Doctoral Degrees (Microbiology)
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- ItemCharacterisation and consequences of urban water resilience at the human-mycobiota interface: risk versus reward(Stellenbosch : Stellenbosch University, 2024-03) Smith, Katrin Mary Geneve; Wolfaardt, Gideon M. ; Stone, Wendy; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Urban waters reflect the confluence of microbial ecology and anthropogenic influences. The interactions of microorganisms, chemical micropollutants (MPs) and the surrounding environment require in-depth delineation with a dual purpose. While microbes and MPs can pose risks to humans and ecology, an understanding of physico-chemical, species dynamics and intrinsic MP resilience at the microbial-contaminant interface can benefit future MP attenuation strategies. Therefore, the goal of this research was to characterise and harness urban river microbial and chemical constituents, and the interplay thereof. Moreover, focus was placed on the assessment of fungi and antifungals (AFs), represented by comparatively less literature than bacteria and antibiotics. An environmental survey of representative antifungals in regional rivers revealed nanogram per litre concentrations in alignment with similarly low-risk levels detected globally. Trends reflected agricultural practises and medical use, showing wastewater treatment plant (WWTP) influence on rivers. Bioassays detected androgen (AR) antagonistic and aryl-hydrocarbon receptor (AhR) activity from agricultural fungicides, and AF-MP mixture responses could be attributed to individual effects of carbendazim, dimethomorph, flusilazole and procymidone. Moreover, concentration factors applied to achieve toxicity quantification were ten- to 100-fold higher than the average MP levels detected in local and global waters. This indicates more beneficial bioassay utility for whole effluent or environmental samples with elevated MPs and metabolites, where concentrations are inherently higher than typical river water. Analyses of fungi in riverine consortia revealed limited clinically- relevant taxa that were associated with elevated pollution. The acclimatisation of consortia to sterile river water, containing MPs, in bioreactors favoured community shifts toward environmental rather than opportunistic taxa. Acclimatised microbiota were thereafter exposed to AF and AF-MP mixtures in reactors, the former containing low and the latter 100-fold higher microgram (per litre) concentrations. Fungal numbers increased despite limiting conditions including the presence of inhibitory AFs. After in vitro exposure, AF susceptibility differences were assessed with two medical and agricultural AFs apiece. Inherent levels of resilience in the consortia were apparent, with enhancement observed post-exposure, particularly toward fungicides. This was more evident in communities exposed to sub-inhibitory AFs, compared to the MP mixture, aligning with reports on exacerbated drug-resistance in environmental taxa subject to consistent contact with low-level antimicrobial (AM) and non-AM mixtures. However, despite an increased risk of AFR, the observation of resilience toward MPs demonstrates advantages for environmental consortia in bioremedial applications, as sub-inhibitory exposure does not limit metabolism or survival. As such, MP degradation potential was investigated in continuous flow reactors under environmental conditions. Significantly higher removal rates were observed for inoculated reactors compared to uninoculated controls. Despite the presence of broad-spectrum AFs, the microbiota exhibited higher removal rates for several AFs compared to other MPs. The biological reactors also significantly reduced estrogenicity to below human toxicity thresholds. Although some MP removal rates were lower than reported in exploratory literature on fungal or mixed microbial WW treatments, the aim of this study was to investigate unmanipulated consortia in energy- and cost-effective conditions representing parameters that remain less explored. Field-scale, feasible bioremediation setups are critical for future remediation of anthropogenic pollution and protection of water resources. Urban waters comprise both the natural interplay of microorganisms and MPs in surface waters, and the harnessing of these interactions in engineered ecosystems, including WWTPs. Environmental and manipulated settings apply direct and indirect pressures on microbiota, the consequences of which may be negative or advantageous. Monitoring for MPs, microbes and drug-resistance is important for characterising potential human and ecosystem health risks. On the other hand, the predisposition of microbiota toward MP resilience, and their adaptability to a wide range of conditions, provides opportunity for microbiological applications in MP attenuation. This research contributes toward an understanding of ecological dynamics at the microbial-contaminant interface to help inform the development of energy- and cost- efficient bioremediation strategies.
