Bioautography-Guided Identification of Antimicrobial Essential Oil Components of Oregano, Cumin and Rosewood against Antibiotic Sensitive and Resistant Staphylococcus aureus, Enterococcus faecium, Escherichia coli and Pseudomonas aeruginosa

The increase in antimicrobial resistance and decrease in antibiotics being developed has resulted in the need for new strategies to treat antibiotic resistant infections. Many Essential Oils (EOs) possess broad spectrum antimicrobial activity, including against antibiotic resistant microorganisms, indicating that antimicrobial formulations could be developed. The aim of this study is to conduct high throughput screening of EOs by direct bioautography to identify antimicrobial components therein, in order to inform the future development of antimicrobial formulations. Rosewood, cumin and oregano EOs were screened for antimicrobial activity against methicillin sensitive and resistant Staphylococcus aureus, vancomycin sensitive and resistant Enterococcus faecium and ciprofloxacin sensitive and resistant Escherichia coli and Pseudomonas aeruginosa using the Kirby-Bauer disc diffusion method. Minimum Inhibitory Concentrations (MICs) were also determined using microdilution; the MICs ranged from 0.29-9.20 mg/ml for oregano EO, 0.29-37.20 mg/ml for cumin EO and 8.80-35.20 mg/ml for rosewood EO. Direct bioautography of the EOs was conducted, whereby a bacterial suspension was sprayed onto eluted two-dimensional thin layer chromatography plates and zones of inhibition of microbial growth were visualized using tetrazolium salts. Antimicrobial spots were isolated and characterised using 1H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectroscopy (GC-MS). NMR and GC-MS analysis identified carvacrol, linalool and cuminaldehyde as the major bioactive components present in oregano, rosewood and cumin EOs, respectively. The MICs of these components were then determined. Carvacrol possessed the greatest antimicrobial activity (MICs 0.99 – 31.62 mM), while the MICs of linalool ranged from 57.05-912.80 mM and cuminaldehyde from 2.10-268.83 mM. The antimicrobial activity of these compounds indicates that they may have the potential to be developed as alternative therapies for infections caused by antibiotic resistant bacteria. Future studies will investigate interactions between these EO components and currently-employed antibiotics with the aim of developing a combination therapy to combat antibiotic resistance.