Quantification and Viability of "Indicator" E. coli by Lab on a Chip Isothermal Nucleic Acid Amplification for Biosecurity in Sustainable Aquaculture

Filter feeding bivalve shellfish, which include mussels, oysters, clams, and cockles, naturally accumulate microorganisms from their environment. These can pose a risk to human health after consumption, including infection with enteric viruses (e.g. Norovirus) and harmful bacteria (e.g. Salmonella Spp. and Escherichia coli), and exposure to bio-toxins produced by marine algae. Therefore, in the UK, bivalve shellfish are routinely tested in accordance with European Union regulations to ensure that they are safe to eat, and which (if any) treatment they should undergo before market. One species, Escherichia coli, is a human pathogen (disease causing microorganism) and a causative agent in gastroenteritis (food poisoning). It is also found naturally in human and animal intestines and is a valuable indicator of the level of faecal contamination in water, and therefore an indirect measure of the threat posed by other pathogens associated with human waste and sewage.

The UK statutory shellfish monitoring programme is based at the Centre for Environment, Fisheries and Aquaculture Sciences' (Cefas) laboratory in Weymouth. Here, E. coli in bivalve shellfish flesh is measured using a regulatory standard technique wherein the bacteria are cultivated in the laboratory before their numbers are estimated. This method can take up to several days, allowing time for the bacteria to grow and replicate to concentrations at which they can be counted and analysed. In contrast, nucleic acid amplification is a method in which a genetic sequence from the target organism is amplified to a point at which it can be detected, analysed and counted in just a few hours and can be adapted to provide a wealth of other information about the organism (e.g. how dangerous it is). One problem with nucleic acid amplification using DNA (genomic) sequences is that these can be present in dead cells as well as those that remain active and still pose a risk to human health. This is circumvented by the use of chemical agents that destroy the DNA from dead cells before the amplification stage. Additionally, it is possible to measure whether the DNA sequence comes from a genome that is in-tact (i.e. the cell is still alive) or measure a different, but very similar molecule, mRNA, which is quickly degraded from dead cells. Most nucleic acid methods require laboratories containing bulky equipment and skilled personnel. In contrast, the Lab on a Chip concept is the miniaturisation of laboratory processes to a point at which they can be easily automated, and carried out in portable (e.g. handheld) or deployed (e.g. in the ocean) devices providing in situ and real-time analysis.

Our objective is to combine nucleic acid methods for the detection of E. coli with state of the art Lab on a Chip technology to (1) provide an automated assay (test) for the measurement of E. coli from shellfish flesh in the laboratory, and (2) to prime the development of a deployable E. coli sensor that will carry out analysis of seawater in proxy to shellfish harvesting areas. This will be beneficial for two reasons. First, the new assays will aim to improve the speed and accuracy of detection. This is crucial as underestimation of the microbiological contamination in shellfish leads to increased risk for the consumer, whereas over estimation can lead to intervention and significant cost to the industry. Second, the development of a deployable E. coli sensor will enable scientists to study the routes of contamination and the environmental / seasonal events that underpin them. Whilst it is not anticipated that this system will be delivered during the lifetime of this project, the development of the Lab on a Chip nucleic acid amplification method will represent a significant step towards its completion. The technology that the proposed research will develop can be easily modified for a wealth of other applications in food safety, medical diagnostics and environmental microbiology.

This project will develop new Lab on a Chip (LOC) nucleic acid amplification assays for the quantification of viable indicator Escherichia coli in shellfish water and shellfish flesh for statutory monitoring. We will achieve this using the isothermal NASBA method, which unlike conventional nucleic acid amplification by PCR, is performed at a single, low temperature (approximately 41 C) and therefore does not require the high power consumption, bulky heating / cooling apparatus and is more suited for integration with miniaturised Lab on a Chip devices. Quantification will be achieved by amplifying a region of the E. coli genome in tandem with a quantification reference sequence. The amplification will be measured in real time by using molecular beacon probes, providing data in less than 60 minutes. To address the problem of cell viability we will cultivate 3 methods. Firstly, we will optimise a cell pre-treatment step using either propidium monoazide or DNase to inactivate / remove genomic fragments left over from non-viable cells. Secondly, we will use mRNA-specific amplification assays. The mRNA, unlike DNA, is susceptible to rapid degradation in environmental matrices and therefore its amplification is a direct indicator of viable, transcriptionally active cells. Thirdly, we will develop genomic fragment amplification assays that will report on the level of intactness of genomic DNA. These assays will be incorporated into a Lab on a Chip platform which will manipulate an extracted sample through microfluidic channels into a reaction chamber containing preserved assay reagents and enzymes. Re-hydration of the preserved components will activate the NASBA-based amplification of the template sequence, which will be measured in real time using our state of the art fluorescence detection apparatus. The proposed LOC assays will hold advantage over the conventional culture based method for E. coli including rapidity and automation to reduce sample-sample variability.

Societal Benefit

The new tools (NASBA assays and Lab on a Chip) that the proposed research will generate will benefit both shellfish producers and regulatory authorities by improving the quality and efficiency of the measurement of indicator E. coli in line with regulatory standards. They will reduce the incidence of under or over estimation of the microbiological contamination in shellfish flesh, reducing risk to human health and unnecessary intervention respectively. Society at large will benefit from improved food safety and reduced costs for regulatory testing (i.e. by minimising the need for attended measurements (through automation) and significantly reducing the time required to perform each assay). The indirect benefits to society will result from the provision of new tools (i.e. the Lab on a Chip NASBA) for environmental microbiology which will assist research into the sources, transport and fate of microorganisms within shellfisheries; improvements to the prediction and management of shellfish contamination events will follow. Wider benefits to society will result from the further development or modification of the new tools to address related microbiological issues including quality assurance in different foodstuffs and, where required, statutory testing for other pathogens (e.g. Salmonella, Norovirus, etc). This impact will be achieved primarily through the Centre for Environment, Fisheries & Aquaculture Science (Cefas) who will benefit from direct access to the new technology. The anticipated development of these tools beyond the scope of this project to produce portable or deployable microbiological sensors enabling in situ and near real time monitoring by minimally skilled operators will broaden the scope of state of the art molecular methods for monitoring and protection in line with societal changes such as the rise in foraging and the expansion of the aquaculture sector.

Economic and Industrial Benefits

Shellfish constitute 39% (£300 M wholesale ~ £1.2 B retail, 2012) of the value of landings by UK fishing vessels with cultivated stocks contributing a further £38 M wholesale: ~70% is exported (http://www.seafish.org/research-economics/market-insight/market-summary). The industry incurs considerable cost when the microbiological quality of shellfish falls below regulatory standards, and therefore, the proposed research will benefit the shellfish industry by providing a tool to improve shellfish monitoring standards by applying state of the art molecular methods. Over time a reduction in the incidence of shellfish poisoning events, which are typically heavily publicised, will further benefit the industry by increasing confidence, both domestically and internationally, of UK-produced shellfish. Additionally, there is a clear benefit to the instrumentation industries from the new tools we will develop. A recent survey by one of our commercial partners TELabs, as part of the Aquawarn EU FP7 proposal, identified an £8.6 Bn market for environmental sensing and monitoring technologies with significant growth potential. The outputs of this project will have significant market value and we anticipate these to be commercialised via licensing or spinout in the medium term. The flexibility of the proposed Lab on a Chip device (i.e. through the incorporation of additional assays) will ensure that the principle technology has a significant range of potential applications to maximise commercial potential.