Disruption of Fusobacterium nucleatum biofilm using innovatitve anti-microbial compounds to prevent and treat periodontal disease

Abstract

Background: Periodontal disease, colloquially known as ‘gum disease’, contributes heavily to the global burden of chronic diseases known to affect between 35% and 50% of the world population. It is defined as a progressive loss of the supporting tissues of the teeth including the gingiva, alveolar bone and periodontal ligaments leading to tooth mobility or tooth loss and ultimately loss of function. It is a complex pathophysiology: characterised by a host inflammatory response against microorganisms in the sub-gingiva and their by-products. One of the key microorganisms that is responsible for the sequential maturation of subgingival plaque is Fusobacterium nucleatum (F. nucleatum). F. nucleatum plays a significant role as a bridging organism between the early colonisers including Gram-positive streptococci sp. and the late colonizers. It is the late colonizers that are disease causing and tissue destructing periodontopathogens including Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. Furthermore, F. nucleatum has been attracting wide attention due to, not only its pivotal role in disease progression, but also its role in other systemic disease such as adverse pregnancy outcomes, arthritis and colorectal cancer. Different subspecies of F. nucleatum have been located around the human body and have been contributing to different diseases processes. This is thought to stem from F. nucleatum subspecies having great genetic diversity. Therefore, this project looked at characterising the biofilm forming ability of four different subspecies, F. nucleatum subsp. nucleatum (FNN), polymorphum (FNP), fusiforme (FNF) and animalis (FNA). Current treatment of periodontitis includes mechanical debridement by a specialist and in severe periodontitis, antibiotics may be indicated. However, antimicrobial resistance is a serious problem due to over prescription of antibiotics and bacteria in biofilms exhibiting up to 1000- fold increase in antibiotic resistance compared to planktonic bacteria. Therefore, it is crucial to investigate other methods to control oral biofilms, such as the use of novel antimicrobial agents to reduce periodontal disease progression. In recent years, novel antimicrobial and antibiofilm compounds have emerged to combat this issue. These include D-amino acids (DAAs), silver nanoparticles (AgNPs) and gold (AuNPs) nanoparticles. DAAs have been shown to have biofilm inhibiting and biofilm dispersion effects. A mixture of D-Leucine D-Methionine D-Tyrosine D-Tryptophan has been shown to reduce biofilm formation and initiate biofilm breakdown. Nanoparticles including silver (AgNPs) and gold (AuNPs) have also been of growing interest due to their antimicrobial activity showing both bactericidal and bacteriostatic effects on Gram-positive and Gram-negative bacteria including multi-drug resistant strains present in the oral environment. Successfully growing and characterising F. nucleatum biofilms is the first step in investigating the effectiveness of novel antimicrobial/antibiofilm agents. Our further understanding will contribute to the development of clinical treatments against F. nucleatum mediated pathologies including periodontitis. Aims: The aim of this project is to grow and characterise biofilms produced by F. nucleatum subsp. nucleatum (FNN), polymorphum (FNP), fusiforme (FNF) and animalis (FNA) to investigate if there are sub-species specific differences in biofilm formation. The second aim is to characterise and investigate the efficacy of novel antimicrobial/antibiofilm agents including DAAs and AgNPs and AuNPs. Methods: Single-species Fusobacterium nucleatum (F. nucleatum) were grown under anaerobic conditions and characterised through growth curves, crystal violet staining, viable cell counts, xCELLigence® platform, SEM and confocal imaging and Imaris software analysis. When the protocol was established, biofilms were treated with novel antibiofilm agents including D-amino acids (DAAs), and antibacterial agents: silver (AgNPs) and gold nanoparticles (AuNPs) to investigate their biofilm inhibiting abilities and biofilm dispersing abilities. Results: There were sub-species specific differences in F. nucleatum growth where FNP and FNA showed increase in sensitivity to oxygen. FNF and FNN consistently showed better growth with minimal sensitivity to oxygen. SEM imaging showed differences in cellular morphology amongst the four subspecies. Furthermore, protocol optimisation to improve F. nucleatum biofilm growth was successful for crystal violet staining and confocal imaging. Unfortunately, F. nucleatum biofilm growth was unable to be successfully detected by the xCELLigence® platform. DAAs reduced the growth of FNP, FNF and FNN with minimal differences between the control and DAA treated growth curves for FNA. FNF and FNN treated with DAAs showed similar results when added at the start of the experiment where over the first 6 hours produced a 2-fold increase in doubling time (log phase) and stationary phase was reached earlier near 4 hours in the DAA treated groups. DAA treated FNP, FNF and FNN groups showed a reduced capacity to grow, reflected in a reduction of the maximum OD600nm reached. On the contrary, minimal differences were observed between the control and DAA treated growth curves for FNA, which was consistently observed throughout the experiment repeated in triplicate. SEM imaging further showed changes in biofilm architecture after DAA treatment where FNP and FNF showed absence of EPS compared to the control groups and FNA produced elongated cells. All four subspecies including FNN showed to have almost vesicular looking irregularities on the surface of the bacteria after DAA treatment. Qualitative results from confocal imaging further confirmed a decrease in bacterial cell numbers after DAA treatment for all 4 subspecies. Interestingly, contradictory to results seen through assessing growth in the spectrophotometer, DAA treated FNP showed minimal changes in biofilm volume, whereas FNF and FNN biofilms showed almost 4-fold decrease in mean volume and FNA showed almost 3-fold decrease in mean volume after quantitative analysis using Imaris Software. Furthermore, while decrease in bacterial cell numbers was observed, no significant difference in viability between DAA treated and control groups were observed through viable cell counts and Imaris analysis of viability (%). Similar results were observed for DAA treatment on established F. nucleatum biofilm where a decrease in biofilm volume due to biofilm dispersal, but no significant difference in viability was observed. Results from these experiments indicate that DAAs are a promising biofilm breaker with minimal effects of viability of F. nucleatum cells after treatment. AgNPs and AuNPs showed a concentration dependant effect where the concentration of nanoparticles was inversely proportional to viability. Confocal imaging showed that all four subspecies of F. nucleatum had a reduction in bacterial cell number after being treated with 60g/ml AgNPs. Due to the Imaris analysis tool’s inability to differentiate between F. nucleatum and AgNPs, quantification analysis of the biofilms was unable to be performed. Conclusion: This project successfully produced a protocol to successfully grow all four subspecies of F. nucleatum in a spectrophotometer. DAAs showed promising results as a biofilm breaker with minimal effects of viability of F. nucleatum cells after treatment. On the other hand, AgNPs and AuNPs both exhibited potential bactericidal effects. Therefore, future studies may conduct investigations on effects of combinations of these novel antimicrobials and antibiofilm compounds on F. nucleatum biofilm. Clinically, incorporating these novel antimicrobials and antibiofilm compounds may have application in periodontitis treatment. This may overcome the issue of increased antibiotic resistance in the community and become a useful adjunct to mechanical removal of subgingival biofilm by the specialist, reducing the number of appointments and cost needed by the patient for periodontitis treatment in the future.