Understanding the role of Enterococcus faecalis in polymicrobial wounds

• Main Author: Davient, Bala
• Other Authors: Kevin Pethe
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Science::Biological sciences::Microbiology; Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/165831
• Institution: Nanyang Technological University

Microbial biofilms are an impediment to wound healing. Current chronic wound therapies involve costly interventions such as wound debridement to remove microbial biofilms, and antibiotics to prevent the re-establishment of microbial biofilms, to facilitate wound healing. Recently, topical application of lactic acid bacteria onto chronic wounds has been shown to improve wound healing by antagonizing the biofilm production and growth of wound pathogens. While Enterococcus faecalis is a lactic acid bacteria frequently detected in polymicrobial chronic wounds, its role in these wounds is unclear. I was therefore motivated to understand the role of E. faecalis in polymicrobial wounds, as the knowledge will guide evidence-based personalized wound therapies, enhancing treatment effectiveness, while reducing treatment costs. My overarching hypothesis was that E. faecalis antagonizes wound pathogens, reducing the wound pathogen bacterial load, and thereby facilitating wound healing. My working hypothesis, as part of the overarching hypothesis, was that E. faecalis antagonizes known wound pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. To test my working hypothesis, I performed pair-wise experiments of E. faecalis (Strain: OG1RF) with S. aureus (Strain: USA300LAC) and P. aeruginosa (Strain: PAO1), characterizing their interactions with regard to biofilm production, growth, and viability. My original contribution to knowledge is that E. faecalis antagonizes the biofilm production, growth, and viability of S. aureus and P. aeruginosa depending on the species ratios and the environment. Using E. faecalis and S. aureus co-cultures as a model, I revealed that E. faecalis antagonizes S. aureus in a contact-independent manner through culture hyperacidification and constitutive secretion of anti-biofilm and anti-growth effectors. In addition, using a library of isogenic E. faecalis transposon mutants, I identified genes within E. faecalis Type VII secretion system (T7SS) gene locus involved in the contact-dependent antagonism of S. aureus. Moreover, I discovered that E. faecalis mannose phosphotransferase system (MPT), extracellular electron transfer system (EET), and a predicted c-di-AMP biosynthesis system, systems that are involved in energy metabolism, were associated with the contact-dependent antagonism of S. aureus. Since E. faecalis T7SS encodes toxins involved in bacterial competition, but not the MPT and EET systems, I hypothesized that a link between the energy metabolism systems and the T7SS may exist. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays, I found that E. faecalis T7SS gene expression was downregulated in an MPT mutant but not in an EET mutant, relative to wildtype E. faecalis, suggesting that the T7SS gene expression was regulated by the MPT system and the EET system may be antagonizing SA independent of T7SS. Interestingly, through data mining RNA-sequencing (RNA-Seq) datasets of E. faecalis under different nutritional conditions, I discovered that the E. faecalis T7SS gene expression was upregulated in a nutrient-rich environment, independent of the gene expression of the MPT and EET systems. Taken together, based on the experiments performed in this dissertation, I propose that the MPT system may not be directly regulating E. faecalis T7SS gene expression, but indirectly, likely through sensing the energy status of the cell. In addition, I also propose that there may be an EET-mediated mode of SA antagonism independent of EF T7SS. Regarding wounds, E. faecalis-mediated antagonism of S. aureus was discovered to be mild and short-lived in murine cutaneous wounds that have been excised. In vitro, I revealed that E. faecalis-mediated antagonism of S. aureus is perturbed by pH, and an alkaline pH abolishes the antagonism of S. aureus. In murine cutaneous wounds that have been excised, I demonstrated that these wounds spontaneously become alkaline, independent of bacterial infection. Therefore, the loss of S. aureus antagonism by E. faecalis in these murine cutaneous wounds can be explained by the spontaneous wound alkalinization. In humans, wound alkalinization occurs as part of the normal wound healing process. However, given that human chronic wounds have an alkaline pH, and almost all E. faecalis-positive chronic wounds are polymicrobial, it is unclear why co-detection of E. faecalis and S. aureus in such polymicrobial chronic wounds is rare. Given the experimental findings in this dissertation, where I demonstrated the multimodal mechanism of E. faecalis-mediated antagonism of S. aureus, I predict that E. faecalis can antagonize S. aureus in chronic wounds through pH-independent mechanisms. Overall, the work in my dissertation reveals that E. faecalis antagonizes wound pathogens such as S. aureus and P. aeruginosa, proving the working hypothesis to be true. The work in my dissertation establishes the foundational knowledge that permits the overarching hypothesis to be tested further. Evaluating wound healing parameters next in E. faecalis-positive polymicrobial wounds will unveil if the species can be of utility in chronic wound healing, circumventing the need for wound debridement and antibiotics, and thereby reducing the cost of chronic wound care through personalized wound therapy.