Browsing by Author "Aaron, Joshua Alexander"

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    Isolation and characterization of bacteriophages infecting UTI-associated bacteria and evaluation of phage-derived proteins as potential therapeutic agents and diagnostic probes
    (Stellenbosch : Stellenbosch University, 2023-12) Aaron, Joshua Alexander; Dicks, Leon Milner Theodore; Perold, W. J. ; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH 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.