Masters Degrees (Medical Microbiology)
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- ItemInvestigation of the contribution of oral bacterial microbiota to health and disease in a South African cohort.(Stellenbosch : Stellenbosch University, 2024-02) Pekeur, Jade Chantel; Hoek, Kim Gilberte Pauline ; Whitelaw, Andrew Christopher ; Erasmus, Rajiv T. ; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.ENGLISH ABSTRACT: Background: Metabolic syndrome (MetS) is a worldwide epidemic. Risk factors for MetS include diet and lifestyle, as well as genetic predisposition; however, evidence suggests that disruption of the oral microbiota is an important emerging risk factor for MetS. Additionally, periodontal disease (PD) is more prevalent in individuals with MetS and both conditions are associated with inflammation and insulin resistance. Porphyromonas gingivalis has been linked to PD which may be a marker for MetS. The aim of the study was to compare the microbial diversity of subgingival plaque of individuals with MetS and PD to that of healthy individuals, and to determine the abundance of P. gingivalis in the oral cavity of individuals with and without PD. An additional aim was to compare amplicon sequencing to quantitative PCR of P. gingivalis in individuals with PD. Methods: Subgingival plaque samples were obtained from 119 individuals from the mixed ancestral community in Bellville South, Western Cape. Individuals were classified as having MetS and/or PD using standardised criteria. Illumina sequencing of the V3-V4 hypervariable region of the 16S rRNA gene was performed and sequences were clustered into operational taxonomic units based on 97% similarity, using the Human Oral Microbiome Database. Microbial community profiles of healthy and diseased groups were compared using alpha and beta diversity measures. Additionally, P. gingivalis DNA in dental plaque was quantified by a SYBR green based real-time quantitative polymerase chain reaction (qPCR). Results: There were no significant differences in microbial community composition or diversity between individuals with and without MetS and between those with PD and controls. No statistically significant health-associated and PD associated OTUs were identified. Only one OTU, classified Peptostreptococcaceae (XIII) (G-1), was significantly more abundant in the MetS group. The real-time qPCR results showed similar levels of P. gingivalis detected in healthy individuals and those with PD. Higher P. gingivalis levels were observed in the severe PD group, however, the sample set was too small to determine adequate statistical differences. Conclusions: The findings suggest that factors (such as lifestyle, environment, and host immunity) other than genus-level oral microbial community composition may be responsible for the progression of MetS or PD in the Bellville South community. The study was limited to small sample sets and therefore, subtle differences in the microbial community may not have been detected. Furthermore, a larger study could reveal the role Peptostreptococcaceae plays in metabolic syndrome. P. gingivalis was not an indicator for PD, but may be an indicator of severe PD. Further investigation is needed regarding the role that other periodontal pathogens, as well as bacterial abundance, play in the initiation and progression of PD.
