Development and validation of novel methods for microbial source tracking based on Escherichia coli as an indicator of water quality

Microbial pollution in water periodically affects human health in Australia, particularly in times of drought and flood. There is an increasing need for the control of waterborn microbial pathogens. Methods, allowing the determination of the origin of faecal contamination in water, are generally referred to as Microbial Source Tracking (MST). Various approaches have been evaluated as indicatorsof microbial pathogens in water samples, including detection of different microorganisms and various host-specific markers. However, until today there have been no universal MST methods that could reliably determine the source (human or animal) of faecal contamination. Therefore, the use of multiple approaches is frequently advised. MST is currently recognised as a research tool, rather than something to be included in routine practices. The main focus of this research was to develop novel and universally applicable methods to meet the demands for MST methods in routine testing of water samples. Escherichia coli was chosen initially as the object organism for our studies as, historically and globally, it is the standard indicator of microbial contamination in water. In this thesis, three approaches are described: single nucleotide polymorphism (SNP) genotyping, clustered regularly interspaced short palindromic repeats (CRISPR) screening using high resolution melt analysis (HRMA) methods and phage detection development based on CRISPR types. The advantage of the combination SNP genotyping and CRISPR genes has been discussed in this study. For the first time, a highly discriminatory single nucleotide polymorphism interrogation of E. coli population was applied to identify the host-specific cluster. Six human and one animal-specific SNP profile were revealed. SNP genotyping was successfully applied in the field investigations of the Coomera watershed, South-East Queensland, Australia. Four human profiles [11], [29], [32] and [45] and animal specific SNP profile [7] were detected in water. Two human-specific profiles [29] and [11] were found to be prevalent in the samples over a time period of years. The rainfall (24 and 72 hours), tide height and time, general land use (rural, suburban), seasons, distance from the river mouth and salinity show a lack of relashionship with the diversity of SNP profiles present in the Coomera watershed (p values > 0.05). Nevertheless, SNP genotyping method is able to identify and distinquish between human- and non-human specific E. coli isolates in water sources within one day. In some samples, only mixed profiles were detected. To further investigate host-specificity in these mixed profiles CRISPR screening protocol was developed, to be used on the set of E. coli, previously analysed for SNP profiles. CRISPR loci, which are the pattern of previous DNA coliphages attacks, were considered to be a promising tool for detecting host-specific markers in E. coli. Spacers in CRISPR loci could also reveal the dynamics of virulence in E. coli as well in other pathogens in water. Despite the fact that host-specificity was not observed in the set of E. coli analysed, CRISPR alleles were shown to be useful in detection of the geographical site of sources. HRMA allows determination of ‘different’ and ‘same’ CRISPR alleles and can be introduced in water monitoring as a cost-effective and rapid method. Overall, we show that the identified human specific SNP profiles [11], [29], [32] and [45] can be useful as marker genotypes globally for identification of human faecal contamination in water. Developed in the current study, the SNP typing approach can be used in water monitoring laboratories as an inexpensive, high-throughput and easy adapted protocol. The unique approach based on E. coli spacers for the search for unknown phage was developed to examine the host-specifity in phage sequences. Preliminary experiments on the recombinant plasmids showed the possibility of using this method for recovering phage sequences. Future studies will determine the host-specificity of DNA phage genotyping as soon as first reliable sequences can be acquired. No doubt, only implication of multiple approaches in MST will allow identification of the character of microbial contamination with higher confidence and readability.

A Web-based system enabling the integration, analysis, and 3D sub-surface visualization of groundwater monitoring data and geological models

The 3D Water Chemistry Atlas is an intuitive, open source, Web-based system that enables the three-dimensional (3D) sub-surface visualization of ground water monitoring data, overlaid on the local geological model (formation and aquifer strata). This paper firstly describes the results of evaluating existing virtual globe technologies, which led to the decision to use the Cesium open source WebGL Virtual Globe and Map Engine as the underlying platform. Next it describes the backend database and search, filtering, browse and analysis tools that were developed to enable users to interactively explore the groundwater monitoring data and interpret it spatially and temporally relative to the local geological formations and aquifers via the Cesium interface. The result is an integrated 3D visualization system that enables environmental managers and regulators to assess groundwater conditions, identify inconsistencies in the data, manage impacts and risks and make more informed decisions about coal seam gas extraction, waste water extraction, and water reuse.

