Vitamin k1

P. gingivalis (ATCC 33277) was cultured as previously described by us (Wang et al., 2020 (link)). P. gingivalis cells maintained as frozen stock were firstly grown on blood agar plates (44 g/L Columbia agar base, Difco, 5% horse blood, Hemostat, 5 mg/L hemin, Sigma-Aldrich, 1 mg/L vitamin K1, Sigma-Aldrich) in an anaerobic atmosphere composed of 10% H₂, 5% CO₂, and 85% N₂ at 37°C. After 7-day culture, a single colony was picked into liquid trypticase soy broth (30 g/L TSB; Difco) supplemented with yeast extract (5 g/L), vitamin K1 (1 mg/L), and hemin (5 mg/L), and it was then cultured in the same anaerobic conditions.

Vitamin K2 extraction samples were analysed on a HPLC (UFLC, Shimadzu, Japan) system coupled with a Micromass Quattro Ultima MS (Waters, USA). Fifty-microliter of each sample was injected on a Symmetry C18, 5 µm, 150 × 3 mm column (Waters, USA). Elution of compounds followed a gradient that started with 100% methanol (solvent A), 0% 2-propanol/hexane 50/50 (solvent B), and changed to 25% solvent A and 75% solvent B in 10 min. Then the gradient changed to 1% solvent A and 99% solvent B in 2 min and remained for another 2 min. The flow rate was 0.4 ml/min and oven temperature was 40 °C. Eluted compounds were detected by the MS system with an atmospheric pressure chemical ionization (APCI) source in the positive mode. The corona current was 5 µA. The APCI source temperature was 120 °C and probe temperature 500 °C. Details of MRM (multiple reaction monitoring) are presented in Additional file 1: Table S1. Standards of MK-1 (Santa Cruz Biotechnology), MK-4 (Sigma), MK-7 (Sigma), MK-9 (Santa Cruz Biotechnology) and vitamin K1 (Sigma) in the concentration range from1 ng/ml to 3 µg/ml were analysed to obtain the calibration curves. Except for the strain screening experiment, vitamin K1 was added at a concentration of 300 ng/ml as an internal standard in each sample.

The minimum inhibitory concentrations (MICs) for moxifloxacin (MXF), erythromycin (ERY), tetracycline (TET), amoxicillin (AMX), metronidazole (MTZ) and vancomycin (VAN) were evaluated by E-test (bioMérieux, Marcy l’Etoile, France) onto pre-reduced BA plates supplemented with 5 mg/L hemin, 1 mg/L vitamin K1 (Sigma Aldrich, Darmstadt, Germany) and 5% defibrinated sheep red blood cells. The MIC values were recorded after 48 h of incubation in anaerobic conditions.
The breakpoint used for ERY and MXF was 8 mg/L, while the breakpoint for TET and AMX was 16 mg/L, in accordance with the CLSI interpretative categories approved for anaerobic bacteria [53 ]. The resistance to metronidazole MTZ and VAN was defined as MIC > 2 mg/L, according to the epidemiological cut-off values (ECOFFs) suggested by the European Committee on Antimicrobial Susceptibility Testing [54 ].
The Wilkins–Chalgren-based agar incorporation method was used as previously described [55 (link)] to re-evaluate strains showing MICs for VAN > 2 mg/L by E-test.

Since is reported that the lack of vaginal H₂O₂ producing lactobacilli is associated with bacterial vaginosis, the Lactobacillus isolates were also characterised for their production of H₂O₂. The capacity of L. crispatus L1 to produce H₂O₂ was tested with a semiquantitative assay on tetramethylbenzidine agar plates [15 (link)] using Brucella agar (Difco) containing 0.001% (w/v) horseradish peroxidase (Sigma), 0.023% (w/v) tetramethylbenzidine (Sigma) and 1% (w/v) starch. This medium was supplemented with 0.5 mg of bovine haemin (Sigma) and 0.1 mg of vitamin K1 (Sigma) in 100 ml of final volume. Serial dilutions of lactobacilli were inoculated in the medium and incubated in anaerobic conditions at 37°C for 72 h. Plates were then exposed to ambient air and H₂O₂-producing colonies were revealed by the appearance of a blue colour. According to the colour intensity, the strains were classified as strong, medium, weak or negative (white colonies) producers [41 (link)].

Fusobacterium nucleatum was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA; #25586). WT Fusobacterium nucleatum and FadA−/− Fusobacterium nucleatum were cultured in Columbia blood agar with 5 μg/mL heme, 5% desalted sheep blood, and 1 μg/mL vitamin K1 (Sigma-Aldrich, St. Louis, MO, USA) in a 37 °C anaerobic glove box containing 85% N₂, 10% H₂ and 5% CO₂ [30 (link)]. Escherichia coli (MG1655, ATCC, Manassas, VA, USA) was propagated in Luria Bertani medium (BD Biosciences, Franklin Lakes, NJ, USA) at 37 °C in an aerobic incubator.

Stool samples from five healthy infants exclusively breast-fed and two months old were selected from a previous study^{65 (link)}. A pooled fecal sample was used to isolate the microbiota as previously described^{66 (link)}. Two grams of feces were homogenized in 18 ml of physiological serum (NaCl 0.9%), placed on top of 3.5 ml Nycodenz 80% solution (Alere Technologies AS) and centrifuged for 1 h at 3,327 xg (Hermle Z383K). The layer containing the microbiota was collected and stored in 20% glycerol at – 80 °C. This microbiota was used to inoculate two ml of basal medium containing bactopeptone (Difco), 2 g/L; yeast extract (Pronadisa), 2 g/L; NaCl 0.1 g/L; K₂HPO₄ 0.04 g/L; KH₂PO₄ 0.04 g/L; MgSO₄·7H₂O 0.01 g/L; CaCl₂·H₂O 0.01 g/L; NaHCO₃ 2 g/L; L-cysteine 0.5 g/L; bile salts 0.5 g/L; Tween 80 2 ml/L; haemin solution 0.05 g/L (Sigma); vitamin K₁ (Sigma), 10 μl/L; and resazurin 0.025% solution, 4 ml/L. The pH of the medium was adjusted to 7.4. Four cultures were initiated with the purified disaccharides, LNB, GNB, 3FN or 6FN at 10 mM. A control culture without carbohydrate added was also included. Cultures were grown at 37 °C under anaerobic conditions using an anaerobic atmosphere generation system (Anaerogen, Oxoid), and at 48 h they were centrifuged, and pellets and supernatants were collected. Three independent cultures were carried out for each oligosaccharide.