Essential Oils And Their Components As Inhibitors Against Multidrug Resistant Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium and nosocomial pathogen that causes a diverse range of infections. There are multidrug resistant (MDR) strains of P. aeruginosa that have acquired resistance to at least three antibiotics. Prior to 2003, MDR infections were rare and still treatable. Now, MDR infections are more prevalent and sometimes untreatable. Antibiotic resistance is often referred to as the silent tsunami facing modern medicine. This highlights the importance of identifying additional treatment methods outside of antibiotic therapies alone. Our study aims to identify compounds within essential oils that are inhibitory against MDR P. aeruginosa so that their efficacy could be explored as potential mutual prodrugs in treatment to improve patient outcomes. We first examined the inhibitory potential of 26 essential oils against control (non-MDR) strain, ATCC 33347, and one experimental (MDR) strain, ATCC BAA 2110. A two-fold serial dilution of the oils from 20% to 2.5% concentrations was performed, using DMSO as our control. DMSO showed no inhibitory affects against either strain of P. aeruginosa, and this was also supported by the literature. We then performed a standard disc diffusion assay in triplicate, over two trials. After 24 hours of incubation, zones of inhibition were measured with ImageJ software. Results indicated that oregano wild, wintergreen, cinnamon branch, cloves bud, tea tree, mugwort, sage, and ajowan oils were potent inhibitors and displayed a dose dependent inhibitory trend against both strains of P. aeruginosa. Based on the HPLC profiles of these oils, we predict that eugenol, linalool, γ-terpinene, and β- caryophyllene are the active, inhibitory compounds based on their shared presence in these oils. Our next step was to develop a method for testing these compounds individually and in combination against both strains of P. aeruginosa to determine which is/are the active component(s). This method involves serially diluting the compound ten-fold from 1M to 0.0001M in 10% DMSO and nutrient broth, along with 100 ul of MDR or non-MDR P. aerusinosa. The mixture was incubated overnight and 50 ul was drop plated on Mueller-Hinton agar, incubated overnight, and assessed for growth. No growth on the plate indicated a bactericidal concentration whereas limited growth represents an inhibitory concentration. Cloves bud oil, which has previously shown high inhibition of P. aerusinosa in our earlier studies, was used to determine the efficacy of this method and has shown a bactericidal concentration of less than 1.25%. β-caryophyllene has been tested using this method and has shown no inhibitory capabilities at any concentration. The next compounds to be tested are eugenol, eugenol acetate, gamma-terpinene, and linalool individually and in combination. Active compounds will then be assessed in combination with multiple antibiotics to determine if the compounds are able to act synergistically and overcome the MDR activity of P. aeruginosa