Magister Scientiae - MSc (Biotechnology)

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Screening and evaluation of bacteria for the biological control of Botrytis cinerea on Vitis vinifera
(University of the Western Cape, 2024) Abrahams, Malikah
Agriculture plays a significant role in food security and the economic sector. However, the agriculture industry is threatened by plant pathogenic microorganisms, with fungi accounting for 42% of plant diseases. Botrytis cinerea is the most notable of all plant pathogenic fungi. B. cinerea is notoriously known to cause grey mould or grey rot in grapevine (Vitis vinifera), which threatens viticulture. Chemical fungicides have been used to control it for many years, however, chemical fungicides have negative effects on the environment and human health. In addition, excessive use of chemical fungicides and the polycyclic nature of B. cinerea led to the development of resistant strains, making it difficult to control. Alternative methods have been investigated to control B. cinerea, such as using microorganisms as biological control agents (BCAs). Therefore, researchers have been searching for BCAs that can control B. cinerea. This study aimed to test bacterial isolates for antifungal activity against B. cinerea and investigate their modes of action, which can aid in preventing grey mould in grapes. The dual-culture assay was used as a preliminary screening method to test antifungal activity against three B. cinerea strains (IWBT FF1, IWBT FF2, and B05.10). The bacterial isolates were identified using 16S rRNA gene sequencing. Various experiments assessed the modes of action behind the inhibitory activity, including testing for cell wall degrading enzymes (glucanases, proteases, chitinases), evaluating competition for space and nutrients via siderophore production on chrome-azurol S agar, and conducting an in vivo assay on wounded grape berries. Antibiosis was evaluated by assessing the effect of the cell-free supernatant on the development of B. cinerea strains. Additionally, polymerase chain reaction (PCR) was used to detect genes related to cyclic lipopeptide (CLP) synthesis, and liquid chromatography-mass spectrometry was used to determine if CLPs were expressed.
In situ green synthesis of red wine silver nanoparticles for the production of antimicrobial cotton fabrics and the investigation of their biomedical effects
(University of the Western Cape, 2024) Erasmus Alexandria
Microbial infections, particularly those caused by pathogenic bacteria, pose a significant barrier in the treatment of infectious diseases. In addition, overuse of antibiotics has resulted in the development of antimicrobial resistance (AMR). AMR is a growing global health concern that can cause serious complications in common infections and chronic wounds infected with AMR microbes. Conventional therapeutic approaches previously used in these conditions (wounds and microbial infections) become ineffective and their life cycle are prolonged. Hence, it is crucial to create innovative and alternative antimicrobial treatment strategies. The field of biomedicine can benefit from the application of nanotechnology to produce alternative or enhanced pharmaceutical products and therapies. Silver nanoparticles (AgNPs) have demonstrated strong antimicrobial properties and have shown to be beneficial in treating AMR infections. Their added benefit in chronic wounds is their ability to promote the wound healing process by stimulating angiogenesis, cell proliferation, and collagen synthesis, among others. These properties are enhanced in AgNPs synthesized by using medicinal plant extracts. Although green synthesis using this route is rapid, easy and cheap; it can often be limited by low solubility and aggregation after purification. In situ synthesis of nanoparticles offers advantages like enhanced stability, better uniformity, reduced contamination, cost-effectiveness, and tailored functionality, especially when nanoparticles need to be directly integrated into a matrix. This synthesis approach has a wide range of applications, including textiles and wound healing dressings. Cotton has been utilised in medical applications since the Middle Ages, primarily as a material for dressing wounds, whereas red wine is recognised for its elevated levels of resveratrol – a phenolic chemical molecule that possesses strong antioxidant and anti-inflammatory qualities. This study reports on the green synthesis of AgNPs using freeze-dried red wine extract (RW) (RW-AgNPs), the fabrication