1. The aim of this research was to determine appropriate microbial indicator systems for use in monitoring the occurrence and source of faecal contamination in aquatic environments. Particular emphasis has been placed on the identification of indicators which might enable faecal contamination of human origin to be distinguished from that of non-human derivation. Two lines of investigation have been pursued; the use of alternative microbial indicator systems for water quality monitoring, and the development of diagnostic molecular methods for identifying the origin of faecal organisms. 2. Field and treatment plant studies were undertaken to investigate the utility of three indicator systems for monitoring water quality impacts in the coastal area surrounding the discharge from Auckland's North Shore City wastewater treatment plant. F-specific bacteriophage (F-phage) and ratios of F-phage to enterococci and faecal coliforms were examined over a twelve month period in which indicator levels at various stages of the wastewater treatment process were measured. These indicators and levels of Clostridium perfringens spores in sediment were measured in four outfall impact studies. 3. F-phage levels in effluent from the treatment plant (median=2.5 x 102 cfu/l00 ml) were too low to enable their use as source-specific tracers of effluent dispersion. However, higher F-phage levels in raw sewage (median=6.2 x l05 cfu/l00 ml) suggested that phage may be an appropriate indicator of raw sewage contamination in the receiving environment. F-phage was detected in coastal waters remote from the sewage outfall, providing presumptive evidence of sewage contamination entering the sea from sources other than the outfall discharge itself. 4. F-phage/enterococci and F-phage/faecal coliform ratios that were determined for sewage and treated effluent derived from the North Shore plant proved to be highly variable and were therefore unreliable for determining the source faecal contamination. 5. The concentration of Clostridium perfringens spores in sediments did not provide a useful means for tracing the distribution of faecal contaminants in the coastal system because the spore levels were found to be similar throughout the study area. This widespread occurrence of C. perfringens spores prevented identification of defined impact zones for any of the potential sources of faecal contaminants examined. 6. Randomly Amplified Polymorphic DNA (RAPD) analysis was used to investigate whether genetic markers could be identified for Escherichia coli isolates derived from different sources. RAPD profiling was found to be a highly discriminative method able to subtype E. coli strains present in both sewage and in the wastewater stream of a mechanical wood-pulping operation. In the pulp mill effluent, RAPD analysis indicated the presence of a restricted range of E. coli strains compared to the range found in the water intake and sewage effluent discharges. The results obtained provide presumptive evidence for the multiplication of E. coli strains within the wastewater stream. 7. Comparison of RAPD profiles derived from a range of human and environmental E. coli isolates led to the identification of a 1.6-kb marker. present in a subset of human isolates, but found only infrequently in isolates from animal origin. Primers based on sequence information obtained from this region of the genome yielded a direct PCR assay for the 'human' marker. The reliability and utility of the assay was improved when fluorescently-labelled primers and an automated DNA detection system was employed for the analysis of the amplified PCR products. 8. The diagnostic polymorphism was localised to the region immediately downstream of the gene encoding the glycine decarboxylase P-protein (gcvP). Comparison of DNA sequences from this region in 'marker-positive' and 'marker-negative' E. coli isolates revealed an abrupt loss of homology immediately downstream of the transcription termination point of the gcvP gene. The region of non-homology extends for at least 130 base pairs beyond the gcvP transcription terminator. Sequences spanning this region in marker-negative isolates exhibited a high level homology with that determined for E. coli K-12. The origin of the sequence for the same region in marker-positive isolates was unable to be identified. 9. The utility of the marker for environmental monitoring was investigated in a field study of a rural stream receiving bacterial contamination of both human and animal origin. The marker was consistently detected both in isolates derived from sewage effluents and in those found immediately downstream from sewage inputs-despite the presence of a high background of E. coli of animal origin. However the utility of the marker as a sewage-specific tracer was limited by the relatively low numbers of marker-positive d. coli in sewage effluents (approximately 8%). 10. In summary, the work described in this thesis has confirmed the need for multiple indicator systems in water quality monitoring and has identified appropriate areas for further research. The most promising area for future research is in the development of molecular tools which might provide both a better understanding of genetic variation in indicator organisms and enable methods to be developed for the detection of host-specific strains. Location of other polymorphisms likely to be present in the E. coli genome should enable further host-specific markers to be identified which could complement the 'human' marker identified in this study. / Whole document restricted, but available by request, use the feedback form to request access.
| Identifer | oai:union.ndltd.org:AUCKLAND/oai:researchspace.auckland.ac.nz:2292/2375 |
| Date | January 1996 |
| Creators | Turner, Susan Jane |
| Contributors | Dr Gillian Lewis, Professor Richard Bellamy |
| Publisher | ResearchSpace@Auckland |
| Source Sets | University of Auckland |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | Scanned from print thesis |
| Rights | Whole document restricted but available by request. Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated., http://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm, Copyright: The author |
| Relation | PhD Thesis - University of Auckland, UoA658032 |
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