Mueller hinton agar (mha)

Bacterial stocks of Gram-positive, Staphylococcus aureus ATCC^®6538, S. aureus ATCC^®43300 and S. epidermidis and Gram-negative Escherichia coli ATCC^®25922 and Pseudomonas aeruginosa PAOI were kept in Brucella Broth (Fluka Chemie, Madrid, Spain) with 20% (v/v) glycerol (Sigma, Madrid, Spain) at −80 °C.
Pre-inoculum was prepared by stocks defrosting at room temperature, suspension in Mueller Hinton Broth (MHB; Liofilchem, Roseto degli Abruzzi, Italy) followed by a 6 h incubation at 37 °C, 220 rpm. Afterwards, bacteria were streaked onto Mueller Hinton Agar (MHA; Liofilchem, Roseto degli Abruzzi, Italy) and incubated overnight at 37 °C. Then, bacteria were harvested with sterile peptone water (Liofilchem), washed twice by centrifugation at 2700 rpm and bacterial pellet suspended in MHB.

Unless otherwise stated, bacteria were grown in Mueller Hinton Broth (MHB, Pronadisa) at 37°C with agitation at 250 rpm. The initial concentrations of tigecycline (Pfizer) and tobramycin (Normon, S. L) that inhibit the growth of P. aeruginosa PA14 under the culture conditions used in the evolution experiments were determined at 37°C.
General susceptibility to a wide range of antibiotics - tigecycline, tetracycline, aztreonam, ceftazidime, imipenem, ciprofloxacin, levofloxacin, norfloxacin, tobramycin, streptomycin, amikacin, colistin, polymyxin B, chloramphenicol, fosfomycin and erythromycin – was examined by disk diffusion in Mueller Hinton Agar (MHA) (Sigma) at 37°C.
The MICs of different antibiotics were determined for the bacterial populations over the evolution period at 37°C in MHA using E-test strips (MIC Test Strip, Liofilchem^®). MICs of colistin and polymyxin B were determined in MHB II by double dilution in microtiter plates. The MICs to the antibiotics of selection and to fosfomycin were repeated twice and in all cases, the results were the same in the replicated assays.

Test microorganisms—Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Candida albicans P37037—strains were provided by the culture collection of the Microbiology Laboratory, Alexandru Ioan Cuza University of Iasi. The methicillin-resistant Staphylococcus aureus (MRSA) isolate was kindly provided by Med. biol. PhD Simona Matiut from Praxis Clinical Laboratory, Iasi, Romania, and included in the same microbial culture collection mentioned above, with the following accession number: prx-MRSA-2018.
All strains were stored in 15% glycerol stocks at −80 °C. Prior to experiments, S. aureus and E. coli strains were transferred to Mueller–Hinton agar (MHA, Liofilchem, Roseto degli Abruzzi, Italy) and C. albicans on Sabouraud dextrose agar (SDA, Carl Roth, Karlsruhe, Germany) and incubated at 37 °C. Subsequently, 10 mL of Mueller–Hinton broth (MHB, Scharlau, Barcelona, Spain) and 15 mL of Sabouraud dextrose broth (SDB, Carl Roth, Germany) were inoculated with one representative colony of each test organism taken from MHA or SDA, cultured overnight (37 °C, 190 rpm and 130 rpm, respectively) and used as source of inoculum for further experiments.

The antibacterial activity of the EOs was evaluated by the disc diffusion assay, following the M2-A8 method as described by the Clinical Laboratory and Standards Institute (CLSI) for bacteria [13 ]. Inoculums were prepared by suspending bacteria in a sterile saline solution to a cell suspension of 0.5 McFarland (1 to 2 × 10⁸ colony-forming units/mL (CFU/mL)). Discs with a diameter of 6 mm were saturated with 10 µL of each EO. Then, the Müeller-Hinton agar (MHA) (Liofilchem, Roseto degli Abruzzi, Italy) plates were inoculated, allowed to dry, and the discs previously prepared were placed over the agar. The inoculated plates were incubated at 37 °C for 24 h. After the incubation, all the plates were visually checked for inhibition zones, the diameters being measured in millimeters. Each experiment was done independently three times [12 (link)].

Antimicrobial susceptibility tests of isolates were performed by using the Kirby–Bauer disk diffusion technique on the Muller–Hinton Agar (MHA) (Liofilchem Ltd., Italy) [26 ] according to the Clinical and Laboratory Standards Institute (CLSI) [27 ]. The antimicrobial agents used include, cefoxitin FOX (30 μg), clindamycin CLN (2 μg), erythromycin ERY (15 μg), chloramphenicol CAF (30 μg), ciprofloxacin CIP (5 μg), amoxicillin/clavulanate AMC (20/10 µg), cloxacillin COX (5 μg), tetracycline TTC (30 μg), gentamicin CN (10 μg), penicillin PEN (10units), trimethoprim-sulphamethoxazole SXT (25 μg), doxycycline DOX (30 μg), rifampin RIF (5 μg), and nitrofurantoin NIT (300 μg). The cefoxitin disk was used to characterize MRSA isolates. A zone of inhibition was measured in millimeters using a ruler around each antimicrobial disc. The results were reported as sensitive, resistant, or intermediate according to the CLSI recommendation.

Pathogenic yeast Candida parapsilosis CBS 883C was from the collection of the Laboratory of Biodeterioration Research, Nature Research Centre (Vilnius, Lithuania). Baker’s yeast S. cerevisiae was obtained from a local market (shelf life not less than 2 weeks as specified by the producer). The antifungal activity of Rh. tomentosum was tested by the agar disc diffusion assay. The tests were carried out employing 100 μL of suspension containing 10⁶ CFU (colony-forming units)/mL of yeast spread on Mueller–Hinton agar (Liofilchem, Roseto, Italy) on a Petri plate (90 mm). Inoculum was obtained from yeasts cultured in Sabouraud dextrose agar (Liofilchem, Roseto, Italy) at 28 ± 1 °C for 48 h. After the absorption of inoculum by agar, the sterile paper discs (6 mm in diameter) impregnated with 10 µL of methanolic solution (10 mg/mL) of Rh. tomentosum EO (8 Sh, October) was placed on the surface of agar layer. The inoculated Petri plates were incubated at 30 °C for 48 h. Nystatin (100 I.U., Liofilchem, Roseto, Italy) was used as a positive control. The diameters of transparent inhibition zones formed in the yeast lawns around impregnated paper discs were used as a measure of antifungal activity. Each assay was repeated twice.