Community engagement for development and application of 3D visualisation models : a two edged

Background: SEQ Catchments Ltd and QUT are collaborating on groundwater investigations in the SE Qld region, which utilise community engagement and 3D Visualisation methodologies. The projects, which have been funded by the Australian Government’s NHT and Caring for our Country programmes, were initiated from local community concerns regarding groundwater sustainability and quality in areas where little was previously known. ----- Objectives: Engage local and regional stakeholders to tap all available sources of information;•Establish on-going (2 years +) community-based groundwater / surface water monitoring programmes;•Develop 3D Visualisation from all available data; and•Involve, train and inform the local community for improved on-ground land and water use management. ----- Results and findings: Respectful community engagement yielded information, access to numerous monitoring sites and education opportunities at low cost, which would otherwise be unavailable. A Framework for Community-Based Groundwater Monitoring has been documented (Todd, 2008).A 3D visualisation models have been developed for basaltic settings, which relate surface features familiar to the local community with the interpreted sub-surface hydrogeology. Groundwater surface movements have been animated and compared to local rainfall using the time-series monitoring data.An important 3D visualisation feature of particular interest to the community was the interaction between groundwater and surface water. This factor was crucial in raising awareness of potential impacts of land and water use on groundwater and surface water resources.

ERTP : Energy-efficient and reliable transport protocol for data streaming in wireless sensor networks

Emerging data streaming applications in Wireless Sensor Networks require reliable and energy-efficient Transport Protocols. Our recent Wireless Sensor Network deployment in the Burdekin delta, Australia, for water monitoring [T. Le Dinh, W. Hu, P. Sikka, P. Corke, L. Overs, S. Brosnan, Design and deployment of a remote robust sensor network: experiences from an outdoor water quality monitoring network, in: Second IEEE Workshop on Practical Issues in Building Sensor Network Applications (SenseApp 2007), Dublin, Ireland, 2007] is one such example. This application involves streaming sensed data such as pressure, water flow rate, and salinity periodically from many scattered sensors to the sink node which in turn relays them via an IP network to a remote site for archiving, processing, and presentation. While latency is not a primary concern in this class of application (the sampling rate is usually in terms of minutes or hours), energy-efficiency is. Continuous long-term operation and reliable delivery of the sensed data to the sink are also desirable. This paper proposes ERTP, an Energy-efficient and Reliable Transport Protocol for Wireless Sensor Networks. ERTP is designed for data streaming applications, in which sensor readings are transmitted from one or more sensor sources to a base station (or sink). ERTP uses a statistical reliability metric which ensures the number of data packets delivered to the sink exceeds the defined threshold. Our extensive discrete event simulations and experimental evaluations show that ERTP is significantly more energyefficient than current approaches and can reduce energy consumption by more than 45% when compared to current approaches. Consequently, sensor nodes are more energy-efficient and the lifespan of the unattended WSN is increased.

Human-specific E. coli single nucleotide polymorphism (SNP) genotypes detected in a South-East Queensland waterway, Australia

The World Health Organization recommends that the majority of water monitoring laboratories in the world should test for E. coli daily since thermotolerant coliforms and E. coli are key indicators for risk assessment of recreational waters. Recently, we developed a new SNP method for typing E. coli strains, by which human-specific genotypes were identified. Here, we report the presence of these previously described specific SNP profiles in environmental water, sourced from the Coomera River, located on South East Queensland, Australia, over a period of two years. This study tested for the presence of human-specific E. coli to ascertain whether hydrologic and anthropogenic activity plays a key role in the pollution of the investigated watershed or whether the pollution is from other sources. We found six human-specific SNP profiles and one animal-specific SNP profile consistently across sampling sites and times. We have demonstrated that our SNP genotyping method is able to rapidly identify and characterise human- and animal-specific E. coli isolates in water sources.

