In Vitro Evaluation of Rosemary Essential Oil: GC-MS Profiling, Antibacterial Synergy, and Biofilm Inhibition

Background: Antimicrobial resistance (AMR) has become precarious, warranting investments in antimicrobial discovery. Aim: To investigate the antibacterial activity of rosemary essential oil (REO), alone and in combination with selected conventional antibiotics. Methods: REO was subjected to antimicrobial susceptibility testing (including minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) determination) and investigation of anti-pre-biofilm and antibiofilm activities. Results: The phytochemical composition of the REO was eucalyptol (42.68%), bornanone (33.20%), endo-borneol (9.37%), α-terpeneol (7.95%), linalool (2.10%), bornyl acetate (1.81%), caryophyllene (1.09%), 4-terpeneol (0.94%), and anethole (0.87%). The antibacterial inhibition zones generally increased with increasing REO concentration (i.e., 10, 20, 50, 100, and 200 mg/mL). The MIC and MBC ranges of REO for all bacteria were 3.13-6.25 mg/mL and 3.12-12.5 mg/mL, respectively. The MICs (in µg/mL) of ciprofloxacin, chloramphenicol, streptomycin, tetracycline, and ampicillin, respectively, were Escherichia coli (0.98, 3.92, 1.96, 7.81, and 250), Klebsiella pneumoniae (1.25, 7.81, 125, 7.81, and 1000), MRSA (62.5, 7.81, 3.91, 7.81, and 250), Streptococcus mutans and Bacillus subtilis (125, 15.68, 250, 31.25, and 1000), Pseudomonas aeruginosa (125, 31.25, 500, 31.25, and 1000), and Salmonella Typhi (0.98, 15.68, 125, 1.96, and 1000). The MBC-MIC ratios of REO against all bacteria were in the range 1-2, indicating bactericidal effects. Mainly synergy (FICI = 0.16-0.37) was observed between REO and the conventional antibiotics. The IC50 values (in µg/mL) of REO against the bacteria, pre-biofilm vs. biofilm formation, were E. coli (1342.00 vs. 4.00), K. pneumoniae (106.00 vs. 3.00), MRSA (134.00 vs. 6.00), S. mutans (7259.00 vs. 7.00), B. subtilis (120.00 vs. 7.00), P. aeruginosa (4989.00 vs. 7.00), and S. Typhi (10.00 vs. 2.00). Conclusions: Rosemary essential oil had significant bactericidal effects on the bacteria tested, and its MIC and MBC values were low. Overall, it was synergistic with known conventional antibiotics and, thus, has encouraging prospects in combination therapy involving conventional antibiotics, even in the treatment of infections with multidrug-resistant bacteria, including biofilm-forming ones.