- ItemAn investigation on the role of oxytocin in chronic neuropathic pain in a Wistar Rat Model(Stellenbosch : Stellenbosch University, 2023-03) de Kock, Michaela; Qulu, Lihle; Chetty, Sean; Ahmed Sherif, Isa; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.ENGLISH ABSTRACT: Introduction Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect with ineffective preventative and curative treatment, due to the condition's complexity perpetuated by the extensive central involvement, including the chronic disruption and subsequent dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. Currently, only Duloxetine has been recommended as effective treatment for CIPN, which has shown individual-dependent, short-term analgesic effects, with limiting adverse effects and poor bioavailability. The neuropeptide, oxytocin, may offer significant analgesic and anxiolytic potential, as it exerts central and peripheral attenuating effects on nociception. However, it is unknown whether the intervention administered in a model of CIPN is an effective therapeutic alternative. Materials and Methods The intervention was divided into two phases. Phase 1 aimed to induce CIPN in adult Wistar rats using the chemotherapeutic agent Paclitaxel. Mechanical (electronic von Frey filament) and thermal (acetone evaporation test and Hargreaves test) hypersensitivity testing was used to evaluate changes due to the neuropathic induction. Phase 2 consisted of a 14-day intervention period with saline (o.g.), Duloextine (o.g.), or oxytocin (i.n.) administered as treatment. Analgesic behavioural testing was assessed throughout the intervention period. Following the intervention, anxiety-like behaviour was assessed using the elevated plus maze (EPM) and light-dark box protocols. Analysis of peripheral plasma corticosterone, peripheral plasma oxytocin, and hypothalamic oxytocin concentrations were assessed using ELISA assays. Results The findings showed that we were able to successfully establish a model of chemotherapy-induced peripheral neuropathy during Phase 1, determined by the increase in mechanical and thermal nociceptive responses following chemotherapy administration. Furthermore, based on this finding, we were able to evaluate the effect of different treatments administered in the presence of CIPN. The animals treated with oxytocin displayed a significant improvement in mechanical sensitivity over the intervention phase, indicative of an improvement in nociceptive tolerance in the presence of neuropathic pain. Animals that received Paclitaxel and treated with oxytocin also displayed significantly greater explorative behaviour during the EPM, indicative of a reduced presence of anxiety-like behavior. Conclusion Our results support the hypothesis that intranasally administered oxytocin may augment the analgesic and anxiolytic effects of duloxetine in a chemotherapy-induced peripheral neuropathy model in a Wistar rat. Administered in conjunction, oxytocin and duloxetine may provide enhanced therapeutic effects in the treatment of CIPN. Further research is necessary to establish optimal treatment and dosage requirements.
- ItemThe role of the accessory gene regulator system on biofilm formation and stress response in Staphylococcus aureus(Stellenbosch : Stellenbosch University, 2023-03) Maleka, Kgomotso Frank; Matukane, Siphiwe; Shima, Abdulgader; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.ENGLISH SUMMARY: Background: Biofilm formation is a key contributor to Staphylococcus aureus virulence and pathogenicity. It is regulated by the accessory gene regulator (agr) operon, which may become dysfunctional due to genetic mutations. These mutations may affect the expression of key genes like RNAIII and icaA that are involved in key pathogenesis pathways. Previous studies have associated agr dysfunctional strains with strong biofilm formation, persistent infections, and treatment failure. Therefore, this study aimed to determine the impact of agr functionality status on biofilm development and antibiotic stress tolerance in clinical S. aureus isolates. Methods: Twelve previously characterized (phenotypically and genotypically) blood culture S. aureus isolates, collected from February 2015 to March 2017 at Tygerberg Hospital were selected for this study. Crystal Violet biofilm assay was then performed to assess biofilm formation over a 24-hour period at different time points in the presence and absence of oxacillin, vancomycin, and rifampicin at sub-minimum inhibitory concentrations [sub-MIC: 0.25 μg/ml (oxacillin and vancomycin), 0,005 μg/ml rifampicin] and clinically relevant concentrations (10 μg/ml). The minimum inhibitory concentration (MIC) was determined using the gradient diffusion assay (E-test). Reverse-transcription real-time PCR was used to measure the expression of RNAIII and icaA genes. Whole genome sequence data were analyzed for genetic differences in the agr locus including the bap, icaA, and icaD regions for the 12 isolates, using online platforms (Prokka, Artemis, and BioEdit 7.2). Result: There was statistically an insignificance increase in the overall biofilm formation levels in agr dysfunctional isolates than in agr functional isolates in the absence and presence of antibiotics, except for when exposed to sub-MIC of oxacillin (p=0.007). Similarly, an increase in the overall biofilm formation level in agr I isolates was observed when compared to agr II and agr III isolates in the absence and presence of antibiotics. Furthermore, overall methicillin-resistant S. aureus (MRSA) isolates produced more biofilm, especially at time point 6 and 8 hours after incubation in the absence of antibiotics; while methicillin-sensitive S. aureus (MSSA) isolates formed more biofilm in the presence of antibiotics overall time points. Furthermore, a significant increase in the expression levels of both RNAIII (p=0.041) and icaA (p=0.020) was observed in agr dysfunctional isolates when compared to agr functional isolates. A significant increase in the expression of icaA (p=0.008) was observed in MRSA isolates; and dysfunctional isolates had more mutations on the agr-related gene than functional isolates. Conclusion: An increase in biofilm formation based on phenotypic agr functionality, agr type, and methicillin susceptibility profile in the absence or presence of antibiotics was not statistically significant. Additionally, mutations observed on the agr locus in agr dysfunctional isolates confirmed the role mutations play on agr functionality. The study analyzed 12 isolates, which may decrease statistical power. Therefore, future studies with a larger sample size should confirm or refute these study findings about the role agr functionality, agr type, and methicillin susceptibility profile have on the ability of clinical S. aureus isolates to produce biofilm in the absence and in the presence of antibiotics.