of RW-AgNP-loaded cotton fabrics (RW-ALC), and the investigation of their antimicrobial, antioxidant, and cytotoxic properties. RW-AgNPs were green-synthesized by optimizing pH, silver nitrate (AgNO3) concentration, and RW extract concentration under hydrothermal conditions and characterized using several physicochemical techniques including Ultraviolet-visible (UV-vis) Spectroscopy, Dynamic Light Scattering (DLS) using a Zetasizer, High-Resolution Transmission Electron Microscopy (HR-TEM), Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared (FTIR) Spectroscopy. The RW-ALC were synthesized in situ under hydrothermal conditions for one hour using RW extract at a concentration of 6.25 mg/ml and pH 10, and AgNO3 at a concentration of 3 mM. Assessment of antioxidant activity of the RW and RW-AgNPs was conducted using the ABTS and DPPH scavenging assays. Varying quantities of RW, RW-AgNPs, and the positive control ascorbic acid (ranging from 0.78 to 100 μg/ml) were used in the assays. The antibacterial activity of the RW-ALC and RW-AgNPs was evaluated using the agar disc diffusion and broth microdilution assays against the human pathogens S. aureus, MRSA, E. coli, E. cloacae, K. pneumoniae, P. aeruginosa, and A. baumannii. In the disc diffusion assay, RW-ALC samples synthesized with varying concentrations of RW (ranging from 1.56 to 25 mg/ml) were employed as the treatment. A standard cotton piece served as the negative control. The positive control was prepared by applying 50 μl of a 15 μg/ml Ciprofloxacin solution to a clean cotton piece, except for the E. coli samples, where a 10 μg/ml Ciprofloxacin concentration was used instead. The Müeller-Hinton agar (MHA) plates were incubated at 37 °C for 24 hours. The effect of pretreatment with 2-mercaptoethanol on the antibacterial efficacy of the RW-ALC was also investigated. For the microdilution assay, bacterial cultures were subjected to treatment for 24 hours using RW-AgNPs at serially diluted concentrations of 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, and 0.195 μg/ml. The negative control consisted of 50 μl of Mueller-Hinton broth (MHB) added to the well. For the positive control, 50 μl of a solution containing 10 μg/ml of Ciprofloxacin was introduced into each well, with the exception of E. coli, for which a concentration of 5 μg/ml of Ciprofloxacin was used. Cytotoxic properties of the RW-AgNPs on five cancerous (MCF-7 Caco-2, PC-3, Panc-1, MIA-Paca-2) and one non-cancerous (KMST-6) human cell lines was assessed using the WST-1 cell viability assay. Cell lines were treated for 24 hours with RW and RW-AgNPs at concentrations ranging from 0 – 100 μg/ml. 5 % DMSO was utilised as a positive control. The RW-AgNPs were spherical with a core size of 8.7 ± 1.3 nm, hydrodynamic size of 104.30 ± 5.51 nm, PDI value of 0.344 ± 0.2 and ζ-potential of -21.3 ± 6.16 mV. RW-AgNPs exhibited minimum inhibitory concentrations and minimum bactericidal concentrations against S. aureus (0.195 μg/ml and 0.78 μg/ml), MRSA (1.56 μg/ml and 3.125 μg/ml), K. pneumoniae (0.78 μg/ml and 1.56 μg/ml), A. baumannii (0.78 μg/ml and 1.56 μg/ml), E. cloacae (3.125 μg/ml for both), E. coli (0.78 μg/ml for both), and P. aeruginosa (3.125 μg/ml and 6.25 μg/ml). Characterisation of the cotton showed polydispersed RW-AgNPs on the surface at a concentration of 80.2 μg/ml. The results of the DPPH antioxidant assay demonstrate that the scavenging activity of RW-AgNPs is dose-dependent within the concentration range of 3.125 – 100 μg/ml. No significant activity was observed at concentrations between 0.78 and 1.56 μg/ml. While RW-AgNPs exhibited considerable antioxidant activity, their efficacy was lower compared to that of RW and ascorbic acid. Conversely, in the ABTS assay, RW, RW-AgNPs, and ascorbic acid (used as a positive control) showed a dose-dependent reduction of the ABTS•+ radical. Notably, within the concentration range of 0.78 to 50 μg/ml, RW-AgNPs exhibited superior scavenging activity compared to the positive control. RW-ALC exhibited significant antibacterial activity, with zones of inhibition ranging from 12.33 ± 1.15 mm to 23.5 ± 5.15 mm in comparison to 10 μg/ml Ciprofloxacin (between 10 ± 3 mm – 19.17 ± 1.39 mm). Preincubation with 2-mercaptoethanol abrogated the antibacterial activity of RW-ALC, indicating that the antibacterial properties are attributed to silver's ability to bind to sulfhydryl groups. Furthermore, RW-AgNPs showed significant cytotoxicity towards the cancer cell lines: MCF-7; PC-3; Caco-2; MIA-Paca-2; Panc-1 cells with half