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Molecular epidemiology of antibiotic resistant ESKAPE and escherichia coli pathogens isolated from bloodstream infections in public sector hospitals in uMgungundlovu district, KwaZulu-Natal, South Africa.
(2024) Hetsa, Bakoena Ashton.; Essack, Sabiha Yusuf.; Mbanga, Joshua.; Abia Akebe , Luther King.; Amoako, Daniel Gyamfi.
Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. (ESKAPE) pathogens and Escherichia coli (ESKAPEEc) are major contributors to bloodstream infections (BSIs) and pose significant treatment challenges. This study describes the molecular epidemiology and genomic profiles of ESKAPEEc isolates from BSIs in two public hospitals in the uMgungundlovu District, South Africa.
Blood samples (n = 195) were collected from adult and paediatric patients with suspected BSIs between November 2017 and December 2018. Isolates were identified using VITEK 2 system, and confirmed by polymerase chain reaction (PCR). Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method and interpreted according to EUCAST/CLSI guidelines. Whole-genome sequencing (WGS) and bioinformatics tools were used to determine the resistome, virulome, mobilome, clonality and phylogenies of selected K. pneumoniae, K. oxytoca, and S. aureus isolates.
Out of 195 presumptive isolates, 159 were confirmed as ESKAPEEc. K. pneumoniae (28.9%) and S. aureus (28.3%) were the most predominant pathogens. In total, 151 (94.9%) of isolates were multi-drug resistant (MDR). K. pneumoniae and K. oxytoca carried β-lactamase genes including the blaCTX-M-15, blaOXA-1, blaTEM-1B, blaSHV, and blaOXY genes. The blaCTX-M-15 and blaTEM-1 genes were associated with Tn3 transposons and insertion sequences (ISs) (ISEc9, IS91). Virulence genes detected in Klebsiella spp. were associated with ISs (IS1, IS3, IS630) and integrase. Six sequenced S. aureus isolates harboured ARGs including blaZ, aac(6′ )- aph(2′′), ant(9)-Ia, ant(6)-Ia, and fosB among others. Most ARGs were associated with ISs (IS6, IS1182) and recombinase. Virulence genes identified in S. aureus isolates were mostly associated with ISs, recombinases and integrases. Phylogenetic analysis of selected K. pneumoniae and S. aureus isolates revealed clear grouping patterns based on Multilocus Sequence Typing (MLST) and geographic origin.
WGS and bioinformatics analysis revealed that Klebsiella strains and S. aureus have a rich repertoire of ARGs, virulence genes, and MGEs in several permutations and combinations, indicating a complex situation of mobilized antibiotic resistance and pathogenic characteristics in clonal and multi-clonal strains responsible for BSIs in this healthcare context.
Tetradenia riparia (hochst.) Codd (lamiaceae) aqueous leaf extract enhanced oxidant production and nf-b mediated apoptosis in hepatocellular carcinoma (HEPG2) cells.
(2024) Gcaba, Seluleko.; Khan, Rene Bernadette.; Khumalo, Hlolisile.
Introduction: Cancer has become a global health problem, contributing almost 20 million new cases and 10 million deaths worldwide in 2020. Ranked third globally, liver cancer accounted for 8.3% of the global cancer-associated mortality. Hepatocellular carcinoma (HCC) accounts for more than 90% of diagnosed primary liver cancer cases. Treatment may include chemotherapy, radiation therapy and liver transplant, but these treatments are expensive and induce serious adverse effects that reduce the efficacy of treatment. Medicinal plants are the suggested alternate treatment option attributed to anti-microbial, antioxidant and anti-inflammatory effects that may ameliorate the harsh side effects, but also demonstrate anti-cancer activity. Tetradenia riparia is a readily available South African shrub with medicinal and anti-cancer potential, but the mechanisms require elucidation. This study aimed to investigate the antioxidant and cell death mechanisms activated by Tetradenia riparia aqueous leaf extract (TRALE) in HCC (HepG2) cells.
Methods: HepG2 cells were treated with several concentrations of TRALE (0–3000 μg/ml) for a duration of 24 hours to assess the cell viability. The methylthiazol tetrazolium (MTT) assay was used to obtain an IC50, which was used to treat the HepG2 cells for all subsequent assays. After treatment, mitochondrial activity was assessed as a measure of cell viability using luminometry for the ATP and mitochondrial membrane potential (m) assays. The cells were assayed for oxidative stress by quantifying free radical mediated membrane damage (TBARS, NOS and LDH cytotoxicity assays, and iNOS gene expression), and antioxidant response using luminometry (GSH, GSSG, ratio of GSH/GSSG), qPCR (SOD2, catalase, GPx1, Nrf2) and western blotting (HSP70). Induction of apoptosis was determined by luminometrically quantifying caspase-3/7, caspase-8, and caspase-9 activities. Furthermore, the Annexin V apoptosis and necrosis assay elucidated the cell death pathway. Protein expression of cIAP (Western blot) and qPCR for NF-κB and BCL-2 were also evaluated.