- ItemCharacterization of atypical class I lanthipeptides from the marine bacterium thalassomonas viridans XOM25ᵀ(Stellenbosch : Stellenbosch University, 2024-03) Vermeulen, Ross; Dicks, Leon Milner Theodore ; Van Staden, Anton du Preez; Trindade, Marla; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The natural products (NPs) that are produced by living organisms have historically served as a pivotal starting point for numerous highly effective therapeutic agents. These compounds often possess distinct chemical structures that enable valuable mechanisms and modes of biological activity. Natural products are thus a promising reservoir that potentially contains the key to the treatment of diseases like cancer, infections, resistance, neurological disorders, and even a range of agricultural and environmental challenges. The rate of novel drug discovery is exhibiting a gradual decline. However, recent advancements in biotechnology, such as next-generation sequencing (NGS) and heterologous gene expression, are empowering researchers to access unexplored realms of biological and chemical diversity. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of NPs that have experienced significant growth by combining NGS and heterologous expression. Within the RiPP group, lanthipeptides are characterized by the post-translational installation of lanthionine rings or thioether cross-linkages. The incorporation of lanthionine rings offers several advantages such as enhanced stability and expanded structural diversity at low genetic cost. However, the process of post-translational modification poses distinct challenges when attempting to heterologously produce lanthipeptides exhibiting previously unobserved and markedly distinct characteristics. To date, the majority of described lanthipeptides play an antimicrobial role which provides a competitive advantage to the native producer by inhibiting the growth of similar or closely related species. Recently, a significant number of cryptic lanthipeptides have been heterologously produced which do not exhibit antimicrobial activity against species related to the native host. Many of these seemingly inactive and atypical lanthipeptides originate from Gram-negative bacteria associated with the marine environment. In this work, a significantly dissimilar lanthipeptide operon was identified in the genome of the marine Gram-negative marine bacteria Thalassomonas viridans XOM25T. The operon was used to construct an Escherichia coli BL21 expression system capable of co-producing the peptide substrates and post-translational modification machinery. The target peptides were subsequently isolated, confirmed to contain post-translationally installed lanthionine rings, and designated as the viridisin operon. Finally, the antimicrobial, stereochemical and pharmaceutical potential of the viridisins were assessed. The viridisins have similar stereochemistries to other lanthipeptides from Gram- negative bacteria, and likewise, they do not have antimicrobial properties. However, this study proved that the viridisin were able to modulate zebrafish larvae behavior. While the results of the zebrafish behavioral screening do not provide definitive proof of the mechanisms at play, such findings allude to the remarkable diversity of biological roles that RiPPs fulfil – intended or otherwise.
- ItemFunctionalised nanofibers for water treatment(2023-12) Cloete, Thomas Eugene; Wolfaardt, Gideon M.; Klumperman, Bert ; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Access to safe drinking water and adequate sanitation is a pressing global issue with significant implications for health, welfare, and economic development. Nanotechnology holds promise in addressing the challenges associated with water purification. Specifically, functionalized nanofibers have emerged as a potential solution for water treatment. In one study, antimicrobial poly (vinyl alcohol) nanofibers were produced by incorporating AquaQure biocide into the polymer solution. These nanofibers demonstrated strong antimicrobial properties, achieving a substantial reduction in populations of various bacteria strains including E. coli, S. aureus, S. typhimurium, P. aeruginosa and K. pneumoniae bacteria. Additionally, immobilized enzymes were explored as a means of breaking down biofilms and preventing their formation. Non‐woven mats made of Polyacrylonitrile (PAN) were successfully fabricated using different electrospinning processes, retaining over 80% of the enzymes' specific activity. These functionalized membranes offer potential applications in the water filtration industry by preventing biological fouling of membranes. A cartridge filter where activated carbon was encased in electrospun nanofibers was manufactured and evaluated. Culturable methods indicated that the filter could produce 3 l of potable water. However, when using PCR assays, it was unable to eliminate Klebsiella spp., Legionella spp., Pseudomonas spp., and Yersinia spp. from the filtered rainwater harvested tank water. This suggested that these bacteria were in the non‐ culturable but viable state and passed through the filter. Furthermore, adenovirus, being small enough to pass through the membrane's pores, was not effectively removed by the cartridge filter. Overall, this research demonstrates the potential of functionalized nanofibers and immobilized enzymes in developing advanced filter media for water filtration. However, challenges remain in effectively removing certain pathogens and viruses, highlighting the need for further improvements and innovations in the field.
