Combined Efficacy of Essential Oil Compounds and Bacteriophage to Control Staphylococcus aureus with Potential Application

Introduction:  Multidrug-resistant (MDR) Staphylococcus aureus are increasingly prevalent and control via standard antibiotic treatment has become challenging. Combinations of different antimicrobials represent an approach for combating MDR bacteria. Antimicrobials with potential food industry application include essential oils (EO) and bacteriophage (phage). 

Purpose: Here, we aimed to investigate the combination of EOs and phages as alternative antimicrobials to control S. aureus.

Methods: Four EO compounds were evaluated by disc diffusion assay to determine inhibitory effects against five strains of S. aureus. Next, a 48-hour growth inhibition assay was performed using a 96-well bioassay. Phage adsorption assays were performed up to 120 h at 6, 13, and 37°C to determine lytic activity, and phage concentration over time was determined by plaque assay. Combinations of phage and EO against S. aureus were also evaluated at 37°C using a 96-well bioassay.

Results: Disc diffusion assays indicate that the zones of inhibition (IZ) of alpha-pinene (IZ=10 – 23 mm) and alpha-pinene + limonene (IZ=11 – 15 mm) have a greater inhibitory effect against S. aureus strains when compared to other EOs tested. Growth inhibition assay at 37°C confirmed these results with 1.5% alpha-pinene inhibiting S. aureus growth up to 30 to 40 h depending on the strain. Phage adsorption assays indicate that phage K has high lytic activity at 37°C when compared to 6 and 13°C possibly impacting applicability in food industry. Results from the combined effect of EO and phage indicate that this combination inhibits S. aureus in vitro at 37°C more effectively than phage or EO alone although there is variability between strains. For application in the food industry, these antimicrobials were evaluated for their efficacy against S. aureus on raw chicken pieces at 6, 13 and 25°C. Results indicate that at 25°C 1.5% alpha-pinene inhibits S. aureus growth better as compared to other antimicrobial combination. At 6 and 13°C, there was no significant effect of EO and phage alone or in combination against S. aureus when applied on the raw chicken pieces. 

Significance: These results indicate that EOs and phage can be used as potential antimicrobials against S. aureus in vitro. For these antimicrobials to work in vivo such as raw meat products, better delivery method of the antimicrobials should be employed for a uniform application on meat.