Development of culture-free methods for selecting host-specific bacteriophages and the characterisation of unusual bacteriophages

Bacteriophages show great potential in many applications. Their abundance exceeds the bacterial population by ten-fold. Studying phages and their characteristics requires culturing of the bacterial host, allowing the phage to propagate by lysing cells forming plaques on a bacterial lawn. However, the number of isolated host-specific phages does not reflect the wide variety of phages in the environment.This study aimed to design a culture-independent method of selecting host-specific bacteriophages from complex mixtures of bacteriophages. Firstly, lipopolysaccharides (LPS) were used to isolate host-specific phages. Pseudomonas LPS was immobilised onto glass beads to work as an affinity sorbent that selects for only Pseudomonas aeruginosa phages. It was observed that phages non-specifically bound to the beads and remained bound even after multiple washing steps. Therefore, another method was devised involving the use of outer membrane vesicles (OMVs) to select for the DNA of host-specific phages. Analysis of the OMV-based method revealed successful selection of a P. aeruginosa phage from a mixture containing different known phages, as assessed by PCR. Further optimisation is required to apply this method to analyse environmental samples containing a complex mixture of unknown phages.During this study, novel bacteriophages infecting P. aeruginosa and E. coli strains were isolated from a horse manure sample by the traditional method of phage isolation (plaque assay). Genomic and biological analysis of these isolated phages revealed the presence of unusual features. One of the isolated phages was, phage MIJ3. This is a jumbo phage which has genome size of 288,170bp; it is capable of infecting most of the P. aeruginosa Liverpool Epidemic Strains along with other multi-drug resistant strains. Bioinformatic analysis of the phage genome revealed that the phage encodes several “host-like” proteins, some of which have not been reported in phages before.Another isolated phage from the horse sample was phage MiEL, which has a genome size of 176,867 bp. Phage MiEL infects a variety of E. coli clinical strains isolated from urinary tract infection patients. It is also able to enter a pseudolysogenic state during poor bacterial growth conditions. Thus, novel phages infecting clinical strains can be isolated from a variety of sources.