Chronic Wound Milieu Challenges Essential Oils' Antibiofilm Activity

The treatment of infected non-healing wounds poses a significant challenge to contemporary medicine. Essential oils (EOs) are being increasingly investigated as potential antibiofilm agents for the management of biofilm-related wound infections. However, their in vitro antimicrobial activity reported in literature does not necessarily reflect the actual in vivo activity due to, among others, methodological imperfections of performed tests. In our study, we cultivated a Staphylococcus aureus biofilm in a novel IVWM (In Vitro Wound Milieu) medium designed to simulate the wound environment and compared it with biofilms cultured in the standard microbiological Tryptic Soy Broth (TSB) medium. We examined and compared critical biofilm properties such as morphology, biomass, metabolic activity, cell count, thickness, and spatial distribution of live and dead cells. Subsequently, staphylococcal biofilms and planktonic cells cultured in both media were exposed to the activity of thyme or rosemary EOs (T-EO, R-EO, respectively). We found that morphology of biofilms cultured in IVWM resembled more the morphology of biofilms visualized in the non-healing wounds than biofilms cultured in TSB. The biomass, metabolic activity, cell number, and the ratio of live to dead cells of S. aureus biofilms were all lower in IVWM compared to the TSB medium. Additionally, while EOs demonstrated overall significant anti-staphylococcal activity, their efficacy varied depending on the medium used. Generally, EOs displayed lower antimicrobial activity (against planktonic cells) in the IVWM than in the TSB medium. Interestingly, T-EO caused a higher reduction of biofilm cells in IVWM than in the TSB medium, in contrast to R-EO. Our findings suggest that EOs hold promise as agents for the treatment of biofilm-related wound infections. However, it is crucial to apply in vitro conditions that closely reflect the wound infection site to gain an accurate insight into the real-world activity of these antimicrobial/antibiofilm agents.