Metabolic Profiling of Bacterial and Cell Cultures

1 ml samples of culture medium supernatant were collected either after 4 h and 24 h from 16HBE14 tissue cell cultures with or without Hi2019 infection or from supernatants of bacterial cultures grown to stationary phase. All samples were preserved and prepared for ¹H-NMR analysis as in [34 (link)] in 3 mm NMR tubes. Proton NMR spectroscopy was performed on a Bruker AV900 900MHz spectrometer and one–dimensional proton spectra were measured essentially as in [83 (link)] but using 256 scans. 1D spectra were processed using Topspin 3.0 (Bruker Biospin) and Chenomx NMR Suite 8.2 (Chenomx Inc., Edmonton, Canada) as in [34 (link)]. Detection of acetate, succinate and formate by high-performance liquid chromatography (HPLC) was carried out as in [26 (link)] using supernatants from cultures grown on defined medium with either glucose (10 mM) or lactate (4 mM) and inosine (7.5 mM) as the carbon sources. Metabolite detection used an Ultimate 3000 HPLC system (Thermofisher Scientific) equipped with a UV detector and an RI detector (Shodex RI-101). Samples were separated (total runtime: 20 min) on a Metab-AAC column (300 x 7.8 mm; Isera, Dueren) at 30°C, a flow rate of 0.8 ml/min and a running buffer of 5 mM sulfuric acid. Data analysis used Chromeleon (Thermofisher Scientific).