- ItemElucidating the duality of biofilms as both a proliferation and survival strategy using novel IoT technology(Stellenbosch : Stellenbosch University, 2023-12) Klopper, Kyle Brent; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Microbes rarely exist and proliferate as individual cells, but rather form complex community structures known as biofilms. Biofilm formation ensures that microbial communities are ubiquitous throughout both natural and man-made environments, where the presence of microbial biofilms can be advantageous or detrimental, depending on the context. Biofilm formation is traditionally viewed as a mechanism for microbial survival, since it provides protection from adverse environmental challenges, including starvation, desiccation, predation and antimicrobials. However, a more contemporary perspective also takes the role of biofilms in microbial proliferation into consideration. The reductionist approach, which classifies a biofilm as either a survival or a proliferation mechanism often leads to poor outcomes when the aim is to transition fundamental biofilm research to the real-world and its associated problems. Owing to the inherently complex and dynamic nature of biofilms, a multi-parameter approach is required to better understand its form-function relationship. The development of the novel BioSpec system provides a simple, reliable, and cost-effective tool to monitor biofilm biomass at high- resolution and in real-time, without disturbing the biofilm structure. Coupling of the BioSpec with the carbon dioxide evolution monitoring system (CEMS), facilitates the simultaneous measurement of two important interrelated biofilm metrics; namely biofilm biomass and metabolic activity. The ability to monitor these parameters non-destructively in real-time provides an unprecedented level of insight into biofilm dynamics, as demonstrated here when challenged with biocides at sub-inhibitory and inhibitory concentrations. The inherent complexity of biofilms requires biofouling mitigation strategies that are based on well-informed chemical treatment dosing regimens and the right combination of biocides and dispersants. In industrial applications, the rapid response of microbial respiration rates to chemical treatment offers a strong utility to monitor the success of antimicrobial application, whereas biofilm biomass measurement shows promise as a direct measure of dispersal or removal efficacy. The role of biofilms as ‘planktonic cell production factories’, even when challenged with antimicrobials, was again highlighted in this thesis and emphasises the need for a change in perspective. Microbial proliferation is of critical importance to multiple facets of society ranging from the food, to the medical and industrial sectors. For too long, the emphasis has been placed on the survival attributes of biofilms, thereby neglecting the equally important role of biofilms as mechanism of proliferation and dissemination. The latter has important implications for sanitation regimes in food and beverage processing facilities, where planktonic cell dissemination and secondary biofilm formation at distant locations within a facility, pose serious challenges and risks.
- ItemInteractions between gut microbiota and the central nervous system, with emphasis on quorum sensing between commensal lactic acid bacteria and human cells(Stellenbosch : Stellenbosch University, 2023-03) Dicks, Leon Milner Theodore; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The human gut hosts close to 4 trillion microorganisms, which is nearly equivalent to the estimated 3.0×1013 human cells in a 70 kg body. Although the composition of gut microbiota changes with age, variation in diet, medication, hormone levels, stress and other environmental factors, a core group of autochthonous bacteria, between 400 and 500 species, are always present. More than 90% of the gut microbiome is represented by Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, with the latter in the majority. Fusobacteria and Verrucomicrobia make up the remaining 10% of the gut microbiome. The human gut microbiome supersedes the number of cells in our bodies ten-fold. Since lactic acid bacteria (LAB) are the predominant gut microbiota, it is safe to conclude that changes in this group will affect the entire microbiome, ultimately leading to adjustments in the behaviour of intestinal epithelial cells (IECs). Changes in the immune system and quorum sensing (QS) signals instigated by an altering gut environment trigger a cascade of hormonal and neurological reactions. Activation of Toll-like receptors, for instance, induce strong immune and inflammatory reactions, but at the same time stimulate the secretion of hormones such as 5-hydroxytryptamine (5-HT, or serotonin), glucagon-like peptide-1 (GLP-1), peptide