Masters Degrees (Microbiology)
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- ItemInvestigating the effects of the plaC accessory protein on disulphide bond formation in plantaricin 423 heterologously expressed in escherichia coli(Stellenbosch : Stellenbosch University, 2024-03) Joos, Carla Grete; Dicks, Leon Milner Theodore; Van Staden, Anton du Preez ; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The discovery of antibiotics has revolutionised society. Antibiotics have been used to treat countless diseases and improved the quality of life for many. Unfortunately, the overuse of antibiotics has led to bacteria developing mechanisms to outmanoeuvre the inhibitory effects of these pharmaceuticals. The emergence of antibiotic-resistant bacteria poses a major threat to human health, industries and the economy. Thus, the search for alternatives to antibiotics has become imperative. Lactic acid bacteria (LAB) have a long-standing association with food preservation, pharmaceutical production and use as probiotics. Amongst the myriad of metabolites produced by LAB, ribosomally synthesised peptides referred to as bacteriocins, have gained interest for their antimicrobial properties. LAB secrete bacteriocins to help outcompete other bacteria inhabiting their surrounding environment. This study focused on class IIa bacteriocins, in particular plantaricin 423 isolated from Lactobacillus plantarum 423. Class IIa bacteriocins are pediocin-like and well-known for their antilisterial properties. The production of class IIa bacteriocins has been greatly improved, particularly by their heterologous expression as fusion proteins. In this study, we further improved the heterologous expression of plantaricin 423 through the co-expression with its accessory protein, PlaC, identified in the plantaricin 423-encoding operon. A thioredoxin-fold and CXXC catalytic motif were identified in the PlaC accessory protein, which are characteristics associated with the thioredoxin superfamily. However, thioredoxin-like accessory proteins are uncommon amongst class IIa bacteriocins. An insulin reduction assay based on the dithiol-disulphide oxidoreductase activities of the thioredoxin system was performed and further confirmed that the PlaC accessory protein is a thioredoxin superfamily member. In addition, a peptidase (PEP) domain was identified in the native secretion machinery of plantaricin 423. The PEP domain shares characteristics with C39 cysteine peptidases. The PEP domain was isolated and its ability to proteolytically remove the leader peptide of precursor plantaricin 423 was evaluated. It was hypothesised that the PlaC accessory protein improved the heterologous expression of plantaricin 423 through promoting disulphide bond formation. Previous studies obtained similar results with another class IIa bacteriocin, pediocin PA-1, with its respective accessory protein, PedC. The formation of disulphide bonds in the periplasmic space of Escherichia coli involves two important enzymes, DsbA and DsbC, which function as an oxidase and isomerase, respectively. Unfortunately, disulphide bond formation is poorly understood in Gram-positive bacteria. To improve our current knowledge of disulphide bond formation in secreted proteins from Gram-positive bacteria, the PlaC accessory protein was expressed in E. coli strains with knockout mutations for DsbA or DsbC. In addition, PlaC was heterologously expressed with two plant antimicrobial peptides, VvAMP1 and VvScorpio, that both require four nonconsecutive disulphide bonds. This is the first study that provides experimental evidence confirming that the thioredoxin-like accessory protein, PlaC, identified in the plantaricin 423-encoding operon serves as a DsbC isomerase homologue during the maturation and secretion of plantaricin 423 by promoting correct disulphide bond formation. This is also the first report for a DsbC-like homologue in Firmicutes.
- ItemDiamond based electrodes for water quality applications: disinfection, micropollutant removal and the suppression of biofilm proliferation.(Stellenbosch : Stellenbosch University, 2024-03) Louw, Carli; Wolfaardt, Gideon M. ; Botes, Marelize; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Rivers in most low and middle income countries face a growing burden of pollution in the form of pathogens and persistent micropollutants caused by waste discharge from informal settlements and poorly maintained wastewater treatment plants, operating above capacity. Farming communities are often reliant on the these rivers for irrigation. This study investigated the potential application of diamond based electro-oxidation in decentralized water treatment systems for disinfection and micropollutant abatement. The diamond-electrode based electrochemical in-situ system (DiaDis) forms ozone and hydroxyl radicals via electro-generation. It was hypothesised that the system would possess strong disinfection properties, but that micropollutant abatement would be dependent on the compounds and organic matter present. The disinfection study included the treatment and prevention of biofilms by monitoring the effects on biofilm metabolic activity during DiaDis treatment. A pure culture of Pseudomonas fluorescens sp. was used for the single culture biofilm disinfection study, and for the mixed species biofilm study the culture was sampled from a polluted river. The disinfection capabilities of the DiaDis was found to be comparable or slightly superior to 1:10 dilution sodium hypochlorite in this study for the single culture biofilms, however, the mixed species biofilms were more effectively treated with sodium hypochlorite. Biofilm-forming species have varying resistance to disinfectants and the results suggest the presence of synergistic adaptation. For the single and mixed species biofilms the treatments studied proved more effective when used preventatively than to treat mature biofilms. Removal efficiency of micropollutants by the mixed species biofilms was comparable to published literature, although lower abatement rates were reported for caffeine and acetaminophen, which could be the result of higher organic matter acting as oxidation scavenger in the environmental water. Negative removal rates were obtained for sulfamethoxazole, benzotriazole and efavirenz, likely due to electro- oxidation facilitated reconstitution of breakdown products to the respective parent compounds. The DiaDis was evaluated as a final disinfection step in an aquaponics, as well as a constructed floating wetland system. It performed better in the floating wetland, possibly due to higher levels of organic matter coming from fish-feed in the aquaponics system. Overall the DiaDis showed promising disinfection capabilities, which will benefit from future work to better control pH reduction and to reduce interference of organic matter with micropollutant abatement.
