McCullough, Bronwyn KirbyKariem, Min-ghah2024-04-092024-05-092024-04-092024-05-092024https://hdl.handle.net/10566/13395>Magister Scientiae - MScDuring the Western Cape drought from 2015 to 2018, the use of greywater to alleviate pressure on the limited potable water available in the province was encouraged. Greywater, however, has the potential to harbour residual antibiotics and pathogenic bacteria, thereby potentially supporting the growth and proliferation of antibiotic resistant bacteria. This study combined traditional microbiology tests with molecular biology to detect resistant microorganisms within domestic greywater systems and their associated biofilms, while Quantitative PCR (qPCR) was used to determine the levels of clinically relevant antibiotic resistance genes (ARGs) (vanA, ampC and aadA), as well as the intI1 gene, which serves as a marker of horizontal gene transfer. Phenotypic resistance was confirmed using the Kirby-Bauer disc diffusion method. Notably, high levels of resistance to ampicillin and kanamycin were found in greywater and biofilm samples, as well as resistance to vancomycin. Bacterial viability was assessed using flow cytometry using the LIVE/DEAD BacLight Bacterial Viability kit, and it was revealed that a large portion of the cell population within greywater was dead or injured. qPCR analysis confirmed the presence of clinically relevant ARGs vanA and ampC, in domestic greywater samples, with abundance fluctuating in response to seasonal change. Additionally, the intI1 gene was detected in all greywater and biofilm samples, which suggests that genetic exchange occurs amongst bacteria in greywater and biofilm samples. Our findings support the hypothesis that greywater systems are colonised by resistant bacteria, which can form stable communities within the water environment which allows for high levels of genetic exchange. These findings support the use of qPCR and flow cytometry, in combination with traditional microbiology tests for monitoring antibiotic resistance in environmental samples. While the use of greywater is a viable method to reduce the demand for potable water, this study highlights the fact that the improper use of greywater poses a risk to the environment and public health. As such, the public should be better educated on how to safely use these systems.enDomestic Greywater SystemsGreywaterWestern Cape DroughtAntibiotic Resistance GenesReal-Time PCRThesisUniversity of the Western Cape