Risk-based approach to the management of on-site wastewater treatment facilities

Background On-site wastewater treatment system (OWTS) siting, design and management has traditionally been based on site specific conditions with little regard to the surrounding environment or the cumulative effect of other systems in the environment. The general approach has been to apply the same framework of standards and regulations to all sites equally, regardless of the sensitivity, or lack thereof, to the receiving environment. Consequently, this has led to the continuing poor performance and failure of on-site systems, resulting in environmental and public health consequences. As a result, there is increasing realisation that more scientifically robust evaluations in regard to site assessment and the underlying ground conditions are needed. Risk-based approaches to on-site system siting, design and management are considered the most appropriate means of improvement to the current standards and codes for on-site wastewater treatment systems. The Project Research in relation to this project was undertaken within the Gold Coast City Council region, the major focus being the semi-urban, rural residential and hinterland areas of the city that are not serviced by centralised treatment systems. The Gold Coast has over 15,000 on-site systems in use, with approximately 66% being common septic tank-subsurface dispersal systems. A recent study evaluating the performance of these systems within the Gold Coast area showed approximately 90% were not meeting the specified guidelines for effluent treatment and dispersal. The main focus of this research was to incorporate strong scientific knowledge into an integrated risk assessment process to allow suitable management practices to be set in place to mitigate the inherent risks. To achieve this, research was undertaken focusing on three main aspects involved with the performance and management of OWTS. Firstly, an investigation into the suitability of soil for providing appropriate effluent renovation was conducted. This involved detailed soil investigations, laboratory analysis and the use of multivariate statistical methods for analysing soil information. The outcomes of these investigations were developed into a framework for assessing soil suitability for effluent renovation. This formed the basis for the assessment of OWTS siting and design risks employed in the developed risk framework. Secondly, an assessment of the environmental and public health risks was performed specifically related the release of contaminants from OWTS. This involved detailed groundwater and surface water sampling and analysis to assess the current and potential risks of contamination throughout the Gold Coast region. Additionally, the assessment of public health risk incorporated the use of bacterial source tracking methods to identify the different sources of fecal contamination within monitored regions. Antibiotic resistance pattern analysis was utilised to determine the extent of human faecal contamination, with the outcomes utilised for providing a more indicative public health assessment. Finally, the outcomes of both the soil suitability assessment and ground and surface water monitoring was utilised for the development of the integrated risk framework. The research outcomes achieved through this project enabled the primary research aims and objects to be accomplished. This in turn would enable Gold Coast City Council to provide more appropriate assessment and management guidelines based on robust scientific knowledge which will ultimately ensure that the potential environmental and public health impacts resulting from on-site wastewater treatment is minimised. As part of the implementation of suitable management strategies, a critical point monitoring program (CPM) was formulated. This entailed the identification of the key critical parameters that contribute to the characterised risks at monitored locations within the study area. The CPM will allow more direct procedures to be implemented, targeting the specific hazards at sensitive areas throughout Gold Coast region.

Understanding Connectivity between Groundwater Chemistry Data and Geological Stratigraphy via 3D Sub-surface Visualization and Analysis

This paper describes the 3D Water Chemistry Atlas - an open source, Web-based system that enables the three-dimensional (3D) sub-surface visualization of ground water monitoring data, overlaid on the local geological model. Following a review of existing technologies, the system adopts Cesium (an open source Web-based 3D mapping and visualization interface) together with a PostGreSQL/PostGIS database, for the technical architecture. In addition a range of the search, filtering, browse and analysis tools were developed that enable users to interactively explore the groundwater monitoring data and interpret it spatially and temporally relative to the local geological formations and aquifers via the Cesium interface. The result is an integrated 3D visualization system that enables environmental managers and regulators to assess groundwater conditions, identify inconsistencies in the data, manage impacts and risks and make more informed decisions about activities such as coal seam gas extraction, waste water extraction and re-use.