- ItemPredatory bacteria: alternative antimicrobial and biocontrol agents(Stellenbosch : Stellenbosch University, 2023-03) Kode, Megan Ann; Khan, Wesaal; Waso-Reyneke, Monique; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: While Bdellovibrio-and-like organisms (BALOs) are ubiquitous in nature, their diversity in environmental niches is underexplored. The isolation and characterisation of novel strains should thus be investigated, as these strains may exhibit an increased predation efficiency on pathogenic bacteria prevalent in the environment. The primary aim of the current study was thus to isolate and characterise BALOs from various water sources, and to investigate the efficacy of Bdellovibrio spp. in combination with solar disinfection (SODIS) and flocculation, for the removal of multi-drug resistant (MDR) human pathogens from artificial rainwater. Chapter one (abbreviated version published in Frontiers in Chemistry, 2022) thus focussed on elucidating the diversity of the BALO families, as well as the potential application of predatory bacteria and their natural products (including BALO and non-BALO strains) as therapeutic, biocontrol, and preservative agents. In Chapter two, BALOs were isolated from various water sources, including rivers, streams, surface runoff, clinical wastewater, and marine water. The isolation of several Bdellovibrio spp. and one Halobacteriovorax sp. was confirmed by polymerase chain reaction (PCR) analysis and sequencing, with phylogenetic analysis indicating that these isolates clustered with other strains in the Bdellovibrionaceae and Halobacteriovoraceae families, respectively. However, it was hypothesised that the isolated Halobacteriovorax sp. strain may belong to a novel species, as it branched independently from the Halobacteriovorax spp. reference strains. Two BALOs, namely Bdellovibrio bacteriovorus (B. bacteriovorus) TWPF3 and Halobacteriovorax sp. GBVP3, were selected for further characterisation, as B. bacteriovorus TWPF3 formed numerous and large plaques, and the Halobacteriovorax sp. GBVP3 was the only isolate from the Halobacteriovoraceae family. The prey range of both predatory bacteria was assessed using twenty-three Gram-negative bacterial strains (i.e., four clinical; seven environmental; twelve laboratory strains), with results indicating that B. bacteriovorus TWPF3 formed plaques when exposed to the clinical strains Escherichia coli (E. coli) MCC2, and Klebsiella pneumoniae (K. pneumoniae) KP3, and the laboratory strains K. pneumoniae ATCC 13883, K. pneumoniae PF, Salmonella typhimurium (S. typhimurium) ATCC 14028, and Shigella sonnei (S. sonnei) ATCC 25931. Halobacteriovorax sp. GBVP3 then formed plaques using four laboratory strains [K. pneumoniae ATCC 13883, Serratia marcescens (S. marcescens) ATCC 13880, S. sonnei ATCC 25931, and Vibrio cholerae (V. cholerae)], and one environmental strain [Vibrio parahaemolyticus (V. parahaemolyticus) GB6] as prey. The antibiogram for all predation-sensitive bacteria was assessed using Kirby-Bauer analysis, where only E. coli MCC2 and K. pneumoniae KP3 were classified as MDR, and K. pneumoniae PF was classified as extensively drug-resistant (XDR). The B. bacteriovorus TWPF3 and Halobacteriovorax sp. GBVP3 were then co-cultured with each of the respective predation-sensitive bacterial strains, with samples collected after 0, 24, 48, 72, and 96 h assessed using culture-based methods. Results indicated that B. bacteriovorus TWPF3 reduced K. pneumoniae ATCC 13883, S. typhimurium ATCC 14028 and S. sonnei ATCC 25931 by a maximum of 1.63 logs, 0.50 logs and 1.31 logs, respectively, while Halobacteriovorax sp. GBVP3 reduced the cell counts of K. pneumoniae ATCC 13883 and V. cholerae by 1.21 logs and 0.98 logs, respectively. Ethidium monoazide bromide quantitative PCR (EMA-qPCR) analysis was subsequently used to assess the co-culture assays where the prey cell counts were reduced by ≥ 2 logs. Overall, significant reductions in the cell counts and gene copies of MDR E. coli MCC2 [2.05 (72 h) and 1.50 logs (72 h), respectively], MDR K. pneumoniae KP3 [2.25 (72 h) and 3.59 logs (48 h), respectively], and XDR K. pneumoniae PF [3.79 (72 h) and 2.29 logs (96 h), respectively], were recorded after co-culture with B. bacteriovorus TWPF3. Similarly, significant reductions in the cell counts and gene copies of S. marcescens ATCC 13880 [2.38 (48 h) and 2.47 logs (96 h), respectively], S. sonnei ATCC 25931 [3.40 (48 h) and 6.19 logs (24 h), respectively] and V. parahaemolyticus GB6 [2.34 (48 h) and 2.61 logs (96 h), respectively], were recorded after co-culture with Halobacteriovorax sp. GBVP3. Based on the results obtained, B. bacteriovorus TWPF3 and Halobacteriovorax sp. GBVP3 could potentially be applied as biocontrol agents or for therapeutic uses, to target MDR and human pathogenic bacteria. Communities that do not have access to piped water supplies