Bhi medium

Anaerotruncus colihominis (DSMZ, DSM no.:17241) and Enterococcus faecalis were grown anaerobically at 37 °C in brain-heart infusion (BHI) medium (Cat. no.: DF0037178; Fisher Scientific). Each bacterial culture suspension was subsequently transferred to 3 Brucella blood agar plates, and colonies from all plates were resuspended in 2.5 mL anaerobic sterile PBS yielding a suspension of live bacteria at a density of OD600 = 1.3. An 8-week-old female C57BL/6J mice were colonized with 200 μL of bacteria suspension via oral gavage 3 days per week beginning 3 weeks prior to disease induction, and bacteria treatment was received for the entire duration of the experiment. Control mice were orally gavaged with anaerobic PBS (vehicle).

Streptococcus mutans was collected from the BHI agar plate and resuspended in BHI medium (ThermoFisher Scientific Ltd., Waltham, USA). Subsequently, the concentration was adjusted to 1 × 10⁹ (Dhir, 2013) colony‐forming unit/ml. The full inoculation was performed by injecting the bacteria suspension into the upper chamber of a gradient culture container and was then incubated anaerobically at 37 °C for 2 hr.
The bacteria suspension was then removed, and the upper chamber was washed with 5 ml of BHI medium. The modified flow system model was used to perfuse BHI medium with 8% sucrose (Sigma‐Aldrich, Inc., St. Louis, Missouri, USA) at a flow rate 0.5 ml/min during the biofilm creation (Figure 1; Crusz et al., 2012). Anaerobic condition was induced in the incubator by using an anaerobic gas pack (ANAEROGEN: ThermoFisher SCIENTIFIC Ltd., Waltham, USA). The S. mutans biofilms were grown for 24 and 48 hr before samples were harvested.

Bovine β-LG and methyglyoxal (40% aqueous solution) were obtained from Sigma-Aldrich (St. Louis, MO, USA). CML and carboxyethyllysine (CEL) (98%, HPLC) were obtained from Toronto Research Chemicals (Toronto, ON, Canada). Both pepsin and trypsin were sourced from Sigma-Aldrich (St. Louis, MO, USA). BHI medium was acquired from Thermo Fisher (Waltham, MA, USA). Acetonitrile and methanol for mass spectrometry were purchased from Merck (Darmstadt, Germany). Milli-Q water was employed in the experiments. All other reagents used were of analytical grade.

Fetal bovine serum (FBS) was purchased from Life Technologies (Auckland, New Zealand). Dimethyl sulfoxide (DMSO) was obtained from Wako Pure Chemical Industries (Saitama, Japan). Triton X-100, trypsin, and sodium dodecyl sulfate (SDS) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), streptomycin, and penicillin were purchased from HyClone Laboratories (Logan, UT, USA). Lactobacilli MRS broth was purchased from Difco Laboratories (Detroit, MI, USA). BHI medium was obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Isolates of B. pseudohinzii were cultured overnight at 37 °C in BHI medium (Thermo Fisher Scientific). Thereafter, bacteria were diluted and cultured until they reached a phase of logarithmic growth, followed by three times washing with minimal essential medium (MEM; Thermo Fisher Scientific) without antibiotics. Bacterial infection dose was determined by plating bacterial suspension on BHI agar plates and counting CFU after 12 h of incubation at 37 °C.

C. glutamicum strains were grown to exponential phase (OD_600nm = 5–7) in BHI medium (Oxoid). Total lipids were extracted in chloroform:methanol (2:1 v/v) and chloroform:methanol:water (1:2:0.8, v/v). After removal of insoluble material by centrifugation (15,000× g, 10 min), extracts were dried under nitrogen and subjected to biphasic partitioning in 1-butanol: water (2:1 v/v). The organic phase was dried, and lipids were resuspended in 1-butanol. Cell surface exposed lipid was extracted by water-saturated 1-butanol at RT for 30 min followed by the residual lipid extraction by chloroform:methanol as described for total lipid extraction. The lipid fraction was analysed by high- performance thin-layer chromatography (HPTLC) using aluminium-backed silica gel sheets (Merck). One-dimensional HPTLCs were developed in chloroform: methanol:13 M ammonium solution:1 M ammonium acetate: water (180:140:9:9:2.3 v/v). Glycolipids were stained and visualized with orcinol/H₂SO₄.