- ItemThe characterization of antimicrobial resistance and virulence in Coagulase-Negative Staphylococci isolated from neonatal blood cultures in the Western Cape(Stellenbosch : Stellenbosch University, 2023-03) Cloete, Stephanie Simone; Whitelaw, Andrew Christopher; Matukane, Siphiwe Ruthy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.ENGLISH SUMMARY: Introduction: Coagulase-Negative Staphylococci (CoNS) are frequently isolated from the neonatal intensive care unit. However, it is difficult to discriminate between invasive CoNS and coincident contaminants since they are commensals of skin, and the nonspecific clinical signs of neonatal sepsis may be even more subtle due to the low virulence of CoNS. The extensive range of virulence factors in CoNS, some of which may be regulated by mobile genetic elements, contributes to their pathogenicity. This study aims to describe the species distribution, antimicrobial resistance, and molecular virulence markers in CoNS from neonatal blood cultures to identify potential pathogenicity markers. Methods: Between February and July 2021, 127 CoNS isolates were collected from neonatal blood cultures submitted to Tygerberg Hospital National Health Laboratory Services microbiology laboratory. Species identification was performed on all isolates using MALDI-TOF mass spectrometry. Antimicrobial susceptibility testing (AST) using Kirby Bauer disc diffusion was performed on 82 isolates representing the two predominant CoNS species from the most represented hospitals. Twenty isolates, representing a range of susceptibility patterns, were chosen for Oxford Nanopore whole genome sequencing (WGS). The assembled genomes were analysed using the Center for Genomic Epidemiology, pubMLST, Virulence Factor Database, ISsaga, and Resistance Gene Identifier. Reverse-transcription real-time PCR was used to explore the potential association of insertion sequence (IS)256 with the expression of the biofilm associated virulence gene icaA. The icaA normalised cycle threshold (ΔCt) was calculated relative to that of the house-keeping gene (tpi). Results: The most common species identified were Staphylococcus epidermidis (80/127, 63 %) and Staphylococcus hominis (29/127, 23 %). Among the 62 S. epidermidis and 20 S. hominis isolates selected for AST, 81.7 % (67/82) were non-susceptible to at least one antibiotic, and 54 % (44/82) were resistant to three or more antibiotic classes (multidrug resistant). The highest rates of resistance were to erythromycin (64.6%), trimethoprim-sulfamethoxazole (56.1 %), and cloxacillin (54.9 %). There was considerable concordance between the observed phenotypes and the predicted genotype, especially for cefoxitin, linezolid and vancomycin. The WGS data predicted all 20 isolates (14 S. epidermidis and six S. hominis isolates) as possible human pathogens, with a probability pathogen score higher for S. epidermidis (=0.93) than S. hominis (=0.89) isolates (p<0.001). In-silico MLST revealed substantial diversity, reflected by nine S. epidermidis sequence types identified. The ica operon was present in 10/14 S. epidermidis isolates, and 7/14 isolates contained the IS256. None of the S. hominis isolates contained the ica operon. The ΔCt values in isolates with and without IS256 were not significantly different, suggesting that IS256 was not related to ica operon expression. Conclusion: There was a high-level of genetic diversity among CoNS isolates, enriched with virulence factors, antimicrobial resistance genes and mobile genetic elements. S. epidermidis harboured a greater array of virulence factors than S. hominis isolates, which require further investigation to be used as markers of clinical significance. Antimicrobial resistance was common among these isolates, which may complicate treatment strategies. There was no effect of IS256 on ica gene expression, which may relate to the distance of IS256 from the ica operon.
