Untargeted metabolomic profiling of the bacterial samples was done using ultra high-performance liquid chromatography (Ultimate 3000 UPLC, Thermo Fisher Scientific, Waltham, MA, USA) coupled with an Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific Waltham, MA, USA). In order to enhance the chemical coverage of the analysis, we used two different but complementary chromatographic columns, consisting in reversed phase chromatography (C18 chromatographic column) and Hydrophilic Interaction Liquid Chromatography (HILIC) for the analysis of hydrophobic and polar metabolites, respectively.

The C18 chromatographic separation was carried out on a Hypersil GOLD C18 column (1.9 µm, 150 × 2.1 mm, Thermo Fisher Scientific) at 30 °C, with flow elution rate of 500 μL/min. The mobile phases consisted of A (100% water + 0.1% formic acid) and B (100% acetonitrile (ACN) + 0.1% formic acid). Elution started with an isocratic step of 2 min at 5% mobile phase B, followed by a linear gradient from 5% to 100% mobile phase B for the next 11 min. These proportions were kept constant for the next 12.5 min before returning to 5% B for 4.5 min. The HILIC chromatographic separation was carried out on a Sequant ZIC-pHILIC column (5 µm, 150 × 2.1 mm, Merck, Darmstadt, Germany) maintained at 15 °C under a elution gradient of mobile phases A and B at a flow elution rate of 200 μL/min. Mobile phase A was 10 mM ammonium carbonate pH 10.5 (adjusted with ammonium hydroxide), and mobile phase B was 100% ACN. Elution was initiated with 80% B phase for 2 min, followed by a linear gradient of 80–40% B from 2 to 12 min. The chromatographic system was then rinsed for 5 min at 0% B, before returning at 80% B and the and the run ended with an equilibration step of 25 min at 80% B.

The mass spectrometer was fitted with an electrospray source (ESI) operating in positive and negative ionization modes for C18 and ZIC-pHILIC, respectively. It was operated with capillary voltage at −3kV in the negative ionization mode and 5 kV in the positive ionization and a capillary temperature set at 280 °C. Temperature of the autosampler compartment was set at 4 °C, and the injection volume was 10 μL. Detection was carried out from m/z 75 to 1000 in both ionization modes at a resolution of 50,000 at m/z 200 as reported by Aros-Calt et al. [61] (each scan taking 0.5 s).

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