maximal inhibitory concentration (IC50) values of 11.47 μg/ml, 10.98 μg/ml, 6.76 μg/ml, 7.89 μg/ml, and 3.76 μg/ml, respectively. Reduced cytotoxicity was observed against the normal human fibroblast KMST-6 cell line (IC50 of 27.7 μg/ml), suggesting that the RW-AgNPs can be effectively employed in the treatment of bacterial infections and cancer with minimal detrimental effects to healthy mammalian cells. This study demonstrated that RWcan be used to synthesize AgNPs and AgNP-loaded textiles in situ under hydrothermal conditions. Furthermore, when evaluating the biological effects of the resultant RW-AgNPs and RW-ALC in vitro, both had potent antibacterial and anticancer effects. The RW-ALC could be useful in treating infectious diseases, including bacteria infected chronic wounds. However, cytotoxic effects of these RW-AgNPs require further investigation to ensure their biocompatibility at concentrations that are minimally detrimental to mammalian cells yet still toxic to bacteria and cancer cells. Further studies are underway to elucidate the anticancer mechanisms of the RW-AgNPs. These could then be transformed into novel affordable remedies for chronic non-healing wounds and cancer.
Heterologous expression of a putative lodAB operon derived from Thalassomonas viridans”
(University of the Western Cape, 2024) Rowland Dugald Joseph
The growing incidence of antibiotic resistance in bacteria to current antimicrobial compounds is a major concern worldwide. Identifying novel antimicrobial compounds is vital to mitigate the increasing prevalence of antibiotic-resistant bacteria and other pathogens. One of the methods used to find novel antimicrobial compounds is genome mining. The genome mining of marine organisms, which have been poorly represented in research, may reveal a potential source of new antimicrobial compounds which can be used as antibiotics. This study focused on the characterisation of an antibacterial protein identified by the genome mining of a marine microorganism. Thalassomonas viridans is a microorganism that is known to produce antibiotic secondary metabolites (Adams, 2019). The genomic annotation of the T. viridans genome indicates that there are two sequences with similarity to the lodA gene that codes for l-lysine ε-oxidase (LodA) - first described as an antimicrobial protein produced by the marine bacteria Marinomonas mediterranea. LodA is an extracellular enzyme that has antibacterial properties associated with the production of hydrogen peroxide (H2O2) as a product of lysine oxidation (Gómez D et al.;2006). The lodA sequences submitted to a wider BLAST analysis showed that similar sequences are present in the genomes of a number of other microorganisms. In this study the heterologous expression of the lodA genes from T. viridans was conducted in Escherichia coli to produce, purify, and characterise the proteins. Multiple methods of E.coli expression to produce the protein were explored, and multiple activity assays wereemployed to characterise the proteins produced. The LodA protein was produced at low concentrations and no activity was detected on a wide range of amino acid substrates
Novel dyp-type peroxidases from actinobacteria: a genome mining approach
(University of the Western Cape, 2024) Groep, Juandré
Twenty-one whole genome actinobacterial sequences were obtained from strains previously isolated from various environments. a genome-mining approach was applied to identify the presence of dye-decolourising (dyp-type or dyps) peroxidases. from genome sequence annotation twenty-six dyp-type sequences were identified and through bioinformatic analysis for the presence of twin-arginine translocation (tat) pathway signal peptides, was classified as belonging to class i (formerly class a) bacterial dyp-type peroxidases. phylogenetic analysis of the dyp sequences showed that the streptomyces derived dyps were predominantly grouping together and the nonstreptomyces derived dyps followed the same pattern. eight dyp-type peroxidases were selected for cloning, with the majority being derived from streptomyces spp., and primers were designed to amplify the dyp-type peroxidase genes. the pcr-amplicons were subjected to restriction digests and were ligated into pet20b(+). the constructs were transformed into escherichia coli jm109. only two of the constructs were successfully transformed: the dyp-type peroxidase genes from nocardia gamkensis czh20t and micromonospora sp. strain 30-1. further analysis using t7-primers confirmed sequence identity of the constructs.
