title: Assessing the role of Candida albicans in commercially relevant oral biofilm models
Creators: name: Young, Tracy Lynn
Publisher: name: University of Glasgow
Year: 2021
Subjects: subject: Oral biofilms, antimicrobial resistance, standardisation, commercialisation, polymicrobial biofilms, Candida albicans.

description: Most organisms in the natural environment and those implicated in infections, exist within a biofilm. These are typically complex polymicrobial entities and are phenotypically distinct from their antimicrobial sensitive planktonic equivalents. The rise in antimicrobial resistance, including tolerance to drugs of last resort, highlights the seriousness of this issue, with biofilms exhibiting an increased tolerance to treatment. Standards for testing of novel agents have thus far failed to reasonably account for biofilms, with the key focus on minimum inhibitory testing of planktonic cells. Despite the growth of commercial biofilm testing, it often fails to account for the polymicrobial nature of biofilms. Similarly, the laborious and time-consuming nature of biofilm development equally hinders testing advancement. Finally, the exclusion and disregarding of fungi from these systems have limited the real-world challenge, leading to treatment failures of antimicrobials and actives in clinical and consumer use. Biofilms in the oral cavity are associated with the onset of diseases such as caries, periodontitis and denture stomatitis. Oral diseases affect up to 90% of the population and are among the most preventable diseases afflicting millions worldwide. Organisms colonise the oral cavity from birth and plaque forms on abiotic and biotic surfaces quickly after cleaning. Therefore, a manageable amount of plaque with diverse composition is required to sustain a healthy microbiome of the mouth. Identification of more than 700 colonising bacteria and 100 fungal species has highlighted the necessity of biofilms which are beneficial to the host. Candida albicans’ role in health and disease within the oral cavity is still to be determined, though it has been described as a possible ‘keystone commensal’. To this end, the aim of this thesis incorporated these aspects: 1) to assess the impact of C. albicans inclusion within two oral biofilm models and to assess the commercial viability of these within a novel biofilm development methodology; and 2) commercially driven studies were undertaken to better understand the impact of denture cleaning habits on a soft tissue associated biofilm and the use of the novel methodology within this, and the use of probiotics as a means of microbial intervention in a novel periodontitis prevention strategy. Biofilm assays often rely on adapted and sometimes crude measurements to provide results, and so similar assays have been used throughout which can be amalgamated allowing for appropriate conclusions to be drawn. Initially, two established biofilm models were grown and matured ± C. albicans to assess changes to markers upon inclusion. Metabolic activity was assessed using resazurin dye and biomass quantified using crystal violet stain. Cell burden and species-specific changes were determined using quantitative PCR and visual representation was evaluated using scanning electron microscopy. Measures of virulence such as pH changes, reactive oxygen species generation, lactate and protease accumulation were measured. Additionally, changes to the susceptibility of the biofilms to a range of actives following the inclusion of C. albicans were also assessed to determine the fungi’s impact on the biofilms. Moving on from this, a novel methodology of biofilm formation utilised similar techniques to those described above to evaluate an inoculum biofilm grown in bulk and stored for three months. The commercial utilisation was assessed using chlorhexidine to establish the response of the bulk inoculum to treatment in comparison to a freshly grown biofilm. Commercial intervention studies were also carried using the described methods alongside Miles and Misra colony counting and live/dead qPCR using propidium monoazide to distinguish classical microbiological techniques to newer more sensitive molecular methods. Confocal microscopy was also used within this study using SYTO 9 and propidium iodide to visually differentiate between live and dead cells on the surface of the denture material. This study also incorporated a repeat using the novel inoculum biofilm method to further confirm its usability and validate this against a prior study. Finally, a novel treatment using Lactobacillus probiotics encompassed all the methods described herein to determine its feasibility in preventative action against periodontitis. Results from this work showed that C. albicans inclusion was of commensal benefit to the biofilms, increasing pH to closer to neutral and reducing ROS. Additionally, the biofilms were found to be more robust and repeatable in a 96-well microtiter plate format with no change to susceptibility and so are recommended to be used as a real-world challenge in the testing of novel agents. It was also confirmed that these biofilms can be created as an inoculum for quicker biofilm development, with hard tissue biofilms able to be stored for 3 months and soft tissue biofilms stored for 1 month without detrimental effects. Furthermore, these biofilms respond similarly under treatment. In the transition into commercial studies, the denture cleaning study identified that daily brushing following a denture cleanser was most beneficial although successful biofilm control was also shown in brushing only treatment arms. C. albicans was shown to thrive while all treatments exhibited non-selective killing of both health associated and pathogenic organisms with significant regrowth after treatment displayed. A repeat using the inoculum biofilm method was also employed and demonstrated similar outcomes completed within a quicker timeframe and at a cheaper cost than the original study, therefore confirming its commercial use. In the final study, probiotic inclusion into a fresh biofilm offered a novel intervention strategy with brushing that altered the composition and decreased overall bioburden, while decreasing the pH due to an increase in lactate. C. albicans was shown to survive and increases were positively correlated with Lactobacillus. To conclude, this thesis has demonstrated the importance of C. albicans within complex oral biofilm models and in doing so will further progress the testing of novel actives in the prevention of oral diseases. An inoculum biofilm method has also been developed to allow biofilm testing to be more readily accessible and a less laborious process. This could further expand the capacity for service laboratories to complete targeted testing. Studies underpinning the importance of mechanical removal of plaque and intervention on a variety of abiotic and biotic surfaces were undertaken and highlighted the need for clear guidance in the cleaning of dentures and an emphasis on the importance of interactions within a complex interkingdom biofilm in the progression of disease. In-vitro studies are instrumental in progressing biofilm research and knowledge, and this body of work serves as a small continuation of that progression.