Escherichia coli bacteriophages as candidates for phage therapy and their involvement in membrane vesicle biogenesis
- Publication Type:
- Thesis
- Issue Date:
- 2020
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The emergence of multidrug-resistant (MDR) bacterial pathogens is one of the biggest threats to global health. The WHO has recently prioritized the carbapenem-resistant Gram-negative bacteria, including 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪, as critical pathogens for which new therapeutic treatments are urgently required. However, there have been no new antibiotics approved in the past few decades and there is a trend of rapid development of bacterial resistance against the available antibiotics. Thus, non-conventional approaches are essential to control such MDR bacteria and therapy-utilising bacteriophages currently experiencing revival as an effective strategy. Bacteriophages are bacterial viruses that infect and multiply in bacteria thereby lysing them. Bacteriophages can only be cultivated in a susceptible bacterial host and may contain potential contaminants from bacterial cell. Membrane vesicles (MVs) are spherical nanostructures produced by bacteria and are also observed within bacteriophage preparations. However, there is no direct evidence linking bacteriophage activity and MVs formation. This Thesis established a collection of three new bacteriophages Syd1, Syd6, and Syd8 infecting one of the high risks MDR 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 Sequence Type 131 (ST131) and determined the source of MVs within bacteriophage preparations.
The genomic analysis determined that Syd1, Syd6, and Syd8 each lacked genes encoding for lysogeny, bacterial virulence, or antibiotic resistance. Electron microscopy and comparative genomic analyses revealed that Syd1, Syd6, and Syd8 were both morphologically and genetically distinct from each other and other previously characterized ST131 infecting bacteriophages. These three bacteriophages each possessed a completely distinct host recognition gene from each other and other closely related bacteriophages, indicating that each bacteriophage targeted a different surface receptor. Host range analysis on a panel of 𝘌. 𝘤𝘰𝘭𝘪 strains showed that all three bacteriophages can infect multiple ST131 strains efficiently and had different host range infectivity patterns, suggesting them as appropriate candidates for therapy. Live-cell super-resolution microscopy of 𝘌. 𝘤𝘰𝘭𝘪 infected with bacteriophages T4 and T7 showed that during bacteriophage infection, bacterial cells lyse explosively and MVs were formed from shattered membrane fragments. Electron microscopy revealed the presence of different forms of MVs within phage lysates, consistent with MVs formation through phage mediated bacterial lysis. A bacteriophage purification method utilising ultrafiltration and Triton X-114 phase separation was optimised to remove endotoxins and MVs from bacteriophage preparations. Validation of the method on a panel of 𝘌. 𝘤𝘰𝘭𝘪 bacteriophages including Syd1, Syd6, and Syd8 showed the possibility of up to 5-log reduction in endotoxin and MVs were not detected from electron microscopy of any of these purified bacteriophages.
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