Engineering robust yeast strains for the conversion of xylose derived from lignocellulosic biomass to xylitol
(University of the Western Cape, 2023) Maneveldt, Amber
To achieve a sustainable and economically viable 2G biofuels industry, biorefineries must coproduce high-value, low-volume bioproducts alongside high-volume, low-value cellulosic ethanol. This can be realised with the co-production of the low-calorie sugar substitute, xylitol which has a well-established market, as well as other chemicals. The construction of a xylitolproducing S. cerevisiae strain represents an economically feasible and environmentally friendly approach to xylitol production. Moreover, the exploitation of natural S. cerevisiae strain isolates as bioengineering hosts has the potential to be superior starting points due to their robustness towards process conditions. In this study, the xylitol-producing activities conferred to three natural isolate host strains via conventional and CRISPR-Cas9-mediated δintegration of three genes encoding a β-xylosidase, β-xylanase and xylose reductase (XR), was evaluated. The effect of over-expressed heterologous protein production on strain robustness and metabolism was also assayed. Our results revealed that the overexpressed XR failed to improve on the xylose reduction ability conferred to our strains, likely by their native GRE3 gene.
Green synthesis and characterization of silver nanoparticles from the cocktail of capparis sepiaria-tabernaemontana elegans extracts and assessment of their biological effects in vitro.
(University of the Western Cape, 2024) MASHILO, CATE MALOPE
Antimicrobial resistance (amr) is a global crisis that develops when bacteria, viruses, fungi, and parasites adapt and multiply in the presence of drugs that once negatively affected them. amr infections are commonly caused by the overuse and misuse of antimicrobial drugs, thus leading to severe illnesses, longer hospitalization, increased healthcare expenses, treatment ineffectiveness, and increased mortality. the increasing incidence of amr poses a serious threat to public health. as a result, alternative strategies that are both effective against amr pathogens and eco-friendly are urgently needed. green nanotechnology, in particular the use of silver nanoparticles (agnps), has been used as a solution in a wide range of applications, including antibacterial, antiviral, and antifungal therapies. over the last decades, several medicinal plant extracts have been used to synthesize agnps; however, combining extracts from two medicinal plants to synthesize agnps with enhanced properties has received less attention. therefore, this study reports on the green synthesis of agnps using a cocktail of capparis sepiaria–tabernaemontana elegans (cste) aqueous extracts as a reducing, stabilizing, and capping agent, and evaluation of their antimicrobial activities.
Green synthesis and characterization of gold nanoparticles from capparis sepiaria-tabernaemontana elegans cocktail and investigation of their biological effects in vitro.
(University of the Western Cape, 2024) MATSHAYA, ALUWANI
Wounds have a significant impact on the global healthcare system, adding heavy load on the economy and society. therefore, there is an urgent need to develop treatments that can facilitate rapid wound healing. to make matters worse wound infection by antimicrobial-resistant bacteria may result in prolonged debility of the patient and increased healthcare costs. avoidance of the development of antimicrobial resistance therefore needs increasing attention in the management of patients, with chronic wound infection. nanotechnology presents a groundbreaking method for synthesizing biocompatible nanomaterials (nms), revolutionizing wound healing with soft, flexible dressings that possess antimicrobial properties, gas barrier, and absorbency. these nms might fall into two categories: those with inherent wound-healing properties and those serving as delivery vehicles for therapeutic agents. gold nanoparticles (aunps) have more consideration due to their stability, oxidation resistance, electrical and optical properties. this study reports on the green synthesis and characterization of aunps synthesized using an aqueous extract of capparis sepiariatabernaemontana elegans (cste) and the investigation antibacterial and wound healing effects of the cste-aunps against amr pathogens, such as staphylococcus aureus, methicillin-resistant staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa. wound healing properties of cste-aunps was also investigated using kmst-6 (fibroblast) cell line.
Identification of a transducin (beta)-like 3 protein as a potential biomarker of prediabetes from rat urine using proteomics
(University of the Western Cape, 2010) Mofokeng, Henrietta Refiloe
Obesity is a globally increasing disease particularly in developing countries and among children. It is mainly caused by intake of diets high in fat and the lack of physical activity. Obesity is a risk factor for diseases such as type 11 diabetes, high blood pressure, high cholesterol and certain cancers. Prediabetes is a condition where blood glucose levels are above normal but have not reached those of diabetes. It is difficult to diagnose, as there are no signs or symptoms. Some type II diabetes patients bear no symptoms at all and the disease is discovered late. Proteomics is a field that can provide opportunities for early diagnosis of diseases through biomarker discovery. The early diagnosis of diabetes can assist in the prevention and treatment of diabetes. Therefore there is a need for the early diagnosis of diabetes.
