Investigating the potential of bacteriophage induction and phage-derived enzymes as alternative antibacterial approaches
Since their discovery in the 1920s, antibiotics have saved generations of people from succumbing to bacterial infections. Antibiotic usage has resulted in increased antibiotic resistance which has become a problem in various fields and industries. Without effective antibiotics, even simple bacterial infections can be fatal and this has the potential to create a post-antibiotic era where fatalities from currently controlled bacterial infections will become normal. This also extends to the poultry industry where Avian Pathogenic Escherichia coli (APEC) are known to affect the market due to colibacillosis and resulting in poor quality meat and egg products. As bans against antibiotics used in the growth of food animals are established there is a necessity for alternative treatment options. One of the potential solutions is harnessing the power of bacteriophages which are viruses that infect bacteria and proliferate within them. Bacteriophage therapy has been developing and the diverse use of bacteriophages in treatment of bacterial infections ranges from using the whole bacteriophage within its packaged virion, to only parts of the bacteriophage such as proteins/enzymes. Some of the challenges of using bacteriophage cocktails for therapy, extended also to the treatment of APECs, are the myriad environmental conditions that bacteriophages require to proliferate in a host. In this thesis, the use of lysogenic bacteriophages and the heterologous expression of bacteriophage endolysins were investigated as alternative treatments against bacteria. Screening PCRs in combination with induction of prophages within lysogenic bacteria were used to determine whether temperate bacteriophages could be used and genetically manipulated to remain in lysis and therefore be used in therapy or treatment. Potential lysogens were induced using UV-, heat- and mitomycin C inducers. Heterologous expression of bacteriophage proteins was performed through both bacterial expression and yeast expression systems. Bacterial expression was achieved using pETDuet-1 and pET28b expression vectors transformed into BL21 (DE3) competent E. coli cells. The proteins expressed in the pET28b vector contained a 6 x Histidine-tag and were subsequently purified on gravity-flow protein purification and fast protein liquid chromatography (FPLC). Yeast expression was performed using yeast expression vectors pINA1317 and pINA1317-CWP and transformed into Yarrowia lipolytica Po1h competent cells. Proteins visualization was done using 12 % SDS-PAGE and identified with LC-MS/MS. The proteins were tested against bacterial cells, both in the presence and absence of permeabilising agents to determine their efficacy. This included commercial products (Biotronic® Top Line and Virukill®) to determine whether the expressed proteins could improve the products. PCR screening for the presence of prophages resulted in a single strain containing cro and cI genes while about one third of screened strains tested positive for the int gene. This makes it likely that this strain harbours an intact lambda phage that is potentially inducible from lysogeny into the lytic pathway. Bacteriophage proteins were successfully produced using the bacterial expression systems and confirmed through LC-MS/MS results. Although the proteins were purified, they did not elicit an antibacterial effect when applied to the permeabilised bacteria. The yeast expression system was not as successful, although integration into the yeast was confirmed using PCR and sequencing. The treatment assays against bacterial cells were not as significantly effective as expected, whether used in combination with permeabilising agents or not even though initial results showed log differences when compared to the control. The heterologously expressed and identified bacteriophage lambda endolysin from this study can be further tested for efficacy against bacteria. Ideally, this study should be expanded to include endolysins and virolysins from bacteriophages that target other bacteria.