Optical Detection of Listeria in the Chilled Food Environment using Bionanosensors

This programme of research involves the development of an innovative bionanosensor with superior performance for the detection of bacterial pathogens in a sensitive, quantitative and multiplexed manner for use in chilled food production. This will involve developing nanoparticle based analytical technology for the detection of multiple bacterial pathogens (L. monocytogenesis and enterobacteriaceae) associated with food contamination in the chilled food industry. Current methods for detecting bacteria are time consuming (1-2 days in the case of bacteria culturing on selective media, but in reality 7 day turnaround for food production facilities where laboratories are located offsite), expensive and require specialised personnel and equipment. Therefore, there is a strong need for faster, simpler and reliable isolation and detection of bacterial pathogens that can be carried out in the production environment. To address this need we will develop a simple, portable detection platform capable of point of use (POU) detection for bacteria.
Successful bacteria detection in food production is crucial for the health of the general public. Within the chilled food sector, Listeria is of particular concern since it is an unusually resilient bacterium which can survive in temperatures from 4 to 42 degrees C. Listeria monocytogenes causes one of the most severe forms of foodborne infection with an overall mortality rate of 30 %, rising to 40 % in vulnerable individuals. Those most at risk of acquiring the disease are pregnant women, the elderly and the immuno-compromised and in some cases listeriosis can develop into meningitis and septicaemia if infection of the blood occurs. However, outbreaks of Listeria are rare due to strict controls on food safety however our industrial partner, Bradgate Bakery, who manufacture sandwiches and salads, require the ability to locate, detect and contain any contamination within food production lines.
The research involves the use of an optical detection technique called Raman scattering which will be developed for the POU detection of bacterial pathogens. If light of a particular wavelength is directed onto a molecule then some of the scattered light will change wavelength. This change in wavelength is related to the structure of the molecules and provides a molecular fingerprint that can be used for definitive identification. However Raman scattering is an intrinsically weak process and the signal can be greatly enhanced if the molecule is coloured and is adsorbed onto a roughened metal surface (surface enhanced resonance Raman). The metal can be thought of as essentially amplifying the Raman scattering from a molecule on the surface and in this case will take the form of metal nanoparticles. Since a fingerprint unique to the molecule is produced, the composition of mixtures can easily be identified without separation.
A novel bionanosensor for the detection of multiple bacterial pathogens, namely L. monocytogenesis and enterobacteriaceae in one assay combined with enhanced Raman detection will be developed. This will use magnetic nanoparticles which have a biomolecule on the surface known as a lectin which will bind to the surface of bacteria. This will allow isolation and separation of bacteria from the surrounding medium upon application of a magnetic. Additionally, silver nanoparticles which are functionalised with a coloured molecule or label, resulting in intense surface enhanced Raman signals, and a biomolecule which will bind specifically to a particular strain of bacteria (antibody or aptamer) will be added. When the correct bacteria are present binding will occur resulting in magnetic isolation and concentration of the bacteria from the matrix. By using a different label for each bacteria, a unique spectrum will be achieved for each bacteria allowing multiple to be detected simultaneously. A portable Raman spectrometer will then be used to detect the bacteria present.

Successful pathogen detection is crucial for food security in the chilled food industry particularly as the threat of infectious disease is dramatically increasing due to bacterial resistance to antimicrobial drugs. Therefore, there is a strong need for faster, simple, and reliable isolation and detection of bacterial pathogens using novel point of use (POU) technology within food production environments. The use of a novel bionanosensor is proposed for the multiplexed detection of bacterial pathogens within the chilled food industry. The technology is based upon the use of surface enhanced Raman scattering (SERS) due to its high sensitivity and multiplexing capabilities. SERS active silver coated magnetic nanoparticles will be functionalised with lectins which are capable of specifically recognising and binding to carbohydrate constituents on the surface of bacteria. These lectin functionalised magnetic nanoparticles will be used to selectively capture and concentrate bacteria from swabs of production lines and mulched food samples. Silver nanoparticles will then be functionalised with a Raman reporter and a biorecognition molecule (antibodies/aptamers) which is specific towards a bacterial strain. A SERS response will only be obtained when the SERS active nanoparticle binds specifically to its bacterial target. The magnetic 'plug' will then be interrogated using small, portable Raman spectrometers that can be used in food production environments. In this way the detection strategy will be fully portable and allow for rapid, point of use detection. Once the multiplexed quantitative SERS signal is generated it needs to be analysed such that the unequivocal detection of a pathogen is made and/or the concentration of the bacteria predicted giving a response using an easily interpretable interface.

For chilled, ready- to eat (RTE) foods, the European legislation specifies either a complete absence of L. monocytogenes in 25 g of sample or a level below 100 CFU per g at any point in the shelf life. There is also a requirement for producers to swab food processing areas and equipment for the presence of L. monocytogenes as part of their sampling and cleaning regime. L. monocytogenes is a particular issue as it is a very resilient bacteria which can survive at low temperatures (down to 4C). Although the number of reported cases of listeriosis is low compared to campylobacter and salmonella, the disease places significant public health and economic burdens on the UK because of its high hospitalisation and mortality rate. Those at increased risk include people with weakened immune systems, pregnant women and their unborn babies, newborn babies, the very young, and the elderly. Most people infected with Listeria are hospitalised and approximately a third die.
Thus developing new technology for the rapid, onsite detection of bacteria in food production is vital to ensure contaminated food products are not consumed by the public. The impact of this technology is far reaching, from the control of Listeria in the manufacturing environment to the testing of food products before reaching the consumer. Currently the surveillance testing carried out in the manufacturing environment takes c. 7 days from test to result. This means that it is generally a week before any remedial action can be taken if there is a positive result where bacteria can continue to proliferate and spread throughout the manufacturing environment, transferring to food products. Reducing this time from test to result to within minutes would significantly reduce the potential for these bacteria to proliferate and contaminate, therefore reducing the risk to consumer health. This will have implications in terms of economic cost savings to the food industry but also on the cost burden on the NHS, by reducing the amount of cases of food poisoning outbreaks, as well as employers, through reduction in employee absence.
The approach being developed will be a portable, POU platform where bacterial identification can be rapidly carried out in food production areas. The need for this approach is clearly evidenced by the involvement and investment of Bradgate Bakery in this Industrial Partnership Award (IPA) application. Therefore we will pursue the commercial impact of the research through collaboration with Bradgate and their industrial network, Bradgate are part of the Samworth Brothers Group of 15 businesses, as well as its wider network of c. 500 suppliers who could all benefit from the outputs of the research. The outcomes will be of interest to many in the food industry so we will pursue opportunities for a consortium or open innovation approach to achieve maximum impact through Bradgate's networks.
The Global need for such rapid, POU diagnostic devices is huge and the outcome of the research will be a world leading position which could lead to company formation and considerable opportunity for wealth generation and employment in the UK. Even within the chilled food sector this new bionanosensor technology can be further extended into other areas e.g. bacterial detection in storage areas or tracing of outbreaks of contamination to source, indeed anywhere there is a requirement for rapid bacterial detection that is simple enough to be carried out by non-scientists is a further benefit of this approach. This proof of concept approach can be extended in future to the detection of other bacteria as well as disease related biomarkers. Therefore, this POU platform has the capability for extension into developing world detection strategies for example for pathogen detection, disease diagnosis and for security/military applications for chemical, biological threats. Therefore the impact of this research is immense and far reaching.