- ItemCharacterisation of follicular helper T (Tfh) cells in early treated HIV-infected children: relationship to immune activation and inflammation.(Stellenbosch : Stellenbosch University, 2022-11-24) Olifant, Paulina; Glashoff, Richard Helmuth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.ENGLISH ABSTRACT: Background: South Africa has a high burden of Human Immunodefiency Virus (HIV) infection. Babies of HIV-positive pregnant women can become HIV-infected or exposed through vertical transmission. Follicular helper T (Tfh) cells have been of particular interest in HIV infection due to their preferential expansion, contribution to the HIV reservoir and dysregulation within HIV-infected individuals. The aim of this study was to investigate the Tfh cell population within children from the Children with HIV Early Antiretroviral Therapy (CHER) trial who started treatment within the first six months of life to determine whether the numbers of these cells is altered as compared to uninfected children and also whether persistent immune activation and inflammation in these children is associated with Tfh cell dysregulation. Methodology: This retrospective cross-sectional observational study consisted of three groups, i.e., early antiretroviral-treated HIT (HIV Infected Treated), HEU (HIV Exposed Uninfected) and HUU (HIV Uninfected Unexposed), of children. Cryopreserved peripheral mononuclear blood cells (PBMCs) were stained with an 11-colour antibody panel designed and optimised for phenotypic identification and quantification of T cell populations using flow cytometry. Tfh cells populations were characterised as CD4+CXCR5+PD-1+ with/without ICOS+. CD4, CD8 and Treg cells were defined as follicular/ follicular-homing based on CXCR5+ expression and activated based on CD38+ and/or CD69+ expression. Secondary data of clinical parameters and inflammatory cytokines for each group were evaluated. Statistical comparisons between groups were made using the Mann-Whitney test to identify significant differences. Significant correlations between Tfh cells and clinical parameters, other T cell populations and inflammatory cytokines were identified using Spearman’s rank order test. Results: Phenotypic results generally indicated significantly increased proportions of CD38+ subsets in HIT group and CD69+ subsets in HEU group. Although there was no significant difference in median CD4+CXCR5+PD-1+ Tfh cell percentage between groups, the ICOS-expressing subset namely CD4+CXCR5+PD-1+ICOS+ Tfh cells was significantly higher in the HIT (33.6% vs 19.2%; p = 0.016) and HEU group (31.6% vs 19.2%; p = 0.006) compared to the HUU group. In the HIT group, CD4+CXCR5+PD-1+ Tfh cells shared significant negative correlations with a majority of activated T cell subsets. A significant positive correlation between CD4+CXCR5+PD-1+ Tfh and CD8+PD-1+ Tc cells, general indicator of immune exhaustion, was demonstrated. Lastly, the HIT group showed the highest level of INF-α and hsCRP inflammatory cytokines and levels of IL-1β and hsCRP significantly correlated with CD4+CXCR5+PD-1+ICOS+ Tfh cells within this group. Conclusions: Overall levels of immune activation were significantly higher in HIT and HEU groups. Several activated T cell subsets inversely correlated with CD4+CXCR5+PD-1+ Tfh cells, suggesting high levels of immune activation can lead to decreased proportions of circulating Tfh cells. Even though no significant difference in the proportion of CD4+CXCR5+PD-1+ Tfh cells was found between groups, the ICOS+ subset was significantly expanded in HIT and HEU children in comparison to HUU children. The significant positive correlation between IL-1β and ICOS-expressing Tfh cells, within the entire study population and HIT group, suggested that increased inflammation resulted in an Tfh cell increase.