Design and deployment of a remote robust sensor network : experiences from an outdoor water quality monitoring network

This paper investigates a wireless sensor network deployment - monitoring water quality, e.g. salinity and the level of the underground water table - in a remote tropical area of northern Australia. Our goal is to collect real time water quality measurements together with the amount of water being pumped out in the area, and investigate the impacts of current irrigation practice on the environments, in particular underground water salination. This is a challenging task featuring wide geographic area coverage (mean transmission range between nodes is more than 800 meters), highly variable radio propagations, high end-to-end packet delivery rate requirements, and hostile deployment environments. We have designed, implemented and deployed a sensor network system, which has been collecting water quality and flow measurements, e.g., water flow rate and water flow ticks for over one month. The preliminary results show that sensor networks are a promising solution to deploying a sustainable irrigation system, e.g., maximizing the amount of water pumped out from an area with minimum impact on water quality.

Continuous monitoring of reservoir water quality: The Wivenhoe Project

The Lake Wivenhoe Integrated Wireless Sensor Network is conceptually similar to traditional SCADA monitoring and control approaches. However, it is applied in an open system using wireless devices to monitor processes that affect water quality at both a high spatial and temporal frequency. This monitoring assists scientists to better understand drivers of key processes that influence water quality and provide the operators with an early warning system if below standard water enters the reservoir. Both of these aspects improve the safety and efficient delivery of drinking water to the end users.

Experimental evaluation of an autonomous surface vehicle for water quality and greenhouse gas emission monitoring

This paper describes the experimental evaluation of a novel Autonomous Surface Vehicle capable of navigating complex inland water reservoirs and measuring a range of water quality properties and greenhouse gas emissions. The 16 ft long solar powered catamaran is capable of collecting water column profiles whilst in motion. It is also directly integrated with a reservoir scale floating sensor network to allow remote mission uploads, data download and adaptive sampling strategies. This paper describes the onboard vehicle navigation and control algorithms as well as obstacle avoidance strategies. Experimental results are shown demonstrating its ability to maintain track and avoid obstacles on a variety of large-scale missions and under differing weather conditions, as well as its ability to continuously collect various water quality parameters complimenting traditional manual monitoring campaigns.

An autonomous surface vehicle for water quality monitoring

This paper describes a novel Autonomous Surface Vehicle capable of navigating throughout complex inland water storages and measuring a range of water quality properties and greenhouse gas emissions. The 16 ft long solar powered catamaran can collect this information throughout the water column whilst the vehicle is moving. A unique feature of this ASV is its integration into a storage scale floating sensor network to allow remote mission uploads, data download and adaptive sampling strategies. This paper provides an overview of the vehicle design and operation including control, laser-based obstacle avoidance, and vision-based inspection capabilities. Experimental results are shown illustrating its ability to continuously collect key water quality parameters and compliment intensive manual monitoring campaigns.

Water Quality Indices from Unbalanced Spatio-Temporal Monitoring Designs

This chapter investigates a variety of water quality assessment tools for reservoirs with balanced/unbalanced monitoring designs and focuses on providing informative water quality assessments to ensure decision-makers are able to make risk-informed management decisions about reservoir health. In particular, two water quality assessment methods are described: non-compliance (probability of the number of times the indicator exceeds the recommended guideline) and amplitude (degree of departure from the guideline). Strengths and weaknesses of current and alternative water quality methods will be discussed. The proposed methodology is particularly applicable to unbalanced designs with/without missing values and reflects the general conditions and is not swayed too heavily by the occasional extreme value (very high or very low quality). To investigate the issues in greater detail, we use as a case study, a reservoir within South-East Queensland (SEQ), Australia. The purpose here is to obtain an annual score that reflected the overall water quality, temporally, spatially and across water quality indicators for each reservoir.

Douglas Shire Water Quality Monitoring Strategy: Final Report: a Report to Douglas Shire Council and the Department of the Environment and Heritage

This report describes results and conclusions from the monitoring component of the Douglas Shire Council (DSC) water quality project. The components of this project that this report addresses are: • Site selection and installation of in-stream and off-paddock automatic water quality monitoring equipment in the Douglas Shire. • Design of appropriate sampling strategies for automatic stations. • Estimation of loads of suspended sediment, total nitrogen and total phosphorus in rivers and also estimation of the changes in nutrient loads from sugar cane under different fertilizer application rates. • Development of a community-based water quality sampling program to complement the automatic sampling efforts. • Design of an optimised, long-term water quality monitoring strategy.