Masters Degrees (Microbiology)
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- 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.
- ItemCalprotectin (S100A8/A9) as a marker of inflammation and treatment monitoring in cases of juvenile idiopathic arthritis in the Western Cape, South Africa(Stellenbosch : Stellenbosch University, 2023-03) Evert, Christine; Glashoff, Richard H. ; Abraham, Deepthi Raju; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Background: Juvenile Idiopathic Arthritis (JIA) is a common rheumatic disease affecting children and is characterised by persistent inflammation of the joints. The socio-economic climate of South Africa can delay access to treatment to achieve remission. Joint inflammation is currently monitored through C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) measurements. Recent studies have shown that these markers may correlate less well with disease activity than calprotectin. Calprotectin is released by activated monocytes or macrophages at the site of joint damage and binds the TLR4 surface receptor. This protein has also been used to detect subclinical inflammation and may predict risk of relapse. This study aimed to compare standard markers of inflammation with calprotectin, and its relation to other inflammatory markers. Methodology: Blood samples were collected from 22 consented JIA participants. Clinical information and history for each participant was obtained from patient files at Tygerberg Hospital. Monocyte distribution and phenotypic marker expression was investigated using whole blood for surface marker flow cytometry. Plasma levels of calprotectin and JIA associated inflammatory markers (including CCL2, CCL11, CD163, CXCL9, CCL3, CCL22, CD25, CXCL10, IL-1β, MIF, IL12, TNF-α and IFN-γ) were assessed by means of ELISA and Luminex™ multiplex assays. Routine CRP and ESR results were collected from the NHLS TrakCare database. For longitudinal follow up, blood samples were collected from the same cohort 6 months later and assays were repeated. A study database was created with all participant results, at both visits, to investigate relationships between calprotectin and JIA disease activity, as well as the effect of treatment over time. Results: The majority (95%) of participants were already undergoing treatment. Calprotectin was within the normal range for children (127 – 1395 ng/mL) in 86% of baseline samples with a median of 628.6 ng/mL (IQR: 406.2 - 979.8). Only ESR changed significantly (p=0.0079) over time and showed the most evidence for changes in inflammation thereby inspiring analysis by stratification. To evaluate impact of disease phenotype (active vs. remission) and inflammatory state based on ESR expression (high vs. low ESR), participants were stratified into respective groups and compared. Expression of intermediate monocytes at baseline was higher than the expected range (2 - 10 %), with a median of 27 % (IQR: 9 - 43). This distribution is characteristic of inflammatory diseases. Calprotectin correlated significantly (p<0.05) with CRP and ESR at baseline in the returning, remission and high ESR analysis groups. Several notable cases displaying high calprotectin expression were linked to a relapse in disease. Conclusions: Despite limitations, this study confirmed the predictive value of calprotectin in risk of disease relapse and the need for such a marker in the clinical setting to allow for a more tailored approach to treatment. The relationship between intermediate monocyte expansion, calprotectin and disease phenotype also needs to be examined further. Future studies which include treatment naïve participants would be beneficial in assessing the usefulness of calprotectin in monitoring response to treatment.
