4. Electronic Theses and Dissertations (ETDs) - Faculties submissions

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    Investigation of Contamination of Community Groundwater Sources with Antibiotics in Informal Settlements of Kisumu, Kenya
    (University of the Witwatersrand, Johannesburg, 2023-09) Karimi, Kellen Joyce; Ahmad, Aijaz; Duse, Adriano; Mwanthi, Mutuku
    Antibiotics have been used to cure diseases, but there are growing concerns about the risk to human health caused by inadvertent exposure to low levels of antibiotics in the environment. Despite extensive reporting from the developed world on antibiotic pollution of groundwater, relatively little study has been conducted on antibiotic contamination of groundwater in the developing countries, particularly informal settlements. Antibiotic usage and misuse have long been seen as clinical events, with little understanding of the role of disposal in the development of environmentally induced resistance. Exposure pathways that contribute to groundwater contamination in informal settlements put residents at odds because they already face inequalities, such as a high disease burden exacerbated by antibiotic resistance; thus, proper antibiotic disposal is critical in protecting human and environmental health. The purpose of this cross-sectional study was to establish the prevalence of groundwater contamination with the common antibiotics’ such as sulfamethoxazole, trimethoprim, and metronidazole, and the related antibiotic resistance and the human health risk of exposure. Ethical clearance to conduct research was obtained from three institutions as follows: - the Health Research Ethic Committee of the university of the Witwatersrand (HREC. Protocol Number M190412); the Kenyatta National Hospital and University of Nairobi Ethics and Research Committee (KNH/UoN-ERC. Ref No. P71910/2018); and the National Commission for Science, Technology, and Innovation (Ref No. NACOSTI/P/19/3232/28732). Each respondent gave informed consent to participate in the study. Anonymity was maintained at all levels of the study to protect the study participants from identification. Antibiotic use, which is connected to antibiotic disposal, was evaluated in a random sample of 447 families. From the 188 mapped groundwater sources, a random sample of 49 groundwater sources was chosen, and water samples were taken for antibiotic concentration analysis utilising a solid-phase extraction and liquid chromatography coupled to magnetic sector high resolution mass spectrometry (SPE-LC-MS/MS). The Kirky-Bauber diffusion method was used to test antibiotic resistance in Escherichia coli. The community's potential groundwater contamination routes were assessed by determining antibiotic use and disposal among households as well as assessing the environmental risk of exposure. In the households visited, 75% (n=337) were female and 25% (n=110) were male. The prevalence of antibiotic use in informal settlements was 43% (n=193), with 70% (n=137) users reporting that they obtained the antibiotics through a prescription from a health practitioner. A significant relationship was observed between having HIV/AIDS and acquiring antibiotics through a prescription; p=0.001. An association was also observed among the informal settlements, where a lower number of MNY B dwellers did not receive a prescription for the antibiotics acquired. There was no statistically significant difference in antibiotic use between males and females; odds ratio=1.33; whereas there was a difference in HIV/AIDS status; odds ratio=0.313; and among informal settlements where the odds of using antibiotics were reduced in NY B; odds ratio=0.42. Respondents who used antibiotics either kept the unused antibiotics for future use 87.1% (n=27) or disposed them. Among the disposals 51.6% (n=16) disposed in pit latrines, 16.1% (n=5) dispose in compost pits, and 6.5% (n=2) dispose the remaining antibiotics by burning. Females completed their antibiotic doses at a higher rate (36.3%; n=117) than males (32.5%; n=39). Significant difference was observed in completion rate among the HIV/AIDS positive and negative respondents as well as among informal settlements; p<0.000 and p=0.001 respectively. On the other hand, groundwater use in these communities is widespread. Respondents used it for a variety of purposes, including drinking (9%; n=39), though they declined to report. Awareness of the health consequences of drinking antibiotic-contaminated water was also low (35%; n=158), especially among households that reported antibiotic use; p=0.003. Only Sulfamethoxazole was detected in 7 out of 49 groundwater samples at a detection frequency of 14.3%; with concentrations ranging from nd to 258 ng/L. Escherichia coli and Cryptosporidium parvum were isolated from all the 49 water samples and E. coli isolates from 3 (6%) water samples were resistant to sulfamethoxazole with Inhibition Zone Diameters of 0.8 mm, 10.5 mm, and 11.5 mm. The 3 water samples were however not among samples where sulfamethoxazole was detected. The Hazard Quotient was 0 (zero), and therefore no risk of exposure to sulfamethoxazole in the environment, but the level of antibiotics that trigger antibiotic resistance is not known. Because of the rising problem of antibiotic resistance due to overuse and incorrect disposal, teaching on safe antibiotic prescription should be incorporated into medical training for all cadres. In addition to educating patients on proper use and disposal, the ministries of health should ensure the antimicrobial stewardship standards are adhered to both locally and worldwide. Follow-up research of antibiotic resistance discovered in three groundwater sources must be done to eliminate the possible sources and prevent further spread. This study is instrumental in informing the inclusion of antibiotics on the list of frequently monitored contaminants during water treatment, as well as serving as a starting point for antibiotic surveillance in Kenya.
