Investigation of bacteriophages as potential sources of oral antimicrobials

Bacteriophages are natural viruses that attack bacteria and are abundant in all environments, including air, water, and soil- following and co-existing with their hosts. Unlike antibiotics, bacteriophages are specific to their bacterial hosts without affecting other microflora. The use of bacteriophages and phage endolysin based therapies to kill pathogens without harming the majority of harmless bacteria has received growing attention during the past decade, especially in the era where bacterial resistance to antibiotic treatment is increasing. The aims of this study were to characterise a putative prophage (phiFNP1) residing in the genome of the periodontal pathogens Fusobacterium nucleatum polymorphum ATCC 10953, investigating the possibility of prophage induction, examining its presence in clinical plaque samples taken from patients with chronic periodontitis cases, and purify its putative lysis module genes to examine their potential antimicrobial activity. In addition, attempts will be made to catalogue phages that are presents in samples from the oral cavity of patients with chronic periodontitis and in wastewater, followed by isolation of lytic phages targeting endodontic and oral associated pathogens, characterisation of the isolated phages, evaluation of the efficiency of phage towards biofilm elimination, and established an animal model to study phage-bacterial interaction in order to develop them for treatments. The results revealed that phiFNP1 prophage are common in subgingival plaque of patients suffering from chronic periodontal disease but attempts to induce phiFNP1 using Mitomycin C were inconclusive, indicating that it might be defective. Bioinformatics revealed that the genome of phiFNP1 contained potential lysin genes, with one being cloned and purified successfully in soluble form, though its activity as antibacterial was not confirmed, and needs to be further explored as phage lytic enzymes against Fusobacterium have not been identified yet. Various phage like particles with different morphology were visualised by direct electron microscopy from oral and wastewater samples, which reflect the richness and diversity of bacteriophage within those samples. Several phages were isolated namely phiSHEF 2,3,4,5,6,7 which belong to the Siphoviridae family which are specific to Enterococcus faecalis and the full chromosome sequence comparisons for three of the isolated phages (phiSHEF 2,4 and 5) revealed that they are lytic in nature reflected by absence of genes associated with lysogenic cycle, therefore place them as suitable candidate for therapy. They exhibit genomic variations corresponded to their difference in phage-host range especially in the tail region. In addition, phiSHEF 2 was capable of complete biofilm eradication on abiotic surfaces and significantly reduced biofilms formed on the surfaces of tooth root slices. Most importantly phiSHEF 2 recovered a Zebrafish larvae from the deadly infection caused by an oral clinical isolate of E. faecalis, indicating the establishment of a successful animal model for testing the efficiency of phage therapy towards E. faecalis infection. Finally, Exopolysaccharide mutants of E. faecalis were not infected by three of our phiSHEF phages tested, indicating that E. faecalis exopolysaccharide capsule and proper bacterial membrane integrity play an important role during the initial stages of phage infection. In conclusion, bacteriophages exhibit an apparent superiority to fight hard to eradicate pathogens such as those associated with recalcitrant endodontic infections thus "bacteriophage based therapy" could represent an innovative alternative to control and eliminate oral infections caused by biofilm forming and antibiotic resistant pathogens.