- ItemInteraction between plastic particles and biofilms(Stellenbosch : Stellenbosch University, 2023-03) Murray, Thomas Everitt Rautenbach; Wolfaardt, Gideon M. ; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The problem with plastic waste is immense and has received much attention in the last 15 years. Plastics break down into smaller plastic particles over time, from degradation that occurs from various sources. The natural environment poses many forces, including physical, chemical, and biological, which quicken the rate of degradation of plastic. These plastic particles are able to move through the environment due to natural forces which have managed to spread them everywhere. Accumulation of plastic particles is thus thought to be a problem which has the potential to effect living organisms on a large scale. There is reason to believe that association with and ingestion of plastic particles may cause health risks due to the size of the particles and harmful chemicals which may interact with them in the environment. Bacteria are amongst the first organisms which will interact when it is introduced into the environment. The formation of biofilms on the plastic may follow and in aqueous environments, may take only hours. These biofilms are robust and will proliferate under harsh conditions that may accelerate the degradation of the plastic polymer backbone and result in the production of plastic particles. Some microorganisms have the ability to produce plastic degrading enzymes which will enhance this process. They can also change their microenvironment enough to make plastic degradation less efficient and greatly alter the fate of plastic particles. The surfaces of weathered plastic particles are conducive to biofilm growth and thus strong relationships exist with biofilms that form on plastic particles. Information is, however, sparce on the inverse relationship which plastic particles may have with biofilms. While plastic particles are common in the environment, biofilms are ubiquitous. There are thus many environments containing biofilms which may be greatly changed by the introduction of plastic particles. In the experimental section of this project a method was developed to study interactions between biofilms and plastic particles in a simulated continuous flow environment. The fluorescently labelled bacteria used in the study, in this case Pseudomonas fluorescens CT07::gfp were selected due to their affinity for growing as a biofilm. This is important as it facilitates a more realistic environment than a conventional planktonic culture would. The method was successful in detecting accumulation of plastic particles under specific conditions and measuring how the relationship of particle accumulation was affected by altering specific parameters of the system, like the concentration of plastic particles and nutrients in the growth medium. It was determined that biofilms cultured in lower nutrient medium concentration of tryptic soy broth (0.3 g/L) resulted in the adherence of significantly more plastic particles in the flowcells than the higher nutrient medium concentration (3.0 g/L). Furthermore, both biofilms resulted in the adherence of significantly more plastic particles in the flowcells than the sterile controls which were performed, and which had no significant difference between each other. The process of bead adhesion was observed and documented to be due to multiple factors, including extracellular polymeric substances (EPS), biofilm cells, and shear forces at the flowcell surface. It was determined that the presence of a biofilm resulted in significantly more plastic particles accumulating in the biofilm than the absence thereof, indicating that there is significant interaction between the biofilm and plastic particles. The binding ratio of each of the biofilms was calculated by determining the total theoretical number of adhered plastic particles in each flowcell and dividing that by the total number of plastic particles that had passed through each flowcell. A constant binding ratio was obtained for each biofilm cultured on the different nutrient medium concentrations which also remained constant when the concentration of plastic particles was changed Furthermore, the potential of biofilms as plastic particle traps was explored due to the observed relationships, but a low efficiency of bead binding was determined.
- ItemThe production of class I lanthipeptides in escherichia coli using a green fluorescent protein fusion system(Stellenbosch : Stellenbosch University, 2022-12) Faure, Lindsay Marie; Dicks, Leon Milner Theodore; Van Staden, Anton du Preez; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Antimicrobial resistance is currently one of the greatest threats to public health. Bacteria have the natural ability to acquire resistance however, the misuse and over prescription of various antimicrobials has accelerated this crisis. Several alternatives to antimicrobials are being explored, one of which are lanthipeptides. Lanthipeptides are ribosomally synthesized peptides characterized by the presence of lanthionine and methyl-lanthionine residues, which are introduced by post-translational modification enzymes. The interest in lanthipeptides has increased not only due to their antimicrobial activity against several clinically relevant multi-drug resistant bacteria but also various other bioactivities including antiviral, anticancer and pain relief. The main stumbling block hindering the applications of lanthipeptides is their production. Their production is complex, due to the difficulty in cultivation and purification from native sources, as well as the enzymes required for post-translational modification, which ultimately leads to low yields. In this study we fused the green fluorescent protein (GFP) to the core peptides (already fused to the nisin leader peptide) of the known class I lanthipeptides, nisin, Pep5, Epilancin 115x and epidermin and co-expressed the fusion with the post-translational modification enzymes NisB, NisC and EpiD using Escherichia coli (E. coli) as the host. The results showed that the post- translational enzymes were not obstructed by the GFP fusion and were able to modify the core peptides. Furthermore, antimicrobial activity was detected for nisin, Pep5 and epidermin following in-vitro leader peptide cleavage using nisin’s native protease, NisP. Preliminary yield estimates detected 2 mg/L of nisin following cleavage and purification. Following the favourable outcome of the GFP-fusion expression system, 16 putative class I lanthipeptides were mined from the genomes of numerous bacteria. The peptides were produced using the system and antimicrobial activity was observed for six of the peptides. The outcomes of the two-part study showed that E. coli is a suitable host to produce lanthipeptides, and an optimized GFP-fusion system can yield functional class I lanthipeptides. Furthermore, the system can be used to produce putative lanthipeptides discovered via genome mining techniques. These outcomes can prove valuable in the production of lanthipeptides for their use in the fight against multi-drug resistant bacteria as well as various other potential bioactivities.