Isolation And Characterisation Of Bacteriophages Infecting Borrelia Burgdorferi Sensu Lato

Bacterial species of the Borrelia burgdorferi sensu lato (s.l.) complex are the main causative agents of Lyme disease. The disease is transmitted to humans by infected ticks. There are currently no vaccines available. Lyme disease can be effectively managed with antibiotics, such as Amoxicillin and Doxycycline, if it is diagnosed early. Phages (viruses that infect bacteria) may complement antibiotics to combat disease. Until now, no B. burgdorferi s.l. lytic phages have been identified, and only one temperate phage induced from B. burgdorferi CA-11.2A has been morphologically studied. The aim of this project is to isolate and characterise phages of B. burgdorferi s.l in order to investigate potential future therapeutic exploitation. Thus, ticks were targeted for B. burgdorferi s.l. and phage isolation. Subsequently, induction of prophages from several B. burgdorferi s.l. strains was carried out. The induced phages were characterized according to their morphological types, ‘potential’ virulent activities and genomes.

B. burgdorferi s.l. carriage rate in ticks collected throughout UK was estimated to be 3.2% (6/187), as determined according to a published 16S rRNA PCR. However, using a novel PCR targeting the terminase gene developed in this thesis, the positive rate was 17% (31/187). This indicates that this method was more than five times more sensitive than the 16S rRNA-based PCR. Single colony isolation coupled with the whole genome sequencing demonstrated that the dominant B. burgdorferi s.l. genotypes in UK ticks were B. garinii, B. afzelii and B. burgdorferi B31. Transmission Electron Microscopy (TEM) analysis of B. burgdorferi s.l. cultures treated with Mitomycin C, Norfloxacin, and UV light revealed the presence of putative phages particles in twelve samples. Specifically, five myoviruses were induced from five B. burgdorferi s.l. cultures of B. afzelii ACA-1, B. garinii S18, B. burgdorferi S19, B. burgdorferi S21, and B. garinii S90. Six podoviruses were induced from six B. burgdorferi s.l. cultures of B. afzelii ACA-1, B. garinii 190P91, B. valaisiana NE218, B. burgdorferi UK, B. burgdorferi Vs185p9, and B. garinii S18 respectively. A further myovirus was morphologically identified in the B. burgdorferi B31 culture without any treatment,which represents a spontaneous phage release. In addition, some of the induced phage samples showed potential ‘anti- B. burgdorferi s.l.’ activity according to a fluorescence live/dead assay. Using of bioinformatic analysis such as PHAge Search Tool Enhanced Release (PHASTER) analysis revealed the presence of “incomplete” prophages in the studied strains. However, the presence of phage particles in the induced cultures indicates that these prophages may be complete and functional. Although progress was made to detect lytic B. burgdorferi s.l. phages, further research effort is needed to optimize the processes. B. burgdorferi s.l. phage purification was attempted in this project; the bottleneck lies in the low phage titer and a lack of ‘plaque assay’ detection method. This study opens a new door for further B. burgdorferi s.l. phage researches; and it presents evidence of B. burgdorferi s.l. phage presence and provides practical considerations for future ‘breakthrough’ in detecting B. burgdorferi s.l. phages which could be used in potential therapeutic applications to treat Lyme disease.