Staphylococcus aureus atcc 43300

The bioassays were carried out using the strains Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300 (MRSA-methicillin resistant), Staphylococcus epidermidis ATCC 14990, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922 all acquired from the American Type Culture Collection (ATCC). Approximately 5 × 10⁵ microorganisms·mL⁻¹ were incubated in Muller Hinton broth in 96-well microtiter plates containing the samples to be tested. The compounds (monensin A, metabolite 1, and metabolite 2) were dissolved in dimethylsulfoxide (DMSO) and diluted into the medium to give 100, 50, 25, 12.5, 6.3, 3.1, 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 μg·mL⁻¹, as their final concentrations. The plates were incubated at 37°C and the bioassays were performed in triplicate. The cell death was determined by a MTT colorimetric method which was described by Andrews [23 (link)] and Furtado et al. [24 (link)]. The DMSO solution and chloramphenicol were used as controls of the experiment.

Diamino-polyethylene glycol, dopamine hydrochloride, Trizma® hydrochloride, glutaraldehyde, glycerol, and diiodomethane were supplied from Sigma-Aldrich. Listeria ivanovii ATCC 19119, Listeria innocua ATCC 33090 and Listeria seeligeri ATCC 35967, Listeria monocytogenes (ATCC 7644), Escherichia coli (ATCC 12435), and Staphylococcus aureus (ATCC 43300) were purchased from the American Type Culture Collection (Rockville, Md.). All other chemicals were of analytical grade and purchased from Merck AG (Darmstadt, Germany). QCM crystal (15-mm diameter with polished Au electrodes diameter = 5.0 mm, thickness = 1000Å, 10.0-MHz AT-cut piezoelectric quartz wafer for EQCM) was obtained from CH Instruments, Texas, 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.

Sodium carboxymethyl cellulose (CMC) (degree of substitution = 0.7, M.W. = 90,000), carvacrol (natural, originated from thyme essential oil, 99%, food grade), calcium chloride, sodium chloride, hydrogen peroxide, disodium phosphate, monosodium phosphate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulphate, potassium ferricyanide, trichloroacetic acid, ferric chloride, iron sulphate, tris(hydroxymethyl)aminomethane, pyrogallol, resazurin, were purchased from Merck Chemical (Saint Louis, MO, USA). Tween 80, glycerol, ammonia water, hydrochloric acid, sodium hydroxide, chloroform, ethyl acetate, ethanol, and methanol were procured from Chempur (Piekary Śląskie, Poland). Mueller–Hinton broth, Mueller–Hinton agar, and agar-agar were purchased from Merck Chemical (Saint Louis, MO, USA). All chemicals were of analytical grade. Escherichia coli ATCC25922, Staphylococcus aureus ATCC43300, and Candida albicans ATCC10231 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA).

Vancomycin and oxacillin were purchased from Sigma-Aldrich (Milan, Italy). Staphylococcus aureus ATCC 43300 (a methicillin-resistant strain characterized by the presence of the mec operon), S. aureus ATCC 29213, P. aeruginosa ATCC 27853, and K. pneumoniae ATCC 13883 were obtained from the American Type Culture Collection (Rockville, MD, USA).