Green synthesis of silver nanoparticles from indian hawthorn (rhaphiolepis indica) and the investigation of their antimicrobial, antioxidant and cytotoxic effects
(University of the Western Cape, 2024) Nsumpi, Aurelie Ngalula
The global health concern pertaining the rapid spread of antimicrobial resistance (AMR) has led to a rise in mortality rate caused by infectious diseases that have become difficult to treat due to the misuse and lack of novel antibiotics. Nanotechnology is one innovative approach that has the potential to eradicate the burden of AMR with silver nanoparticles (AgNPs) well known for their antibacterial properties. Currently, AgNPs can be synthesised using a greener approach which makes use of the phytoconstituents in plants providing more advantages over the conventional chemical synthesis methods (i.e rapid, cost-effective, safer for the environment and sustainable) also enhancing the antioxidant properties of AgNPs for the treatment of degenerative diseases caused by oxidative stress. Indian Hawthorn (Rhaphiolepis indica) berries have been used to treat cardiovascular diseases, high blood pressure and heart failure and are known to have high content of polyphenols which are involved in bioreduction of metal-based NPs (MNPs) with enhanced bio-activities.
Engineering antimicrobial strains of saccharomyces cerevisiae as industrial platform for non-sterile bioprocesses
(University of the Western Cape, 2023) Van Lill, Gert Rutger
Microbial contamination is a major challenge in fuel ethanol facilities, especially during the fermentation stage. Bacterial contaminants compete with yeast for fermentable sugars and nutrients, which obstructs starch to ethanol conversion. This leads to a reduction in ethanol yield. At large production scales, prior sterilization of the feedstocks is economically unfeasible. Therefore, antibiotic supplementation is common practice to limit the growth of contaminating bacteria. In addition to the high cost of the antibiotics, concerns and evidence are mounting that antibiotic use in non-clinical settings is driving the emergence of drug resistant microbes. This study focused on heterologous expression of antimicrobial peptides (AMP) as an alternative strategy for combating microbial contamination. This work aimed to engineer an industrial Saccharomyces cerevisiae strain that produces AMPs active against lactic acid bacteria and Enterobacteriaceae which are some of the main bacterial contaminants found in industrial biofuel fermenters. Seven candidate AMPs were selected from curated online databases, and their amino acid sequence was analyzed using bioinformatic tools. AlphaFold, Clustal Omega, and SNAP2 were used to predict AMP tertiary structures, construct a phylogenetic tree, and analyse mutation effects, respectively.
Biogenic silver nanoparticles synthesized using eucomis autumnalis bulb aqueous extract, their characterization and in vitro antibacterial activity
(University of the Western Cape, 2023) WILLIAMS, LETOYA SHEILA
Antimicrobial resistance (AMR) is a growing global problem that poses a significant threat to public health. AMR arises when microorganisms become resistant to drugs that were initially designed to kill them. The application of antibiotics has proven effective in eliminating or inhibiting the growth of harmful microorganisms. Conversely, the primary cause of AMR is overprescribing and the misuse of antibiotics which is further increased by a reduced novel antibiotics discovery rate. AMR bacteria lead to hard-to-treat infections, causing longer hospital stays and higher healthcare costs, and is associated with a substantial risk of morbidity and mortality. Consequently, the development of infections caused by multi-drug resistant (MDR) microorganisms has resulted in a considerable reduction in the number of effective treatment options available. Thus, novel therapies are urgently required, considering the ineffectiveness of current therapy, moreover, further exacerbated by a reduced discovery rate of novel antibiotics. Alternative strategies are therefore urgently needed to treat AMR bacterial infections.