Results: After a 24-hour exposure, TRALE conferred a dose-dependent reduction in cell viability that was associated with a notable drop in ATP at the IC50. An increase in ΔΨM led to increased levels of ROS, which increased lipid peroxidation and RNS production despite downregulated iNOS. Elevated SOD2 in response to ROS production enhanced the HepG2 cell’s ability to convert superoxide radicals into H2O2, while catalase and Gpx1 were upregulated to prevent harmful ROS formation and protect macromolecules from oxidative damage. Increased GPx1 was associated with depletion of GSH and the GSH/GSSG ratio, but Nrf2 was downregulated and HSP70 was similar to the control. The downregulation of Nrf2 was associated with increased NF-κB. Initiator caspase-8 activation corresponded with downregulated cIAP, while downregulated BCL-2 contributed to caspase-9 activation. In addition, activation of caspase-3/7 facilitated phosphatidylserine externalisation. Necrotic markers and LDH were decreased, suggesting that TRALE induced apoptosis in HepG2 cells.
Conclusion: The decreased cell viability was associated with depletion of ATP, while m contributed to ROS production that increased RNS and caused lipid peroxidation. Although GSH was depleted and Nrf2 was downregulated, the upregulated SOD2, catalase and Gpx1 suggest a response suggested a response to ameliorate oxidative stress as implied by unchanged HSP70. However, oxidants were still elevated and associated with increased NF-κB. Interestingly, iNOS expression was downregulated, thus NF-B contributed to initiation of apoptosis that was associated with decreased BCL-2 and cIAP, and increased caspase activity. In addition, necrosis was not evident. Therefore, the TRALE-treated HepG2 cells were more susceptible to apoptosis. Further studies are required to elucidate the role of NF-κB in TRALE-induced apoptosis.
An in-silico and in-vivo investigation on the effects of acute fumonisin b1 exposure on inflammation and epigenetics in C57BL/6 mice hearts.
(2024) Gounder, Selwyn Kyle.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Fumonisin B1 (FB1), a mycotoxin produced by Fusarium species, is a significant contaminant in cereal grains, posing serious health risks. This pilot study investigated the cardiotoxic effects of acute FB1 exposure on inflammation and epigenetic modifications in C57BL/6 mice. Given the pilot nature of this study, acute exposure was prioritised to establish initial findings. Molecular docking was employed to predict the binding interactions of FB1 with key inflammatory proteins, including tumour necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) subunits. In vivo experiments involved treating mice with 5 mg/kg FB1 for 24 hours, followed by heart tissue analysis using quantitative polymerase chain reaction (qPCR), Western blotting, nitric oxide synthase assay (NOS assay), and enzyme linked immunosorbent assay (ELISA) to assess selected gene and protein expression levels of inflammatory markers and DNA methylation. Docking results indicated that FB1 binds to inflammatory proteins TNF-α, iNOS, NF-κB (p65), and NF-κB (p50), potentially altering their function. Gene expression analysis revealed significant downregulation of pro-inflammatory cytokines (TNF-α, interleukin-6 (IL-6), interleukin-1β (IL-1β)) and the anti-inflammatory cytokine interleukin-10 (IL-10), while protein analysis showed an upregulation of these cytokines, suggesting a complex regulatory mechanism. Additionally, FB1 exposure led to increased levels of reactive nitrogen species and significant upregulation of DNA methylation, indicating epigenetic modulation.
This study elucidates the cardiotoxic effects of FB1 on mice, emphasizing the intricate interplay between inflammatory pathways and DNA methylation. Molecular docking studies suggest that FB1 may bind to key residues on TNF-α, iNOS, and NF-κB subunits, potentially modulating these proteins' activity and triggering inflammatory responses. In vitro analysis demonstrated significant dysregulation of inflammatory and DNA ethylation-related genes, with a notable upregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and anti-inflammatory mediators (IL-10, Transforming growth factor beta 1 (TGF-β1)) at the protein level. The observed disparity between gene and protein expression could be attributed to several factors, including post-transcriptional and post-translational modifications. These modifications are crucial in biological processes and can cause differences between mRNA and protein levels. Translational regulation, which involves the recruitment of various mRNA species to the ribosome, can lead to a decreased correlation between mRNA and protein amounts. Additionally, the study found contradictory DNA methylation results: global DNA methylation levels were upregulated, indicating hypermethylation, while DNA methylation gene (DNMT) expression was decreased. This suggests a complex interplay between DNA methylation and gene expression, potentially influenced by other regulatory mechanisms like microRNAs. These findings highlight the need for further validation using additional tests, such as Northern blots and microarray assays. Overall, the study underscores FB1's ability to activate inflammatory pathways and cause cardiac distress through cytokine dysregulation and epigenetic changes. Further research is essential to fully understand the mechanisms of FB1- induced cardiotoxicity and the potential therapeutic role of DNA methylation in mitigating these effects.
An investigation into the inflammatory, oxidative stress and DNA methylation status of Fumonisin B1 in the human U87MG glioblastoma cell line.