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    Antibiotic targeted cement rods in chronic osteomyelitis, short-term outcomes from a level one South African trauma centre
    (University of the Witwatersrand, Johannesburg, 2022-09) Nicolaou, Caterina; Sekeitto, Allan Roy; Urrea, orcJuan David; Milner, Brenda
    Background: Chronic osteomyelitis requires complex management plans. Meta-analyses and systematic reviews have not found a gold standard of treatment for this disease. However, the literature reviews various treatment options. This study assesses the efficacy of an alternative treatment protocol to what is generally used in South Africa. This treatment protocol involves debridement, reaming and irrigation; as well as targeted antibiotics, used in a cheaply, effectively, and easily made cement rod spacer. Methods: A retrospective record review was performed on 38 patients, diagnosed with chronic osteomyelitis secondary to a long bone fracture, whom were treated via a specified treatment protocol at Chris Hani Baragwanath Academic Hospital between 01 January 2017 and 31 December 2018. This treatment protocol involved staged surgery with the use of antibiotics and antibiotic tailored cement rods. Data were collected and analysed accordingly using Stata 16.0, 2019. Results: The results indicate that chronic osteomyelitis is most prevalent amongst young males with a median age of 36 years. A multitude of organisms were cultured from bone reamings suggesting targeted antibiotic therapy is required. This treatment protocol demonstrated a 75.7% microbiological resolution to a negative culture. A good clinical outcome of 84.2% overall was demonstrated in terms of sinus resolution, skin changes, pain and function. All inflammatory markers decreased post-treatment with C - reactive protein showing a statistically significant decrease (p = 0.0043). Conclusion: Chronic osteomyelitis remains a complex disease to treat. Our treatment protocol demonstrates favourable microbiological, serological and overall clinical outcomes. Our study highlights antibiotic targeted cement rods as a feasible treatment option in managing chronic osteomyelitis secondary to long bone fractures.
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    Essential oil compounds in combination with conventional antibiotics for dermatology
    (University of the Witwatersrand, Johannesburg, 2024) Simbu, Shivar Bram; Van Vuuren, Sandy
    Skin and soft tissue infections represent a heterogeneous array of clinical entities with varying severity, causative pathogens, and rates of progression. The slow development and overuse of antimicrobial agents have perpetuated the spread and severity of antimicrobial resistance. Natural products such as essential oils and their compounds are often investigated for their pharmacological properties, with particular interest in their antimicrobial properties. This study aimed to investigate the effects of combining six essential oil compounds (α-pinene, γ-terpinene, ±linalool, eugenol, carvacrol, and cinnamaldehyde) with eight conventional antimicrobials (amoxicillin, ciprofloxacin, erythromycin, gentamicin, meropenem, tetracycline, miconazole, and nystatin) against six commonly encountered skin pathogens (Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Cutibacterium acnes ATCC 11827, and Candida albicans ATCC 10231) to elucidate the interactive profiles, toxicity, and anti-inflammatory properties. The antimicrobial analysis involved determining the minimum inhibitory concentrations (MIC) of the conventional antimicrobials and essential oil compounds, singularly and in combination, using the broth microdilution assay. The sum of the fractional inhibitory concentrations (ΣFICs) was calculated to investigate the interactive profile of the combinations. Synergistic interactions were further analysed at varying ratios and depicted on isobolograms. Eight synergistic interactions were identified, with seven against Gram-positive bacteria (ΣFIC 0.07– 0.42) and one against P. aeruginosa (ΣFIC 0.32). In addition, it was demonstrated that when in combination, the selected combinations resulted in reduced toxicity (Brine-shrimp lethality assay). The combination of amoxicillin and eugenol demonstrated the lowest toxicity (LC50 = 1081 μg/mL) and the highest selectivity index (14.41) when in a (70:30) ratio with the antibiotic in the higher ratio. Based on the synergistic results from the antimicrobial analysis, a selection of essential oil compounds with conventional antimicrobials were assessed for cytotoxicity and anti- inflammatory properties. The cytotoxicity properties were determined using the MTT assay on HaCAT keratinocytes. The anti-inflammatory properties were determined using lipopolysaccharide (LPS) activated RAW 264.7 macrophages, and the reduction in nitrate (NO) production was measured. Cinnamaldehyde demonstrated the highest cytotoxicity (IC50 = 28.63 μg/mL, p < 0.05) and the greatest