4.4. Antibacterial Activities of the Pinaceae Essential Oils

The antibacterial activities were investigated by using different methods, the Minimal Inhibitory Concentration (MIC), the Minimal Bactericidal Concentration (MBC), the agar diffusion method, and Vapor Phase Test (VPT).

Five bacterial strains from the culture collections of the Plant Cytology and Biotechnology Laboratory of Tuscia University were tested to evaluate the antibacterial activities of P. cembra L., P. mugo Turra, A. alba M., and P. abies L. essential oils: Escherichia coli ATCC 25922, Pseudomonas fluorescens ATCC 13525, and Acinetobacter bohemicus DSM 102855 among Gram-negative and Kocuria marina DSM 16420 and Bacillus cereus ATCC 10876 among Gram-positive. All tested bacterial strains were maintained on LB broth (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter, autoclaved at 121 °C for 20 min) with agar. Bacteria cultures were maintained at two different temperatures: 26 °C for P. fluorescens, A. bohemicus, and B. cereus and 37 °C for K. marina and E. coli. All inocula were prepared with fresh cultures plated the day before the test.

The MIC is defined as the lowest concentration of antimicrobial agent that completely inhibits the growth of the microorganism as detected by the unaided eye and was carried out according to the microwell dilution method. Briefly, 12 dilutions of the four essential oils in LB broth (for P. cembra from 53.12 to 0.01 mg/mL; for P. mugo from 52.16 to 0.01 mg/mL; for A. alba form 51.28 to 0.01 mg/mL and for P. abies from 53.12 to 0.01 mg/mL), a control with the same percentage of DMSO (from 6.25% to 0.003%) in Lysogeny broth, a growth control without treatments, a positive control with gentamicin diluted from 100 to 0.05 µg/mL, and a sterility control without bacteria were plated on 96 microwell plates. Then, 50 µL of bacterial inoculum, 10⁶ CFU/mL, were added in each well, except for the sterility control, and the plates were incubated for 24 h at the corresponding temperature. The visualization of the inhibition activity was obtained by 20 µL of a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (200 µg/mL, MTT) added to each well. The assay was carried out in triplicate. The MBC/MIC ratio was reported to interpret the activity of the essential oil, and an antimicrobial agent is considered bacteriostatic when the ratio MBC/MIC > 4 and bactericidal when the ratio MBC/MIC is ≤4 [31].

To verify the lowest concentration at which the tested essential oils kill the bacterial cells, which is defined the Minimum Bactericidal Concentration (MBC), 10 µL of the last four dilutions from microwell dilution method in which no bacteria growth was observed were plated on a Petri plate with LB agar and incubated for 24 h. The concentration at which no growth on agar was observed defined MBC values. The assay was carried out in triplicate.

To determine the diameter of the halo inhibition of the bacteria growth induced by P. abies, A. alba, P. cembra, and P. mugo essential oils, the bacterial strains were suspended in LB broth to obtain a turbidity of 0.5 McFarland (approximately 10⁸ Colony-Forming Unit/mL—CFU/mL) and then plated on LB broth with agar in a Petri plate. Sterile disks (6 mm diameter, Oxoid) were placed on the agar and impregnated with 10 µL of samples. Two µL of gentamicin from a stock solution (10 mg/mL) was used as a positive control After 24 h, the inhibitory activities of each essential oil were recorded as mm of halo diameter without growth [58] using a vernier caliper rule. The mean and the respective standard deviation (SD) of the measured halo in three independent experiments were recorded.

The antibacterial activity of the Pinaceae essential oils in the vapor phase was evaluated by the modified disk volatilization method [66,67]. LB agar were poured into an 80 mm plastic Petri dish and a lower amount into its cover. Each bacterial suspension containing 10⁸ CFU/mL was plated on the LB agar medium. Then, 10 µL of tested essential oils were added to a 6 mm sterile disk and placed on agar in the covered Petri plate. Liquid LB agar was put in the space between the cover and the base of the Petri dishes to facilitate the sealing and to prevent any vapor leakage. The Petri plates were incubated for 24 h in an inverted position, and afterwards, the inhibition halos were measured. Negative controls were carried out without the essential. All VPTs were carried out in triplicate.