Microbial Biofilms: An Evaluation of Ecological Interactions and the Use of Natural Products as Potential Therapeutic Agents

Biofilms are communities of microorganisms associated with surfaces encased in a protective extracellular matrix. These communities often pose clinical and industrial challenges due to their ability to tolerate biocidal treatments and removal strategies. Understanding the ecological interactions that take place during biofilm establishment is a key element for designing future treatment strategies. In this work, I utilized unique methods for studying factors contributing to cooperative antibiotic detoxification in a polymicrobial biofilm model. Subsequently, I tested a novel compound mixture that exhibited promising antibiofilm properties. Escapin is an L-amino acid oxidase that acts on lysine to produce hydrogen peroxide (H2O2), ammonia, and equilibrium mixtures of several organic acids collectively called Escapin intermediate products (EIP). Previous work showed that the combination of synthetic EIP and H2O2 functions synergistically as an antimicrobial toward diverse planktonic bacteria. To test the combination of EIP and H2O2 on bacterial biofilms, Pseudomonas aeruginosa was selected as a model, due to its role as an important opportunistic pathogen. Specifically, I examined concentrations of EIP and H2O2 that inhibited biofilm formation or fostered disruption of established biofilms. High-throughput assays of biofilm formation using microtiter plates and crystal violet staining showed a significant effect from pairing EIP and H2O2, resulting in inhibition of biofilm formation relative to untreated controls or to EIP or H2O2 alone. Similarly, flow cell analysis and confocal laser scanning microscopy revealed that the EIP and H2O2 combination reduced the biomass of established biofilms relative to controls. Area layer analysis of biofilms post-treatment indicated that disruption of biomass occurs down to the substratum. Only nanomolar to micromolar concentrations of EIP and H2O2 were required to impact biofilm formation or disruption, which are significantly lower concentrations than those causing bactericidal effects on planktonic bacteria. Micromolar concentrations of EIP and H2O2 combined enhanced P. aeruginosa swimming motility compared to either EIP or H2O2 alone. Collectively, these results suggest that the combination of EIP and H2O2 may affect biofilms by interfering with bacterial attachment and destabilizing the biofilm matrix. INDEX WORDS: Biofilms, Escherichia coli, Pseudomonas aeruginosa, escapin, hydrogen peroxide, natural products, biofilm inhibition, biofilm dispersal MICROBIAL BIOFILMS: AN EVALUATION OF ECOLOGICAL INTERACTIONS AND THE USE OF NATURAL PRODUCTS AS POTENTIAL THERAPEUTIC AGENTS