The chronic wound milieu changes essential oils' antibiofilm activity-an in vitro and larval model study

Essential Oils (EOs) are currently being researched as potential antibiofilm agents to combat infections related to chronic wound biofilms. As documented in the literature, EOs' in vitro antibacterial properties are often assessed using standard microbiological media and conditions that do not accurately reflect the actual environment of a chronic wound. To address this issue, In vitro Wound Milieu (IVWM) medium, which closely resembles the environment of a chronic wound, was applied for culturing S. aureus biofilms (n = 12) in this research. Biofilms cultivated in the standard Tryptic Soy Broth (TSB) medium served as a control for the experiment. Key biofilm features were analyzed and compared. Subsequently, staphylococci were exposed to the activity of thyme or rosemary EOs (T-EO and R-EO, respectively). As proof of concept, the cytotoxicity of T-EO and its antimicrobial in vivo activity were assessed using a G. mellonella larvae model. Key features of biofilm-forming cells were lower in the IVWM than in the TSB medium: biomass (up to 8 times), metabolic activity (up to 9 times), cell number (up to 100 times), and the live/dead cells ratio. Conversely, biofilm thickness was higher (up to 25%) in IVWM. These differences translated into varied responses of the biofilms to EOs exposure. The application of T-EO led to a greater reduction (up to 2 times) in 67% of biofilm-forming strains in IVWM compared to the TSB medium. Conversely, exposure to R-EO resulted in a higher reduction (up to 2.6 times) of 83% of biofilm-forming strains in TSB than in IVWM. The application of T-EO was not only non-toxic to G. mellonella larvae but also increased the survival of larvae infected with staphylococci (from 48 to 85%). Our findings suggest that EOs not only show promise as agents for treating biofilm-related wound infections but also that providing conditions reflecting the specific niche of the human body is of paramount importance in influencing the results obtained. However, before clinical application, challenges related to the methods of assessing their activity, microbial intra-species variability, and different levels of activity of various EOs should be analyzed and standardized.