reduction (77.44%) in nitrite production, which was also concentration dependent. The combination of ciprofloxacin and cinnamaldehyde demonstrated the lowest cytotoxicity (88.42% ± 3.72 cell viability; combination index of 0.12) and the highest reduction in nitrite production (77.42%; ΣFa = 0.44). Further investigations on the interactive properties of ibuprofen were undertaken. The antimicrobial, cytotoxicity, and anti-inflammatory properties of ibuprofen were analysed singularly and in combination with all essential oil compounds and conventional antimicrobials against reference and clinical skin pathogens. For the MIC results, four synergistic interactions were identified between ibuprofen and conventional antimicrobials (ΣFIC 0.33 - 0.50). For the cytotoxicity (MTT assay), none of the combinations demonstrated a cytotoxic effect (cell viability of 93.6-100%) and significant reduction on nitric oxide production. Additionally, higher order combinations involving the synergistic combinations were investigated with the inclusion of the essential oil compounds. Three synergistic interactions were identified (One against C. acnes and two against A. baumannii). The triple combinations were slightly cytotoxic (cell viability of 77.59 - 90.44%; combination index of 0.95 -1.10) on the HaCAT cell line and did not reduce nitric oxide production. Based on the overall results from this study, combinations of essential oil compounds and some conventional antimicrobials demonstrate promising therapeutic approaches to attenuate antimicrobial resistance. These results demonstrated that combinations that comprise cinnamaldehyde have noteworthy antimicrobial and anti-inflammatory properties which may warrant further investigation. Combining ibuprofen with conventional antimicrobials and essential oil compounds may also offer potential advantages in managing resistant infections through direct and indirect antimicrobial mechanisms.
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    Elucidating the Structure-Function Relationships of Enterococcus faecium Nicotinate-Nucleotide Adenylyltransferase through X-Ray Crystallography, Computational Modelling and Binding Studies
    (University of the Witwatersrand, Johannesburg, 2024) Jeje, Olamide Adetomi; Pandian, Ramesh; Achilonu, Ikechukwu A.
    Nicotinate nucleotide adenylyltransferase (NNAT) is a vital enzyme at the heart of NAD biosynthesis, catalysing a crucial reaction that leads to the formation of pyridine dinucleotides. NAD+ is an essential coenzyme in numerous metabolic processes, DNA repair, and cellular signalling. Given its pivotal role, NNAT has emerged as a compelling drug target, particularly for its potential to disrupt the survival mechanisms of bacterial pathogens. By inhibiting NNAT, it is possible to undermine the metabolic integrity of these pathogens, making NNAT a promising focal point in the fight against bacterial infections and antibiotic resistance. However, understanding the structure-function relationship of Enterococcus faecium NNAT (EfNNAT) has remained elusive. Hence, this study aimed to address this gap bycharacterising EfNNAT and validating its potential as a druggable target. EfNNAT was overexpressed and purified using the Escherichia coli system and IMAC purification technique. Subsequently, biophysical characterisation was performed, followed by the determination of the three-dimensional structure in both apo and liganded forms using X-ray crystallography. High-throughput virtual screening, along with SP and XP docking, was conducted using a library of synthesizable flavonoids. Molecular dynamic simulation and fluorescence studies were employed to establish and validate the binding of identified inhibitors to EfNNAT. Successful expression and purification of EfNNAT yielded approximately 101 mg per 7.8 g of wet E. coli cells, with a purity exceeding 98%. High-resolution crystal structures of EfNNAT in native, adenine-bound, and NMN-bound forms were determined at 1.90 Å, 1.82 Å, and 1.84 Å, respectively. These structures provided insights into EfNNAT's substrate preference and revealed a potential allosteric site at the dimer interface of the NMN-bound structure. Virtual screening identified quercetin 3-O-beta-D-glucose- 7-O-beta-D-gentiobioside as the only potential inhibitor from the flavonoid library used. A 500 ns atomistic molecular dynamics simulation showed the compound interacted through hydrogen bonding and water bridges, albeit unstable within the receptor. ANS and mant-ATP fluorescence spectroscopy confirmed quercetin binding, while thermal shift assay revealed minimal impact of the inhibitor on the protein stability and structure. This study establishes a pipeline from expression and purification to structure solution and potential inhibitor identification for EfNNAT, validating its druggability. The mechanistic insights offer a foundation for advancing drug discovery efforts targeting EfNNAT and other bacterial NNAT enzymes.