Cell culture and antibiotic treatment
The S. aureus strains ATCC 25923 (MSSA) and ATCC 43300 (MRSA) were obtained from Huashan Hospital (Shanghai, China). Minimum inhibitory concentration (MIC) is determined by broth dilution method to establish the level of resistance and to determine an appropriate dosage. Both strains were cultured in Muller Hinton broth (Sigma-Aldrich, St. Louis, MO) with a series of concentrations of oxacillin at 35 ºC in an ambient air incubator for 24 h. MIC was determined as the lowest concentration of oxacillin in the medium without visible bacterial growth.
For antibiotic treatment, ∼1 × 106/mL bacterial cells were seeded in 50 mL of fresh culture medium and 1/8 of the MIC oxacillin were given to both MRSA and MSSA. Accordingly, MRSA and MSSA were treated with oxacillin at the concentrations of 2.0 and 0.0625 μg/mL, respectively. For both strains, control samples were seeded with the same density of bacterial cells and cultured under the same conditions but without any oxacillin treatment. Each group (oxacillin treated and untreated) has four biological replicates. When OD600 value (optical density at 600 nm) reached 0.7, the S. aureus cells were harvested.
The S. aureus cells were harvested by centrifugation at 5000 × g for 10 min at 4 oC. Cell pellets were collected and washed with 0.6% NaCl aqueous solution. 0.1 g wet cell pellets were suspended in 1 mL chilled extraction buffer (methanol/acetonitrile/water, 2:2:1, v/v/v). The cell disruption was done by fast cooling using liquid nitrogen, sonication at 4 ºC for 5 minutes and storage at -20 ºC for 1 h. The extraction mixture was centrifuged, the supernatant was condensed by SpeedVac and stored at -80 ºC before LC-MS/MS analysis.
The ultrahigh performance liquid chromatography system (Waters, USA) coupled to a TripleTOF 4600 mass spectrometer (Sciex, Singapore) was used to analyze the metabolome extract samples. Both HILIC column (ACQUITY UPLC BEH HILIC, 1.7μm, 2.1× 150mm, Waters) and C18 column (ACQUITY UPLC BEH C18, 1.7μm, 2.1× 100mm, Waters) were used and the flow rate was set at 0.3 mL/minute, and the injection volume was 10.0 μL. When it is HILIC column, the mobile phase consisted of two components: (A) water/acetonitrile (95/5, v/v) with 5 mM ammonium acetate, (B) acetonitrile/water (95/5, v/v) with 5 mM ammonium acetate. The gradient began at 0 % solvent A, increased to 2 % in 3 min, 2 % to 7 % in 7 min, 7 % to 15 % in 4 min, 15 % to 20 % in 21 min, 20 % to 33 % in 3 min, 33 % to 63 % in 12 min, retained at 63% B for 1 min, followed by 4 min 100 % solvent B. For C18 column analysis, mobile phase A (0.06% acetic acid in water) and B (pure acetonitrile) were used. The LC gradient began at 5% solvent B and maintained for 3 min, increased to 25% in 8 min, 25% to 50% in 5 min, 50% to 100% for 20 min, retained at 100% B for 3 min and then back to 5%. The parameters of MS in positive ionization mode were applied for both chromatographic modes: IonSpray Voltage to 5000 V; Ion Source Gas flow to 35 L/h; Curtain Gas to 30 L/h, Source Temperature to 450 ºC; Collision Energy to 35 V, Collision Energy Spread to 15 V.
Before the data acquisition, the mass spectrometer would be cleaned and adjusted to its best performance. The mass spectrometer is calibrated using commercial PPG solution. PPG solution was directly infused into the mass spectrometer at every six injection intervals to minimize the mass deviation. MS data were obtained by data-dependent acquisition (DDA) and the same setting for both HILIC and C18 analysis. The mass range for both TOF MS scan and Product Ion scan was set at 68–1300 mass/charge (m/z), the accumulation time was set at 0.1 second for TOF MS scan and 0.05 second for Product Ion scan. For the Switch Criteria, exclude isotopes with no exclusion were chosen for both scan types. The dynamic background subtract was selected for the whole data acquisition.
All the extracted metabolic samples were dissolved in 50 µL acetonitrile aqueous solution (1:1, v/v). Pooled samples for quality control purpose were prepared by mixing 10µL of each redissolved metabolic sample. QC samples were injected to monitor and overcome analytical drifts at regular intervals in UPLC–MS/MS data acquiring. Features with coefficient of variation (CV) < 30% were considered reproducible, as suggested elsewhere .
All the UPLC-MS/MS files were firstly converted to mzXML format via ProteoWizard software , and subsequently the generated mzXML data files were submitted to the XCMS online at https://xcmsonline.scripps.edu/ . Parameters for feature detection were set according to the MS data overview . The mass tolerance was set at 15 ppm, peak width from 0.1 to 3 min, prefilter intensity at 10 and noise filter at 3. Bandwidth was set at 5 for alignment, and analysis of variance (ANOVA) test was used for statistical analysis. The other parameters were set as the default. XCMS analysis was carried out in two steps. First, mzXML data files of three groups (control, oxacillin treated and QC) were submitted to XCMS for variations assessment. Then, mzXML data files of control and oxacillin treated groups were submitted to XCMS for statistical analysis (ANOVA). The overlapped features with unique m/z and retention time, as well as CV < 30%, were defined as metabolic features. Volcano plot of the metabolic features was generated by MetaboAnalyst. Metabolic features with p < 0.01, fold change > 2 or < -2 were considered as significantly changed metabolites due to the oxacillin treatment. Systematic pathway analysis was done by XCMS using the tentative identified significantly changed metabolites. The MS2 spectrums of significantly changed metabolites were extracted for further structure confirmation using MetFrag based on the METLIN metabolite database, Human Metabolome Database (HMDB) and LIPID MAPS structure database (LMSD) .
Data Availability Statement
The converted LC-MS data in mzXML format has been deposited to MassIVE . The data files could be downloaded via ftp://[email protected] (User name: MSV000087501 _reviewer, password: 3371) while the dataset is private. After publication, the data should be accessible at ftp://massive.ucsd.edu/MSV000087501/.