Screening for novel cold-adapted nitrile hydratases in Antarctic metagenornic libraries
(University of the Western Cape, 2024) Williams, Wesley Trevor; Cowan Don
Nitrile hydratases (NHase) catalyse the hydrolysis of nitriles to their corresponding amides, the first step in the nitrile degradation pathway. The second step is the conversion of the amide to a carboxylic acid, catalysed by an amidase. The NHases published to date have been isolated from mesophilic and thermophilic bacteria. The aim of this study was to identify a novel cold-adapted NHase from psychrophilic and psychrotrophic bacteria. The identification of a cold-adapted NHase is of interest for its potential use in chemical and pharmaceutical production. Enzymes isolated from psychrophilic bacteria typically have a high specific activ ity and low thermal stabi lity to cope with the inhibition of chemical reactions caused by low temperatures. However, culture based screening for these activities tend to reisolate already characterised isolates amiable to culturing. To access the majority of the bacterial biodiversity that the uncultured bacteria represent, the metagenomic approach was used to screen for nitrile degrading activity. Metage-nomic libraries constructed from soil taken from the McMurdo Dry Valleys in Antarctica were used for both functional and sequence based screens of NHases. Functional screens that were trialled were based on pH change and oligotrophic selective growth.
Metagenomic bioprospecting: discovery and characterization of a novel esterase for biomass conversion
(University of the Western Cape, 2024) Ohlhoff Colin W.; Tuffin Marla
Due to an increasing human population and ever-expanding industrial economies, fossil fuel reserves are rapidly being depleted. In order to reduce our dependence on non-renewable energy resources much global interest has been directed towards the development and production of biofuels. Plant biomass has been identified as a possible feedstock for alternative fuel production and typically consists of cellulose, hemicellulose and lignin. Although these components can serve as valuable substrates in the bioconversion process, they are generally recalcitrant to enzymatic degradation. Current hydrolytic methods limit the depolymerization of the lignocellulose component of plant biomass, which in turn decreases the yield of potential fermentable sugars. As microorganisms represent an intriguing, underexploited resource for the discovery of novel biocatalysts, this study employed a metagenomic approach towards the identification of enzymes for lignocellulose hydrolysis. A large-insert metagenomic fosmid library was constructed using total DNA extracted from thermophilic compost samples. The library comprised 150 000 clones, with an average insert size of 31 Kb, representing approximately 1300 prokaryotic genomes. Initial emphasis was placed on the assessment of the bacterial phylogenetic make-up of the metagenomic library and its validity for the mining of functional enzymes.
Production and characterization of therapeutic peptides with potential antimicrobial properties
(Univeristy of the Western Cape, 2024) Ratshilume, Raphaella; Keyster, Marshall
Bacterial drug resistance is a current and growing serious health problem worldwide because of the long-term and negligent use of antibiotics. Finding new antibiotics and shepherding them through the clinical trials and approval process is becoming increasingly difficult. In addition, the declining number of new innovative antibiotic candidates has worsened this burden, necessitating the development of new classes of antimicrobial agents. Antimicrobial peptides (AMPs) are protein molecules that often form part of the innate immune system and cellular defense mechanisms found in many organisms. AMPs are promising antibiotic alternatives for the treatment of drug-resistant infections. They are, however, constrained by their high manufacturing costs. Genetic engineering and heterologous recombinant peptide expression are promising approaches for producing such molecules at a low cost. In this study, peptide Gene 1 and peptide Gene 2 obtained from the bioinformatics analysis on marine metagenomes sampled from the TARA Oceans Project were produced using the heterologous recombinant peptide expression method. The peptide Gene 1 and peptide Gene 2 were cloned into the pPIC9 vector, transformed, and integrated into the P. pastoris genome. The expression was observed in BMMY medium, pH 6, 1% methanol, and 48 hr, 72 hr, 96 hr with a band at a molecular mass of 6.05 kDa for peptide Gene 1 and 11.37 kDa for peptide Gene 2 following SDS-PAGE analysis.
Effect of green synthesized metal nanoparticles on gene expression in an in-vitro model of non-alcoholic steatohepatitis
(University of the Western Cape, 2024) Nendouvhada, Livhuwani Portia
Metabolic dysfunction-associated liver disease (MASLD) occurs due to an excessive accumulation of fat in the liver (steatosis), independent of secondary causes such as excessive alcohol consumption, viral hepatitis, or certain medications. MASLD is a common chronic liver disease that can lead to end-stage liver disease eventually requiring a liver transplant. It is associated with metabolic syndrome (MetS), especially obesity, diabetes, hyperlipidemia, and hypertriglyceridemia. MASLD can advance to metabolic dysfunction-associated steatohepatitis (MASH), which causes inflammation and damage leading to scarring of the liver (cirrhosis). Only 43-44% of patients with steatosis progress to MASH, with 7-30% of MASH patients progressing to cirrhosis. To date, the mechanism of MASLD and its progression is not well understood, and there are no therapeutic strategies that are specifically tailored for MASLD/ MASH. The anti-obesity and anti-diabetic pharmacological approaches currently used were shown to reduce the MASLD progression, although they remain ineffective to completely treat or reverse its progression.
