Abstract
M.Tech. (Biomedical Technology)
Diseases caused by microorganisms, such as diarrhoea, have become a major public health concern. This disease has plagued the world, affecting children, the aged and immune-compromised individuals in both developed and developing countries. There are a variety of microbes responsible for the disease, most of which are indicative of faecal contamination in water sources. The presence of these microbes in water bodies suggests a malfunctioning treatment system as well as direct contamination by faecal matter. Amongst the faecal organisms released into water sources, Escherichia coli (E. coli) is the most widely accepted indicator organism and is used to determine the possible health impact and level of contamination in source waters. Since E. coli contains pathogenic and non-pathogenic strains, adequate identification is needed to determine the implications and effects of the presence of this bacterium. Most culture based methods are phenotypic based which can detect, but not differentiate between commensal and pathogenic E. coli strains. In recent times, advanced molecular based techniques such as polymerase chain reaction (PCR) identify microbes by their genetic profile, providing rapid results and eliminating the need for extensive microbiological analysis. This study was designed to assess the value added by using molecular biology techniques to test for the presence of E. coli pathogenic genes and to determine if this process could be streamlined for industrial applications.
Water samples were collected from environmental and treated effluents from selected wastewater treatment plants in the Gauteng province (South Africa). Samples were enumerated using the Colilert® Quanti-Tray®/2000 method by an accredited laboratory. The positive E. coli wells on the Colilert® Quanti-Tray®/2000 were extracted in triplicate and subjected to DNA extractions on a semi-automated 96 well plate manifold, using the DX Universal Liquid Sample DNA Extraction kit (QIAGEN®). The extracted DNA was amplified by an optimised 11-gene E. coli multiplex PCR (mPCR).
The semi-automated DNA extraction method proved reliable and enabled the analysis of large numbers of samples, reducing the experimental timeframe. The incorporation of the molecular methods to the microbiological analysis reduced the number of samples to be analysed, thus reducing the time and cost factor. The mPCR adequately identified the presence of the selected E. coli virulence genes in a single step with the two PCR controls ensuring reliable results. The results showed enteropathogenic E...