Candida albicans and Enterococcus faecalis interkingdom interaction: implications for management of endodontic infections

Alshanta, Om Alkhir Mohamad (2022) Candida albicans and Enterococcus faecalis interkingdom interaction: implications for management of endodontic infections. PhD thesis, University of Glasgow.

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Endodontic disease is inflammation or infection of the pulp tissue lying within the root canal space of teeth and the associated periapical tissue, also known as periapical periodontitis. It is estimated that around 52% of adults worldwide have at least one tooth with periapical periodontitis. Root canal infections are usually debilitating, associated with pain, discomfort and decreased quality of life. The role of microorganisms in the pathogenesis of periapical periodontitis is well established.

Root canal infections are characterised by biofilms, i.e. polymicrobial infections encased in polymers that are isolated from infection sites. Candida albicans and Enterococcus faecalis are common endodontic pathogens that have been linked with persistent endodontic infections. Deeper understanding of their response to endodontic procedures and how they interact with each other may enhance our understanding of their role and lead to better treatment outcomes.

Chapter 1: reviews the literature in relation to endodontic infections and their related microbiology, basics of endodontic treatment, C. albicans and E. faecalis as endodontic pathogens and C. albicans and E. faecalis in mixed biofilms.

Chapter 2: is presented in the traditional chapter format. It aimed at assessing a panel of C. albicans oral clinical isolates for their ability to form biofilms using the crystal violet assay (biomass). Following the selection of representative low and high biofilm forming isolates, the response of different C. albicans phenotypes to NaOCl was assessed using XTT (metabolism), crystal violet and microscopic imaging. C. albicans showed an ability to tolerate NaOCl treatment and was able to regrow when a nutrient source is provided. In addition, C. albicans heterogeneity influenced the response to NaOCl treatment, where low biofilm forming isolates showed increased tolerance compared with high biofilm forming isolates.

Chapter 3: is presented as a published journal article. It further analysed the response of single species of C. albicans biofilms to NaOCl. It also assessed the effect of sequential treatment with sodium hypochlorite followed by EDTA on C. albicans biofilms viability using XXT and biofilm biomass using crystal violet assays. In this analysis, the immediate post treatment effect as well as the regrowth potential over 72h of re-incubation with fresh media were investigated. Treatment with NaOCl alone resulted in a significant regrowth of the treated population while regrowth was significantly inhibited with sequential treatment.

Chapter 4: is presented as a published journal article. In this chapter the complexity of the used model was increased by incorporating E. faecalis into the subsequent analysis. C. albicans and E. faecalis mono- and dual-species biofilms were assessed against sequential (NaOCl followed by EDTA) or continuous chelation protocols (NaOCl combined with HEDP) using qPCR and microscopic examination. Sequential treatment with NaOCl and EDTA were more effective in inhibiting C. albicans and E. faecalis regrowth compared with continuous chelation. In terms of the difference between mono- and dual-species, C. albicans was more susceptible to endodontic irrigants when co-cultured with E. faecalis, while E. faecalis remained unaffected. The regrowth of C. albicans was significantly inhibited when cocultured with E. faecalis.

Chapter 5: is presented as a published journal article. Firstly, the effect of E. faecalis on C. albicans biomarkers was assessed using crystal violet, microscopy and real time PCR. Afterwards, RNA seq was employed to investigate the molecular basis of the interaction between C. albicans and E. faecalis. The expression of EntV across twelve E. faecalis strains was also measured and correlated with the degree of C. albicans biofilm inhibition. Finally, the effect of pH modified and ultrafiltered E. faecalis supernatant on C. albicans hyphal morphogenesis and biofilm formation was assessed. The results show that E. faecalis inhibited C. albicans growth, hyphal morphogenesis and biofilm formation. The transcriptional analysis showed rapid and significant changes in the C. albicans transcriptome characterized by upregulation in amino acid biosynthesis and metabolism genes. This guided further analysis on EntV and pH dependent mechanisms where C. albicans biofilm inhibition were shown to be related to an E. faecalis induced environmental pH drop.

In conclusion, this thesis highlights the importance of considering the tolerance potential of the treated microorganisms and the role it may have in the aetiology of disease persistence. It also contributes to our knowledge of the interaction between microorganisms in polymicrobial infections and how the ultimate outcome of this interaction may impact disease progression and prognosis.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: R Medicine > R Medicine (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Funder's Name: Biotechnology and Biological Sciences Research Council (BBSRC)
Supervisor's Name: Ramage, Professor Gordon
Date of Award: 2022
Depositing User: Theses Team
Unique ID: glathesis:2022-83019
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 07 Jul 2022 11:15
Last Modified: 07 Jul 2022 11:22
Thesis DOI: 10.5525/gla.thesis.83019
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