Bactiter glo assay

E. coli strain MG1655 was grown at 37 °C with shaking (200 rpm) in lysogeny broth (LB). Overnight (~16 h) cultures of E. coli were centrifuged at 10,000 rpm for 5 min and then resuspended in sterile water. Cultures were then added to 1000 ml of water for probe testing. Dilution of the water and E. coli mix provided a range of concentrations for testing with the sensor. All dilutions were then quantified by BacTiter-Glo™ assay (Promega), standard plate count (on LB agar), or both these techniques.

Mtb growing in Sauton’s media was treated with the indicated concentration of C10 for 24 h before the OD₆₀₀ of each culture was measured and samples were removed. Mtb samples were inactivated at >95°C for 20 min and stored at −20°C until analyzing the ATP levels using the BacTiter Glo assay (Promega) as previously described (19 (link)). Samples were diluted 1:10, then mixed 1:1 with the BacTiter Glo reagent in a white, opaque 96-well dish, and the luminescence was read on a Synergy HT plate reader with a 1-s integration. The relative luminescence units (RLUs) were calculated by subtracting the luminescence of a media-only control from the luminescence value of each sample. The RLU/OD₆₀₀ was determined to account for differences in bacterial density, and the fold change in each sample was calculated relative to the average of the 0 µM C10 control from that experiment to facilitate the combining of multiple experiments onto a single graph.

The potential impact of the selected extract on bacterial growth was investigated by using freshly collected fecal pellets of wild type mice (C57B6/J OlaHsd, Envigo, 50% male). Samples were diluted with isotonic sodium chloride (0.9%, 100 μL/mg) and homogenized. Fecal suspensions were incubated with either the extract (5 ng/μL), patulin (22 µM) or DMSO as solvent control for 10 min at room temperature with mixing every 2 min. After additional appropriate dilution of the samples, they were spread on 3M^TM-Petrifilm^TM plates specific for Enterobacteriaceae or Lactobacteriaceae (3M Deutschland GmbH, Neuss, DE), 1 mL each. Finally, the plates were incubated for 20 h at 37 °C, colony forming units counted, and normalized to values obtained for DMSO-control. Finally, 1:100 dilutions of the homogenate were additionally used in technical duplicates for viability measurement using the BacTiter Glo assay (Promega, Mannheim, Germany).

M. tuberculosis or M. smegmatis was inoculated into 7H9 medium with or without the test compounds at an ODλ₆₀₀ of 0.1 and incubated with shaking at 37°C for 24 h (M. tuberculosis) or 12 h (M. smegmatis). An aliquot of the culture was heat inactivated at 95°C for 20 min and diluted 1:100. Diluted samples were mixed with the BacTiter-Glo assay (Promega) reagent at a 1:1 ratio, and luminescence was quantified on a Tecan M200 Pro plate reader (integration = 1 s). Relative luminescence units (RLU) were normalized to the number of log₁₀ CFU in the sample to account for differences in bacterial number.

Serial dilutions of working drug stocks (10× to 20×) were prepared in 7H9 medium and then added to a 96-well assay plate (Nunclon Delta; Thermo Scientific) containing 7H9 medium to achieve the desired final concentration. M. tuberculosis culture was diluted to 0.05 in 7H9-GAT and then added to assay plates at a 1:10 dilution (final OD, approximately 0.005). For M. tuberculosis–lux, luminescence reads were recorded daily, as described below. For BCG-mCherry, fluorescence was recorded daily using an Envision multimode plate reader (Perkin-Elmer), with excitation and emission filters of 531 nm and 615 nm, respectively. For wild-type H37RvC and H37Rv-pncAdel, cells were incubated for 4–5 days, and 20 μL of each sample was assayed using the BacTiter-Glo assay (Promega), according to the manufacturer’s instructions. Where indicated, wild-type H37RvC and H37Rv-pncAdel assays were cultured in 7H9-GAT medium adjusted with hydrochloric acid to a pH of 6.0, using an Accumet AB electrode pH meter (Fisher Scientific).

Overnight cultures were diluted 1/40 into 40 ml of M9 medium with carbon source and that lacked casamino acids. Bacteria were shaken at 250 RPM and at 37°C for 2 hours in a 50-ml conical tube. Cultures were then concentrated 2× in fresh diluted (3:1) M9 medium that contained the carbon source and casamino acids. One hundred microliters of these cultures was added to the wells of an opaque walled 96-well plate, overlaid with two Breath-Easy filters (Sigma-Aldrich, St. Louis, MO, to prevent evaporation), and was shaken at 250 RPM/37°C for 1 hour whereupon they reached OD₆₀₀ = ~0.075. [ATP] was measured using a BacTiter-Glo assay (Promega, Madison, WI) according to the manufacturer’s recommendations. ATP (quantified using luminescence) and OD₆₀₀ were measured in a microplate reader. Using a lower initial density of bacteria (1/400) to measure [ATP] does not affect the concentration of ATP measured (fig. S2—note that these bacteria were concentrated 10× before the measurement of ATP to account for differences in density). A standard curve prepared using pure ATP (ATP disodium salt hydrate, Sigma-Aldrich) was used to quantify the ATP concentration. Additional details on measuring ATP during heat shock, in the presence of protease inhibitor and in stationary phase, can be found in SM Methods.