Abstract
D.Tech. (Biomedical Technology)
Escherichia coli (E. coli) was used as model organisms for this study because it is regarded as a highly specific microbial indicator of faecal pollution from humans and warm-blooded animals. E. coli can also be considered a model pathogen because it contains non-pathogenic commensal E. coli (ComEC) (used as indicator organisms) and diarrhoeagenic E. coli (DEC) types. Currently, routine water-testing laboratories test for ComEC and not DEC and the extent to which DEC co-occur with ComEC in various types of water sources is not well understood. The consequence is that we are looking at E. coli only as an indicator of faecal pollution and not at potential pathogen, even if low E. coli numbers are observed.
Standard culture methods used was not developed to differentiate between ComEC and DEC and may need special additional media to detect and isolate the DEC. These methods often require additional confirmation step that can take several days before results are obtained. Furthermore, standard culture methods are unable to detect E. coli cells if they have entered a viable but non-culturable state. Molecular biology techniques, such as the use of the Polymerase Chain Reaction (PCR), have been used to detect and characterize DEC types although they are mostly performed on cultured bacterial cells. Therefore, the aim of this study was to develop a culture independent PCR based method to analyse samples for detection and characterization of ComEC and DEC in samples.
In this study a DNA extraction protocol was optimised to concentrate bacterial cells from the water samples with subsequent DNA extraction. Thereafter, a 11-gene multiplex PCR (m-PCR) and absolute quantitative Real-Time PCR (q-PCR) was optimised for detection, characterization and quantification of E. coli. Once these methods were optimised final effluent samples (from wastewater treatment plants), environmental water samples (rivers and dams) and potable water samples (tap) were tested to compare the results obtained with these methods with that obtained from culture based methods.
The results obtained showed that the optimised DNA extraction protocol had good DNA recovery, repeatability and reproducibility and produced good quality...