- 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; 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.
- ItemBiodegradability of organic carbon following hydrothermal carbonization treatment(Stellenbosch : Stellenbosch University, 2021-12) Mangashena, Hazel; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Hydrothermal carbonization (HTC) is a ‘wet pyrolysis process’ that opens up a field of potential for feedstocks such as the non-traditional renewable and abundant wet agricultural residues, and municipal wastes for char production. It reduces waste and substitutes primary fuels as a source of energy. The product, known as hydrochar, has received attention because of its potential as precursors of activated carbon in wastewater remediation, soil remediation applications, solid fuels, and other carbonaceous materials. However, persistent priority and emerging micropollutants are consistently detected in numerous wastewater treatment plants that could potentially serve as feedstock for the process- this is a cause for concern. The goal of this study was to assess the impact of HTC on organic micropollutants. The working hypothesis was that the combination of high temperature and pressure would sterilize the biomass and transform micropollutants into benign products, while any residue would be readily biodegradable. Experiments indicated that char resulting from belt press sludge carbonized at 240 (with a corresponding°C pressure of 3,3MPa) was sterile, exhibiting a high fuel ratio of 0,72 and HHV 21,40 Mj/kg. Subsequently, 4 test compounds that are; carbamazepine, chloramphenicol, methylparaben, and bisphenol-A were studied for their biodegradability following HTC. Biodegradation of the test compounds extracted from the hydrochar ranged between 84% and 100% while only methylparaben indicated complete biodegradability from the liquid phase. Results of the study prove that with optimization, the question presented by residual micropollutants persisting in the natural environment following HTC technologies can be mitigated.
- ItemAssessing the capacity for micropollutants to induce changes in the biofilm EPS composition and yield(Stellenbosch : Stellenbosch University, 2021-12) Rossouw, Johann Herman; Wolfaardt, Gideon M.; Stone, Wendy; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: A consequence of widespread chemical manufacturing and usage is the increasing presence of a new class of contaminant: micropollutants. Despite the investment of significant resources into the development of novel approaches to wastewater treatment, the removal efficiency of micropollutants has been varied and conflicting between different studies. A notable gap in current research efforts is assessing the capacity for chronic micropollutant exposure to alter the main mechanisms contributing to their removal in secondary wastewater treatment. The aim of this study was three-fold. First, to investigate the comparative homogeneity of the Slime-EPS matrix composition for multi- versus single-species biofilms. Secondly, to attempt to quantify the adsorptive capacity of the Slime-EPS fragment utilizing a published dye-probing analysis protocol. Finally, to assess the capacity for chronic (7-day) micropollutant exposure to influence the composition and yield of the Slime-EPS fragment for a known, biofilm producing Pseudomonas species. Single-species biofilms exhibited a more consistent Slime-EPS composition in terms of their protein: carbohydrate ratio. Dye-probing analysis efforts indicated the capacity for toluidine blue dye to exhibit altered spectral absorbance as a result of increased dimerization – which was found to be influenced by both the introduction of the Slime-EPS itself and increasing concentrations of NaCl. Increasing concentrations of TB dye was shown to induce hypsochromic (blue-) spectral shifts. Ciprofloxacin was found to significantly reduce the biofilm Slime-EPS yield (p < 0.05) following 7 days of continuous exposure, whereas exposure to diclofenac for the same interval had no significant effect on Slime-EPS yield. Neither ciprofloxacin nor diclofenac had a significant effect on the Slime-EPS protein: carbohydrate ratio following 7 days of exposure.