tyrosine tyrosine (PYY), glucose-dependent insulinotropic peptide (GIP), cholecystokinin (CCK), ghrelin, leptin, pancreatic polypeptide (PP), oxyntomodulin and neurotensin. Serotonin act as neurotransmitter but also regulates diverse functions such as platelet aggregation, bone development, immune response, cardiac function and gut homeostasis, and control enteric motor and secretory reflex. Gut bacteria also synthesize, or regulate, the production of serotonin and other neurotransmitters such as glutamine (Glu), gamma-amino butyric acid (GABA), dopamine (DA), norepinephrine and histamine. These molecules communicate with the central nervous system (CNS) via afferent fibers in the Vagus nerve (VN), autonomic sympathetic and parasympathetic nervous systems, but also the hypothalamic-pituitary-adrenal axis (HPA). Intermediate compounds such as short-chain fatty acids (SCFAs), tryptophan and secondary bile acids produced by gut bacteria also communicate with the CNS. Signals received from the brain are sent back to entero-epithelial cells (EECs) via the HPA and efferent VN fibers to complete the circle of communication referred to as the gut-brain axis (GBA). The modulation, development, and renewal of neurons in the enteric nervous system (ENS) are controlled by gut microbiota, especially those with the ability to produce and metabolize hormones. Minor activation of the ENS and VN results in drastic changes in the production of neurotransmitters, which also affects digestion, intestinal permeability, gastric motility, and immune regulation. GABA, in addition to other metabolites, play an important role in anti-inflammatory responses and help alleviate psychiatric symptoms stemming from inflammation. Treatment of schizophrenic and bipolar patients with probiotics alleviated symptoms associated with irritable bowel disease (IBD), and autistic children benefitted from probiotic treatment. Obsessive compulsive disorder (OCD)-like behavior could also be controlled by treatment with LAB. Inter- and intra-species signalling systems have been well studied, but far less is known about interkingdom quorum sensing (QS), especially between gut bacteria and intestinal epithelial cells (IECs). Although the auto-inducer 3 (AI-3)/epinephrine (Epi)/norepinephrine (NE) QS signalling system described for pathogenic Escherichia coli, Salmonella typhimurium and Ctirobacter rodentium are widely used by Gram-negative pathogenic bacteria, not all species have receptors that recognize these signals. Instead, they have developed “broad-range” “solo” LuxR-type receptors such as SdiA (a LuxR homolog) and QscR to improve their communication abilities. Despite our knowledge on QS, the effect of these signalling molecules on the CNS is ill-researched. Several QS peptides (QSPs) have the ability to diffuse through the intestinal mucosa and enter the circulatory system, from where they may penetrate the blood-brain barrier (BBB). It may be that LAB communicate with the CNS using small linear or cyclized oligopeptides (QS peptides, QSPs) of 5 to 17 amino acids long, as reported for other Gram-positive bacteria. In our own research we have shown that bacteriocins can indeed transverse epithelial (Caco-2) and endothelial (HUVECs) monolayers without changing the integrity of the membranes and with no toxic effect. Once in the blood stream, bacteriocins may cross the BBB, similar to that reported for the heptapeptide PapRIV produced by Bacillus. Our understanding of exactly how gut microorganisms control cognitive behavior, mood, and neuropsychiatric disorders remains limited. However, the more we discover about the gut microbiome, QS, neurotransmitters and the GBA, the greater the chance of developing novel therapeutics, probiotics and psychobiotics to treat gastro-intestinal disorders such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), but also improve cognitive functions and prevent or treat mental disorders. This calls for in-depth deciphering of the complex, everchanging network between cells and neurons. Research on the quenching of QS signals need to be prioritised. We need to understand how quorum quenching (QQ) therapy will affect beneficial gut microbiota. Biomarkers need to be developed to identify differences in the gut microbiome of individuals suffering from psychological disorders. Interactions between drugs used in treatment and gut microbiota need to be studied in greater depth. We need to understand the effect psychiatric medication may have on the composition of the gut microbiome. Are intestinal microbiota able to metabolise these drugs? Studies should include multi-omics of gut and oral microbiota to have a better understanding of the mutual interplay between phyla. Will it be possible to develop probiotics to treat dysbiosis and neuropsychiatric abnormalities?