Development of gold nanoparticles based lateral flow assay for detection of food and water-borne pathogens
(Universty of the Western Cape, 2024) Mabhude, Yandiswa; Sibuyi, Nicole
Food and water are two of the necessities required for survival. With increasing global population, urbanization, and climate change, to name a few, the strain on these two precious resources puts pressure on health as well as the economic sector. Microbial contamination of food and water can lead to lethal infections that are detrimental to human health. More than 90% of reported food-borne illnesses were caused by bacterial species such as Staphylococcus, Salmonella, Campylobacter, Listeria, Bacillus etc. In the last decade, Salmonella was the leading cause of bacterial food-borne illnesses; followed by Campylobacter and Listeria. Listeriosis cases were reported in 2017 in Gauteng province, and the number of infected persons rapidly escalated nationwide prompting an investigation wherein a total of 937 cases were identified. Early detection of pathogens in food and water could prevent or slow down the spread of infections and deaths caused by consuming contaminated food and water. The laboratory and on-site approaches that are currently used for quality control technologies are precise and sensitive, but they have drawbacks such as being laborious and time-consuming. Therefore, there is an urgent need for approaches that may be utilized to detect microbial contaminations at a point-of-care (PoC), that are user-friendly, quick, robust, and sensitive. The use of gold nanoparticles (AuNPs)-based lateral flow assay (LFA) for the detection of these pathogens is gaining traction among researchers as an alternative to conventional methods for on-site testing in places where there is no proper infrastructure, particularly low-resourced areas. The current study aimed to develop an aptamer-AuNPs-based LFA for the detection of food and water pathogens at PoC.
Domestic greywater systems: a potential reservoir of antibiotic resistance genes transfer
(University of the Western Cape, 2024) Kariem, Min-ghah; McCullough, Bronwyn Kirby
During the Western Cape drought from 2015 to 2018, the use of greywater to alleviate pressure on the limited potable water available in the province was encouraged. Greywater, however, has the potential to harbour residual antibiotics and pathogenic bacteria, thereby potentially supporting the growth and proliferation of antibiotic resistant bacteria. This study combined traditional microbiology tests with molecular biology to detect resistant microorganisms within domestic greywater systems and their associated biofilms, while Quantitative PCR (qPCR) was used to determine the levels of clinically relevant antibiotic resistance genes (ARGs) (vanA, ampC and aadA), as well as the intI1 gene, which serves as a marker of horizontal gene transfer. Phenotypic resistance was confirmed using the Kirby-Bauer disc diffusion method. Notably, high levels of resistance to ampicillin and kanamycin were found in greywater and biofilm samples, as well as resistance to vancomycin. Bacterial viability was assessed using flow cytometry using the LIVE/DEAD BacLight Bacterial Viability kit, and it was revealed that a large portion of the cell population within greywater was dead or injured. qPCR analysis confirmed the presence of clinically relevant ARGs vanA and ampC, in domestic greywater samples, with abundance fluctuating in response to seasonal change. Additionally, the intI1 gene was detected in all greywater and biofilm samples, which suggests that genetic exchange occurs amongst bacteria in greywater and biofilm samples. Our findings support the hypothesis that greywater systems are colonised by resistant bacteria, which can form stable communities within the water environment which allows for high levels of genetic exchange. These findings support the use of qPCR and flow cytometry, in combination with traditional microbiology tests for monitoring antibiotic resistance in environmental samples. While the use of greywater is a viable method to reduce the demand for potable water, this study highlights the fact that the improper use of greywater poses a risk to the environment and public health. As such, the public should be better educated on how to safely use these systems.