(2025) Foolchand, Ashmika.; Chuturgoon, Anil Amichund.
A frequently overlooked global health issue includes fungal and mycotoxin contamination, which infects many staple foods across the globe. Owing to its ubiquitous nature, long-term exposures to varying doses of mycotoxins pose major health concerns in humans and animals. Fumonisin B1 (FB1) is a toxic secondary metabolite commonly found in contaminated maize, known to induce various toxicities. This study investigated the effects of FB1 on inflammatory responses, oxidative stress, and global DNA methylation in human glioblastoma U87MG cells after a 24-hour exposure.
The MTT assay was performed to determine the FB1 IC50 value in U87MG cells, which was used for all subsequent experiments. Following FB1 treatment, DNA, RNA and protein were extracted from U87MG cells and standardized. An ELISA was then conducted using the standardized DNA samples to assess DNA methylation status. cDNA, synthesized from standardized RNA, was used to measure gene expressions of DNA methyltransferases (DNMTs), methyl-CpG binding domain 2 (MBD2), superoxide dismutase 2 (SOD2), catalase (CAT), glutathione peroxidase (GPx), nuclear factor erythroid 2-related factor 2 (NRF-2), 8-oxoguanine glycosylase 1 (OGG1), interleukin (IL)-6, IL-10 and nuclear factor-kappa B (NF-kB), via qPCR. Standardized protein samples were diluted in sample buffer and analysed by western blot to evaluate protein expressions of DNMTs, MBD2, SOD2, CAT, mitochondrial transcription factor A (TFAM), IL-1β, tumour necrosis factor-alpha (TNF-α), brain-derived neurotrophic factor (BDNF) and cyclooxygenase-2 (COX-2).
Inflammation is a key mechanism of host immune defense, which is triggered by pro-inflammatory cytokines and maintained by anti-inflammatory cytokines. In U87MG cells, FB1 significantly reduced the expression of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, and NF-κB while enhancing the expression of the anti-inflammatory cytokine IL-10. Additionally, it decreased the pro-inflammatory marker COX-2 and increased the anti-inflammatory brain marker BDNF.
Oxidative stress, caused by the imbalance between free radicals and antioxidants, is a known cellular toxicity mechanism of FB1 which results in DNA damage, protein degradation and neurodegenerative diseases. Here, FB1 decreased lipid peroxidation and antioxidant responses of SOD2, CAT, GPx, and NRF-2, while also inhibiting oxidative DNA damage through regulation of the OGG1 gene. Mitochondria are susceptible to reactive oxygen species (ROS) attack due to oxidative stress, which leads to mitochondrial dysfunction and mutations in mitochondrial DNA. Although FB1 prompted the upregulation of Sirtuin 3 (SIRT3), lon protease 1 (LonP1), and heat shock protein (HSP60) to support mitochondrial health in U87MG cells, it simultaneously caused alterations in mitochondrial DNA by downregulating mitochondrial transcription factor A (TFAM).
The regulation of gene expression by DNA methylation is suggested to influence biological processes including neurogenesis and the pathogenesis of brain disorders. FB1-treated U87MG cells displayed global DNA hypermethylation, evidenced by increased levels of 5-methylcytosine and elevated expressions of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), alongside a significant decrease in demethylase expression (MBD2), providing an alternative mechanism for FB1 toxicity.
Taken together, this data suggests that FB1, over a short period of time, may initially act as a selective neuroprotective agent by modulating inflammation, oxidative stress and mitochondrial responses, while also highlighting its potential neurotoxic effects through mitochondrial dysregulation and global DNA methylation.
An investigation into adult learner dropout at a community learning centre in KwaZulu- Natal.
(2024) Maphumulo, Precious Primrose Bongiwe.; Harley, Anne.
Adult education is essential to national development. Education is the bedrock of any nation’s quest to stem poverty. Adult education affords individuals previously denied access to education another chance at learning and self-development. It helps bridge the gap between the learned and the unlearned and further accelerate the achievement of set national goals and objectives. Thus, this research examines adult education in general, but from the propensity for dropout amongst adult learners. Specifically, the research investigated the adult learner dropout at Khulubuse Community Learning Centre. The study involved thirteen (13) participants who were purposively selected. These participants include five learners who had dropped out, five learners who had completed their studies, and three educators from this adult learning centre. Three research questions guided the study: What are the causes of learner dropout from the AET program at Khulubuse Community Learning Centre CLC? How does teaching and learning at Khulubuse Community Learning Centre CLC contribute to learner dropout? How can learner dropout be prevented at Khulubuse Community Learning Centre CLC? Data were collected using semi-structured interviews. The interviews were thematically analysed. The theoretical framework (Tinto’s theory) was used as a lens for analysing data. This study shows personal learner issues at home such as work obligations, personal circumstances, class related issues and logistical issues impacted on learners’ decisions to drop out. Teaching and learning at the centre, however, had far less impact. The study concluded with recommendations on strategies that could reduce learner drop out at the centre.