often rely on alternative water sources such as harvested rainwater, for potable and domestic uses. Various microbial pathogens have however, been detected in these water sources and while treatment methods such as chlorination, filtration, and SODIS, have been implemented for their removal, K. pneumoniae and Pseudomonas aeruginosa (P. aeruginosa), amongst many other microorganisms, have been found to persist. The aim of Chapter three (published in the Journal of Environmental Chemical Engineering, 2022) was thus to investigate the efficacy of combination treatments for the eradication of MDR K. pneumoniae and XDR P. aeruginosa from artificial rainwater. The combination treatments included a 72-h predatory bacteria pre-treatment using B. bacteriovorus PF13 (isolated by a member of the Water Resource Laboratory) or B. bacteriovorus TWPF3, or a dual-predatory pre-treatment using both B. bacteriovorus PF13 and TWPF3. The different predatory bacteria pre-treatments were followed by SODIS (for 6 h), and flocculation (for 1 h) using Moringa oleifera (M. oleifera) seed extract. Overall, the use of the dual predatory pre-treatment, followed by SODIS, significantly reduced the MDR K. pneumoniae cell counts and gene copies (as determined by EMA-qPCR) by 8.46 logs and 4.40 logs, respectively, to below the detection limit. Contrastingly, while none of the predatory pre- treatments were found to reduce the XDR P. aeruginosa cell concentration, SODIS with no pre- treatment was able to significantly reduce the cell counts and gene copies of XDR P. aeruginosa by 6.40 and 3.81 logs, respectively. The M. oleifera flocculation treatment (following SODIS) did not however, significantly reduce the cell concentrations of the prey strains. Analysis of the antibiogram of the MDR K. pneumoniae and XDR P. aeruginosa using the VITEK® 2 Compact System, then showed that no difference in the organisms’ antibiotic-resistance profiles was observed after the implementation of the various treatment stages. Results obtained in the current study thus indicated that MDR K. pneumoniae was significantly reduced to below the detection limit by the dual-predatory bacteria pre-treatment followed by SODIS, and future research should elucidate the interaction and predation kinetics when multiple predatory bacteria are used to target bacterial pathogens.
- ItemIsolation 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.
- ItemMicroalgae as a feedstock for ethanol production(Stellenbosch : Stellenbosch University, 2023-12) De Villiers, Dewald; Van Zyl, Willem Heber; Viljoen-Bloom, Marinda; Cripwell, Rosemary Anne; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Microalgae are increasingly considered a source for high-value products in various markets, with applications in the health food, medicinal and industrial sectors. More recently, microalgae have gained interest as a feedstock for biofuel production due to their high starch content. Various studies investigated the challenges of using microalgae in industry, such as low biomass production, complicated harvesting methods and high lipid/carbohydrate content. Although solutions and mitigation strategies have been proposed, the process must be economically feasible to compete with fossil fuels and other biofuel feedstocks. This could be achieved by optimisation of the growth conditions to maximise the biomass and starch yields and/or through enzymatic treatment to release the starch for fermentation to bioethanol. In this study, the growth conditions for two microalgal strains known for their starch - producing capabilities, Chlamydomonas reinhardtii and Chlorella sorokiniana, were optimised for enhanced biomass and starch accumulation, specifically by evaluating different carbon and nitrogen sources. The two strains were grown under mixotrophic conditions, i.e. photosynthesis in the presence of additional carbon sources (glucose and acetic acid). The C. sorokiniana strain displayed the highest biomass production (3.89 g/L) and starch accumulation (0.67 g/L) when grown in Bold Basal Media (10 g/L glucose) with a modified carbon-to-nitrogen ratio (C:N of 8:1). The C. sorokiniana strain was evaluated in a consolidated bioprocessing (CBP) process for starch-ethanol fermentation by optimising the harvest methods and pretreatment options. The study found that enzymatic pretreatment coupled with freeze-drying provided the best results. The C. sorokiniana biomass was pretreated enzymatically with pectinase and xylanase to release the internal starch granules. Consolidated bioprocessing with an amylolytic Saccharomyces cerevisiae strain (co-expressing an α-amylase and glucoamylase) yielded 4.02 g/L ethanol from a 10% microalgal substrate loading at 30°C. This study is one of only a few that observed microalgae growth in a standardised, mixotrophic growth setting where macronutrients were evaluated for optimised starch production.