Bacillus subtilis atcc 6633

Bacillus subtilis ATCC6633 and Bacillus cereus ATCC14579 strains were purchased from the American type culture collection (ATCC). Escherichia coli BL21(DE3) was obtained from a pET Expression System 30 kit (Novagen) and Saccharomyces cerevisiae was the baker’s yeast l’Hirondelle (Lesaffre, France). Pseudomonas aeruginosa CIP104116 and Acinetobacter baumanii CIP70.10 were obtained from the Collection of Institut Pasteur (CIP).

Escherichia coli ATCC 35218, Bacillus subtilis ATCC 6633, S. aureus ATCC 6538P (methicillin susceptible S. aureus, MSSA), S. aureus ATCC 43300 (MRSA), Enterococcus faecalis ATCC 29212, and E. faecalis ATCC 51299 (VanB phenotype) were obtained from the American Type Culture Collection (ATCC). E. faecalis 9160188401-EF-34 (VanA phenotype) is a clinical isolate, which was kindly provided by Laboratorio Microbiologia Clinica – Ospedale di Circolo, Varese, Italy. E. coli and B. subtilis were propagated overnight in Luria Bertani medium (LB, 2% tryptone, 2% yeast extract, and 1% NaCl), and the S. aureus and E. faecalis strains in Müller Hinton broth 2 (MHB2, 0.3% beef infusion solids, 1.75% casein hydrolysate, and 0.15% starch) with continuous shaking at 200 rpm and 37°C. For exponential growth, overnight cultures were transferred to fresh medium: inocula were prepared to start the cultures with an optical density at 600 nm (OD_{600 nm}) of 0.1 in the final medium. For long-term preservation, bacterial cultures were stored at -20°C in 20% glycerol. Media were acquired from Sigma-Aldrich, Milan, Italy, unless otherwise stated.

S. azurea DSM 44631, S. viridis DSM 43017, S. glauca DSM 43769 and S. cyanea DSM 44106 strains used in this study were purchased from Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures, while S. azurea SZMC 14600 was originated from Szeged Microbiology Collection (SZMC). Bacterial cultures were stored as a suspension in Luria Bertani (LB) broth with 20% (v/v) glycerol at − 80 °C. Culture conditions were carried out according to Valasek et al. (2016 (link)). Briefly 1 mL bacterial cell suspension was inoculated into 50 mL seed medium containing 3% (w/v) soy flour, 4.2% (w/v) water soluble starch, 0.36% (w/v) NaCl, 0.6% (w/v) CaCO₃, 0.5% (w/v) sunflower oil, pH 8.0, and incubated for 2 days at 37 °C in an orbital shaker at 200 rpm. Subsequently, 1 mL of accurately homogenized seed culture was transferred into 35 mL of fermentation medium containing 4% (w/v) soy flour, 4% (w/v) water soluble starch, 0.3% (w/v) NaCl, 0.5% (w/v) CaCO₃, 0.3% (w/v) stearic acid, 0.1% (w/v) KH₂PO₄, 0.6% (w/v) sunflower oil (pH 9.5), and cultivated for 7 days at 28 °C in an orbital shaker at 200 rpm. Bacillus subtilis ATCC 6633 used for agar well diffusion assay was purchased from American Type Culture Collection (ATCC).

Analytical grade solvents, i.e., n-hexane, chloroform, ethyl acetate, methanol, butanol, dichloromethane, and acetone were purchased from R&M Chemical (Selangor, Malaysia). The solvents were used in the extraction and chromatography processes. Deionized water was purified using a Milli-Q purification system (supplied by Milipore, Bedford, MA, USA). The deuterated solvents used in the NMR analysis were chloroform-d1 (CDCl₃), supplied by Merck (Darmstadt, Germany). Sulfuric acid (H₂SO₄) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Media used for the antibacterial activities included: Mueller–Hinton broth (MHB), Mueller–Hinton agar (MHA), and nutrient agar (NA), which were provided by Oxoid Ltd. (Basingstoke, UK). Chlorhexidine (CHX) was purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Bacillus cereus ATCC33019, Bacillus subtilis ATCC6633, Bacillus pumilus ATCC14884, and Bacillus megaterium ATCC14581 were obtained from American Type Culture Collection (Gaithersburg, MD, USA).

Escherichia coli ATCC 35218, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538P (MSSA), Staphylococcus aureus ATCC 25923 (MSSA), Staphylococcus aureus ATCC 43300 (MRSA), Enterococcus faecalis ATCC 29212, and E. faecalis ATCC 51299 (VanB phenotype) were obtained from the American Type Culture Collection (ATCC). Enterococcus faecalis 9160188401-EF-34 (VanA phenotype) and Staphylococcus epidermidis strain 4 are clinical isolates, kindly provided by Laboratorio Microbiologia Clinica – Ospedale di Circolo, Varese, Italy. Staphylococcus haemolyticus 3902 is a teicoplanin-resistant clinical isolate (Beltrametti et al., 2003 (link)), received from FIIRV (Fondazione Istituto Insubrico Ricerca per la Vita), Gerenzano Varese, Italy. For long-term preservation, bacterial cultures were stored at -80°C in 10% v/v glycerol.
E. coli and B. subtilis were routinely grown at 37°C with continuous shaking at 200rpm (revolutions per minute) in LB broth. S. aureus, S. epidermidis, S. haemolyticus, and E. faecalis strains were propagated in the same conditions in MHB2. For exponential growth, overnight cultures were diluted in fresh medium at an optical density at 600nm (OD_600nm) of 0.1 and incubated as above.