Biochemical characterisation of an alpha-amylase with pullulan hydrolase type III characteristics derived from a hot spring metagenomics library
(University of the Western Cape, 2024) Boersma, Bianca; Trindade, Marla
The field of metagenomics has provided biotechnological researchers with new enzymes and insight into previously unknown enzyme families with potential for novel modes of action and being able to function under extreme conditions, such as extremes of temperature, acidity or alkalinity. The enzymes can also often act on multiple substrates and show increased affinity for certain substrates. In this study, the aim was to biochemically characterise a putative type III pullulan hydrolase (PHTIII). A sequence-based metagenomic screen from previous performed by Xiao Ping Hu (2010) was used to identify novel glycosyl hydrolase enzymes in shotgun metagenomic sequence data from the Mphizi hot spring in Malawi. An identified ORF, named Pull3.1, displayed low sequence identity to a characterised α-amylase (62.71%) and 61.66% to a characterised pullulan hydrolase type III, and was, therefore, hypothesized to be an α-amylase (EC 3.2.1.1) with pullulan hydrolase type III (EC 3.2.1.x) characteristics. Further sequence analysis indicated that Pull3.1 had a glycogen binding site and that it belongs to the GH13_20 family as well as having a family 48 carbohydrate-binding module. Pull3.1 displayed the highest substrate conversion rates for starch, pullulan and glycogen. The products released were of the following degrees of polymerisation 3, 2 or 1, in accordance with the products released from the PHTIII from Thermococcus aggregans. Pull3.1 is a thermozyme that shows maximum activity at 75°C and an optimum pH of 7. It was thermostable up to 80°C and had a half-life of 11.6 min at 50°C. Further kinetic characterisation showed that Pull3.1 had a high affinity for starch. The KM value of 0.048 mg/ml is within the range reported for other α-amylase enzymes, and Pull3.1 had a Vmaxof 0,729 (μmol mL−1). All of this data indicates that Pull3.1 is a good candidate for a starch conversion process that requires thermozymes.
South African medicinal plant extracts used in the treatment of fungal infections.
(University of the Western Cape, 2023) Ndlovu, Banele Michelle; Rahaman Fisher, F
The incidence of fungal skin infections is increasing at an alarming rate, especially in people with underlying immunosuppressive conditions. Currently, the most used methods of treatment are in the azole group of synthetic treatments. A major problem with these synthetic treatments’ is the increase in azole-resistant strains spreading worldwide. Additionally, an estimated 65–80% of all fungal infections are biofilm related, with biofilms found in Candida species such as Candida albicans, Candida dubliniensis, Candida glabrata and Candida tropicalis which infect the skin. There is an increased demand for alternative treatments that could be antifungals and medicinal plants could be the potential solution, as some plants possess antifungal properties. The overall aim of this study is to investigate the potential effect of South African medicinal plants used for the treatment of fungal infections against pathogens of dermatological relevance. The objectives were 1) to conduct a comprehensive literature search to identify indigenous medicinal plant extracts used to treat fungal skin infections, 2) to perform a cross-sectional study using an interview-based questionnaire to investigate the knowledge and practices of Cape bush doctors and validate their use of selected medicinal plants for treating dermatophyte infections in the Cape Metropole communities and 3) to investigate the in vitro bioactivity of selected plant extracts identified from the literature search and interviews. Fifteen medicinal plants were found using an in-depth literature search and all 15 plants were validated during the interviews as plants commonly used for fungal skin infections.
Valorisation of brewers’ spent grain: enzymatic hydrolysis in the production of xylooligosaccharides
(University of the Western Cape, 2023) Arries, Chelsey; den Haan, Riaan
The beverage industry constitutes approximately 26% of all food wastes, making it one of the largest contributors in this waste segment. By utilising waste or by-products from agriculture and food production in manufacturing value added compounds, the concepts of waste mitigation and green chemistry can contribute to establishing a circular bio-economy. In a biorefinery, bio-catalytic, thermal, chemical and physical techniques are used to extract valuable compounds from food and agricultural wastes. Brewers’ spent grain (BSG) is an ideal candidate for such a biorefinery approach. This high moisture, nutrient-rich by-product from beer production is either disposed of in landfills or used as an animal feed. However, high value products, such as xylooligosaccharides (XOS) can be extracted from BSG, thereby valorising this brewery waste. Xylooligosaccharides are sought after for their ability to function as a low caloric sweetener while exhibiting prebiotic effects in stimulating the growth of probiotic bacteria in the mammalian gut.

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