title: Microbial interkingdom interaction in endodontic biofilms and the quest for novel antimicrobial treatments
Creators: name: Albashaireh, Khawlah Zakareyya
Publisher: name: University of Glasgow
Year: 2022

description: Candida albicans and Enterococcus faecalis are two commensal microorganisms in the human microflora. Both are opportunistic pathogens that are frequently coisolated from nosocomial, bloodstream, and root canal infections. Biofilm formation is one of the key features of both microorganisms. Inter-species interaction can induce behavioural changes in microorganisms residing within a polymicrobial biofilm, which will eventually affect the overall pathogenicity of the biofilm. Interkingdom interactions are complex and can exacerbate infection. Moreover, these complex communities can result in enhanced tolerance to antimicrobial agents. With the rise of antimicrobial resistance worldwide, coupled with tolerant biofilm communities, then there is a great demand for finding alternative treatment modalities that can manage complex infection. Endodontic infection is a polymicrobial biofilm disease, with C. albicans and E. faecalis frequently coisolated from clinical samples of this infection. It is believed that E. faecalis exert an antagonistic impact on C. albicans by inhibiting its hyphal formation, which results in preserving commensalism within normal microbiome niches. However, their common coisolation in different disease entities indicates a complex interaction beyond antagonism. One of the aims of the experimental work reported in this thesis was to investigate this interkingdom interaction and employ different tools to unravel key behavioural changes that would help in understanding cross-kingdom communications. Interkingdom interaction was first investigated phenotypically by assessing metabolic activity using XTT assay, then by assessing biofilm biomass using a crystal violet assay, and by microscopic analysis wherein dual-species biofilm was visualised. Molecular analysis using qPCR was employed to quantify cell number and assess growth of microorganisms in dual-species biofilm. Gene expression analysis was then performed using real-time quantitative polymerase chain reation (RT-qPCR) to assess key virulence gene expression in C. albicans and E. faecalis at different biofilm formation stages. Next, whole C. albicans RNAsequencing was implemented to investigate differential gene expression of the microorganism in response to E. faecalis presence at 8 hours and 24 hours. Finally, novel therapeutic agents, including nanodiamonds and small molecules screened from FDA approved drug library, were employed to target virulence mechanisms and assess microorganism behaviour within dual-species biofilm compared to single species biofilm. E. faecalis was shown to induce phenotypic changes on C. albicans. It inhibited hyphal morphogenesis, growth and reduced its biofilm biomass. Phenotypic results showed antagonism against C. albicans but E. faecalis remained unchanged. At molecular levels, E. faecalis downregulated key genes involved in C. albicans virulence, whilst C. albicans upregulated genes involved in E. faecalis adhesion and biofilm formation. In addition, the transcriptome of C. albicans was significantly altered in the presence of E. faecalis. Stress responses, oxidoreductase activity, amino acid biosynthesis and the fungal biofilm matrix were the most significantly upregulated pathways in C. albicans. Glucose sensation and transportation, glycerol biosynthesis process and arginine catabolic pathway were the most significantly downregulated pathways in C. albicans. Finally, the coexistence of both microorganisms in dual-species biofilm altered their response to novel therapeutic agents compared to each species alone by increasing each microorganism’s tolerance to different types of treatments. This research can be seen as a starting point to investigate further mechanisms relevant to C. albicans and E. faecalis complex interkingdom relationship using advanced tools. In addition, this research provides a significant input to the current knowledge that repurposing drugs and nanomaterials can be suitable candidates to be used as broad antimicrobial agents.