4.8. Extraction and Examination of the Composition of Glycolipids, Phospholipids, and Fatty Acids in Staphylococcal Membranes

Standards of phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The other chemicals were acquired from Sigma-Aldrich (Darmstadt, Germany) and Avantor (Gliwice, Poland). All chemicals used were high-purity-grade reagents.

A suspension of S. aureus ATCC 43300 (OD₅₃₅ = 1.8) in TSB/Glu prepared from fresh 24 h old culture was added (6 mL) to bacteriological Falcon-like tubes (Medlab, Raszyn, Poland) and centrifuged at 3000 rpm for 10 min. Bacteria in the sediment were then exposed to acetonic and ethanolic extracts from V. opulus fruits and bark at a concentration of 500 µg/mL (tested samples) for a 24 h at 37 °C with gentle shaking. Bacteria in culture medium alone (TSB/Glu) were prepared as control 1 and bacteria in culture medium TSB/Glu containing 2.5% ethanol were used as control 2. After exposure, the samples were centrifuged as described above, bacteria were suspended in 6 mL TSB/Glu, and each sample was added (2 mL) to three Eppendorf tubes (Medlab, Raszyn, Poland) for lipid and fatty acid extraction and analysis. The whole experiment was conducted twice.

Lipids from S. aureus cells were extracted according to our previous method [25] with modifications. The bacterial biomass was moved into 2 mL Eppendorf tubes containing glass beads, 0.33 mL methanol, and 0.66 mL chloroform. Then, the samples were homogenized for 1 min with the use of FastPrep (MP Biomedicals, Shanghai, China). After, the samples had been centrifuged (2000× g, 2 min), the supernatants were transferred into 1.5 mL Eppendorf tubes, and 0.2 mL of saline was added. The lower layer of each sample was collected and evaporated. The obtained extracts were dissolved in 0.75 mL methanol.

The lipid content was determined using an Agilent 1200 HPLC (Santa Clara, CA, USA) and a 4500 QTRAP mass spectrometer (Sciex, Framingham, MA, USA) equipped with an ESI source operated in positive or negative ion mode. Ten microliters of the lipid extract were injected onto a Kinetex C18 column (50 mm × 2.1 mm, particle size: 5 μm; Phenomenex, Torrance, CA, USA) at a flow rate of 0.5 mL/min and a temperature of 40 °C. The gradient elution was applied with mobile phases of water (A) and methanol (B) (both consisted of 5 mM ammonium formate). The solvent gradient was initiated at 70% B, increased to 95% B over 1.25 min, and maintained at 95% B for 6 min before returning to the initial solvent composition over 3 min. The data analysis was performed with Analyst™ v1.6.2 software (Sciex, Framingham, MA, USA).

At first, the untargeted approach was performed with precursor ion scanning at m/z 153 (in negative mode) or neutral loss scanning of m/z 300 (in positive ion mode) to detect PG and Lysyl-PG, respectively, triggering enhanced product ion experiments. Sodium adducts of glycolipids were searched using enhanced mass spec experiments. On the basis of the untargeted analysis, a comprehensive list of the multiple reaction monitoring (MRM) transitions was generated, and a quantitative analysis was performed.

To Eppendorfs with bacterial lipid extract (0.375 mL), toluene (0.05 mL) and 8.0% HCl solution in methanol (0.075 mL) were added [72]. The tubes were vortexed and then incubated at 45 °C for 16 h. After cooling to room temperature, 0.5 mL hexane and 0.5 mL water were added for the extraction of fatty acid methyl esters (FAMEs). One microliter of each extract sample was analyzed using a gas chromatography system (Agilent Model 7890 gas chromatograph, equipped with a 5975C mass detector). The separation was carried out in a HP 5 MS methyl polysiloxane capillary column (30 m × 0.25 mm i.d. × 0.25 mm ft). The column temperature was maintained at 60 °C for 3 min, and then increased to 212 °C at a rate of 6 °C/min, followed by an increase to 245 °C at a rate of 2 °C/min, and finally, to 280 °C at a rate of 20 °C/min for 10 min. Helium was used as the carrier gas at a flow rate of 1 mL/min. The injection port temperature was 250 °C. Bacterial fatty acids were identified by comparison with the retention times of the authentic standards (Sigma-Aldrich, Darmstadt, Germany), and the results are expressed as a percentage of the total amount of fatty acids.