plates were sealed with Nunc sealing tape (Thermo Scientific), placed in a humidified plastic box (Sterilite), and incubated for 4 days (M. avium) or 3 days (M. abscessus) with shaking at 90 rpm at 37°C. Growth inhibition was measured by reading optical density (OD₆₀₀) using TECAN infinite M200Pro microplate reader (TECAN). MIC values, defined as the concentration that inhibited 90% of bacterial growth compared with drug-free control unless stated otherwise, were deduced from the resulting dose response curves. Growth curves were determined in broth via OD₆₀₀ measurement of cultures growing in 1-L roller bottles (Corning) at 37°C (starting OD₆₀₀ = 0.05) using Ultrospec 10 cell density meter (Biochrom, Holliston, MA, USA). GraphPad Prism 8 (GraphPad Software, Inc.) was used to determine the dose response and growth curves.

Real time quaking‐induced conversion (RT‐QuIC) assay was performed as described previously (Groveman et al., 2018 (link); Kurzawa‐Akanbi et al., 2021 (link)) with modifications. Black 96‐well plates with a clear bottom (Thermo Fisher Scientific) were preloaded with six silica beads (1 mm, Thistle Scientific) per well. Seeded α‐synuclein aggregation was conducted by loading 15 μl of 0.2 μm filtered PBS (negative control), 15 μl of α‐synuclein fibrils (positive control; 5 mg/ml – 1:20,000 dilution), and EVs samples (1.5E+07 particles for all samples as determined by TRPS) to the wells containing 85 μl of the reaction mix to give final concentration of 40 mM phosphate buffer (pH 8.0), 175 mM sodium chloride, 0.1 mg/ml recombinant full‐length α‐synuclein (Proteos) and 10 μM thioflavin T (ThT). Recombinant human α‐synuclein was filtered through a 100 kDa MWCO filter immediately prior to use (Amicon, MERCK). The plate was sealed with a Nunc sealing tape (Thermo Fisher Scientific) and incubated at 37°C in a BMG FLUOstar Omega plate reader with cycles of 1 min shaking (400 rpm double orbital) and 1 min rest throughout the indicated incubation time. ThT fluorescence measurements (448 nm excitation and 482 nm emission; gain 1000, bottom read) were taken every 30 min.

Ninety-six well plate assays were prepared by adding 100 μL of synthetic medium with 20 g/L glucose, Tween-80 (420 mg/L) and ergosterol (10 mg/L). The initial pH of the medium was adjusted using 2 M HCl and 2 M KOH. (NH₄)₂SO₄ was used as the nitrogen source and the SO₄²⁻ concentration was kept constant at 38 mM by addition of K₂SO₄ to compensate for the decrease in SO₄²⁻ from (NH₄)₂SO₄. Cells were inoculated in each well to a starting OD₆₆₀ of 0.1. Plates were covered with Nunc™ sealing tape (Thermo Scientific) and incubated at 30 °C with constant shaking at 200 rpm. OD660 was measured regularly in a GENios pro plate reader (Tecan Benelux, Giessen, The Netherlands).