Isolation and characterization of bacteriophages infecting UTI-associated bacteria and evaluation of phage-derived proteins as potential therapeutic agents and diagnostic probes

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dc.contributor.advisorDicks, Leon Milner Theodoreen_ZA
dc.contributor.advisorPerold, W. J. en_ZA
dc.contributor.authorAaron, Joshua Alexanderen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Microbiology.en_ZA
dc.date.accessioned2023-08-28T13:38:46Z
dc.date.accessioned2024-01-08T12:21:29Z
dc.date.available2023-08-28T13:38:46Z
dc.date.available2024-01-08T12:21:29Z
dc.date.issued2023-12
dc.descriptionThesis (MSc)--Stellenbosch University, 2019. en_ZA
dc.description.abstractENGLISH ABSTRACT: Healthcare faces two major problems today, the increased emergence of antimicrobial resistance and the need for rapid diagnostic testing of pathogenic bacteria. The over and or improper prescription of antibiotics has further exacerbated this, also leading to major disruption of the gut microbiome in individuals overcoming diseases. Bacteriophages (phages) can provide the solutions to these current challenges as they solely infect their specific host bacteria. Utilizing whole phages or phage proteins in therapeutics and diagnostics has increased rapidly over the years, offering unique strategies of addressing today’s problems. In this study bacteriophages that specifically target uropathogens were isolated from wastewater treatment plants. Several of these phages were characterized on a genomic and physiological basis. Focus was drawn to a new species of Proteus mirabilis phage belonging to the Novosibovirus genus. The newly identified Proteus_virus_309 was found to drive the emergence of phage insensitive mutants (PIMs). The wild type, phage susceptible, P. mirabilis and phage insensitive mutants were sequenced using the Oxford nanopore sequencing platform which assisted in identifying multiple small nucleotide polymorphisms (SNPs) that may be responsible for the observed phage resistance. Whole genome sequencing of several phages provided an ample source for identifying genes with therapeutic and diagnostic potential tail associated genes from Proteus_virus_309 and Proteus_porphage_301 were selected for protein production and further analysis. Concurrently, previously characterized receptor binding proteins (RBPs) genes from Salmonella bacteriophage vB_SenM-S16 and Staphylococcus aureus phage φ11 were also selected and synthesized. All the selected phage genes were successfully cloned, expressed and the proteins fused with a green fluorescent protein (GFP) were his-tag purified. It was confirmed a putative tail spike protein, TSL309 (ORF57), from Proteus_virus_309 possess depolymerase like activity, evaluated using spot tests, Transmission Electron Microscopy (TEM) and sodium dodecyl sulfate (SDS)-gel analysis of crude capsule extracts. The depolymerase activity observed in TSL309 hampers the biofilm formation and increases de-fouling of Proteus cells on polyvinyl chloride tubing. One aspect of this study shows the potential use of phage derived depolymerases as aiding therapeutics, prompting the requirement for further research into the synergism of phage depolymerases with antibiotics and immune responses in overcoming bacterial infections. The second aspect of the study screened Salmonella and Staphylococcus phage RBPs, Gp38 and Gp45, fused with GFP for their binding capacity toward various bacterial isolates. Bacteria-RBP interactions were evaluated with confocal super-resolution fluorescent microscopy and the fluorescently labelled RBPs. Phage based probes GFP-gp38 and GFP-45 were found to bind to their respective bacterial species, with gp45 binding to a range across Staphylococcus and Enterococcus species. This study highlights a pipeline of identifying, producing, and screening potential phage probes with possible incorporation into a rapid Point of Care (PoC) biosensor device with the aim of providing accurate detection of pathogenic bacteria.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Gesondheidsorg het vandag twee groot probleme, die verhoogde voorkoms van antimikrobiese weerstand en die behoefte aan vinnige diagnostiese toetsing vir patogeniese bakterieë. Die oor- en/of onbehoorlike voorskrif van antibiotika het dit verder vererger, wat ook gelei het tot groot ontwrigting van die dermmikrobioom by individue wat aan siektes lei. Bakteriofage (fage) kan die oplossings vir hierdie huidige uitdagings verskaf aangesien hulle slegs hul spesifieke gasheerbakterieë beinvloed. Die gebruik van die heel faag of faagproteïene in terapeutika en diagnostiek het oor die jare vinnig toegeneem en bied unieke strategieë om vandag se probleme aan te spreek. In hierdie studie is bakteriofage uit afvalwaterbehandelingsaanlegte geïsoleer wat spesifiek uropatogene teiken. Verskeie van hierdie fage is op 'n genomiese en fisiologiese basis gekarakteriseer. Die fokus is gevestig op 'n nuwe spesie Proteus mirabilis-faag wat aan die Novosibovirus-genus behoort. Daar is gevind dat die nuut geïdentifiseerde Proteus_virus_309 die opkoms van fage-sensitiewe mutante (PIM's) dryf. Die wildetipe, fage vatbare, P. mirabilis en faag- onsensitiewe mutante is gevolg deur gebruik te maak van die Oxford Nanopore volgordebepaling platform wat gehelp het met die identifisering van veelvuldige klein nukleotied polimorfismes (SNPs) wat verantwoordelik kan wees vir die waargenome faag weerstand. Die totale genoomvolgordebepaling van verskeie fae het 'n ruim bron verskaf om gene met terapeutiese en diagnostiese potensiaal te identifiseer. Gene van Proteus_virus_309 en Proteus_prophage_301 is geïdentifiseer en in silico-analise het ooreenkomste met hidrolase en faag-geassosieerde depolimerases getoon. Tweedens is gene van bekende reseptorbindende proteïene (RBPs) wat aan Salmonella- en Staphylococcus-fae behoort, gesintetiseer. Hierdie faag gene is suksesvol gekloon, uitgedruk en die proteïene gesuiwer met groen fluoresserende proteïen (GFP) as 'n samesmeltingsvennoot. TSL309, Proteus_virus_309, het depolimerase-aktiwiteit teen sy gasheer- Proteus-stam getoon wat met behulp van koltoetse, transmissie-elektronmikroskopie (TEM) en SDS-gel analise van kapsule-afbraak produkte geëvalueer. Die depolimerase-aktiwiteit wat in TSL309 waargeneem is, belemmer die biofilmvorming en verhoog die ontkoppeling van Proteus- selle op polivinielchloriedbuise. Die RBP's, Gp38 en Gp45, is gekies vir hul bindingskapasiteit teenoor verskeie bakteriese isolate. Bakterie-RBP-interaksies is geëvalueer deur gebruik te maak van konfokale super-resolusie fluoresserende mikroskopie. Hierdie studie toon die gemak van die vervaardiging van faagverwante peilers vir die moontlike inkorporering in 'n vinnige punt-van-sorg (PoC) biosensor toestel met die doel om akkurate opsporing van patogeniese bakterieë te verskaf.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentviii, 189 pages : illustrations (some color)en_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/128821
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshBacteriophagesen_ZA
dc.subject.lcshBacteriophages -- Diagnostic useen_ZA
dc.subject.lcshBacteriophages -- Therapeutic useen_ZA
dc.subject.lcshBacteriophages -- Geneticsen_ZA
dc.subject.lcshPathogenic bacteriaen_ZA
dc.subject.nameUCTDen_ZA
dc.titleIsolation and characterization of bacteriophages infecting UTI-associated bacteria and evaluation of phage-derived proteins as potential therapeutic agents and diagnostic probesen_ZA
dc.typeThesisen_ZA
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