Understanding the ecology of Listeria and its interactions with microbiomes in food processing facilities to inform biocontrol strategies

There are over one million cases of food poisoning in the UK each year with an estimated economic burden of £1.5 billion. Listeria monocytogenes is one of the five major causes of food poisoning. It is a particular problem with chilled foods such as unpasteurised milk, soft cheeses, salads and many of the ready-to-eat, chilled foods. L. monocytogenes can cause meningitis, still births and abortion, in vulnerable groups including pregnant women, the elderly, and the neonates. In Europe, 2161 confirmed cases of listeriosis were reported in 2014, a rise of 30% on the previous year. In comparison to other food-borne diseases the incidence of listeriosis is relatively low, however the disease is associated with significant public health and economic burdens because of its high mortality rate of up to 30%.

This project will provide a knowledge base of factors that influence survival of Listeria in the food processing environment where it is able to colonise many sites, particularly damp, moist conditions such as drains, floors, wash areas and food handling surfaces. Despite robust cleaning and sanitising procedures, Listeria spp. persist in the environment. While we know a great deal about the genetics and persistence of Listeria spp. originating from clinical samples, Listeria spp. present in food factory environments have not been studied to the same degree, and there remains a significant gap in our understanding of: (a) the genetic makeup of Listeria strains that are found in food processing factories and (b) how these Listeria spp. interact with diverse bacterial species of the factory microbial communities. This proposal will address these gaps in our knowledge. We will work closely with the food processing sector and analyse swabs obtained from different types of surfaces within food processing factories. These samples will then be used to identify both the Listeria spp. and associated factory bacteria. The whole genome sequence will be determined to track the pathogen through different parts of the processing factory and this will provide unique insight into routes of contamination within the factory and the persistence of particular Listeria spp. in specific areas of the factory. To survey the complete diversity of bacteria in the factory environment we will use advanced DNA based fingerprinting methods to identify which other types of bacteria are present in different factory sites and if there is a connection between these bacteria and presence/absence of the Listeria strains at a particular location. Whole genome sequence data will also provide insight into the genes associated with biofilm formation, biocide resistance and association as complex communities with other bacteria.

Strong evidence suggests that Listeria persists in the environment because of its ability to form biofilms (where bacteria grow as communities on solid surfaces). We will therefore determine the ability of the Listeria isolates to form biofilms, both individually and also in the presence of other bacteria isolated from the factory surfaces to assess how these bacteria impact biofilm formation by Listeria spp. and their persistence in the factory. These biofilm studies will be examined in a 'model factory' that we will set up in the IFR lab to recreate diverse surfaces and simulate factory environmental conditions.

To undertake this multidisciplinary programme we have assembled a collaborative team of technical experts from the food industry and academic research scientists. This research will provide improved understanding of the behaviour and survival of Listeria spp. in food processing environments. Our ultimate aim is to provide detailed, extensive knowledge to inform the industrial development of new methods to reduce Listeria spp. levels in the food processing environment. These improved methods will be shared as 'best practice' with the wider chilled food industry, eventually leading to significant health / economic benefits.

L. monocytogenes remains a major cause of food poisoning with major public health and economic burden globally. Listeria spp. can be isolated from a wide range of sources including water, soil, silage, food and food processing environments. There is a significant gap in our knowledge of the genetics and epidemiology of the Listeria spp. present in the food factory environment and, in particular, their interactions with other members of the factory microbiome and how this impacts on their survival and persistence. We will test the hypothesis that this interaction plays a key role in the ability of Listeria spp. to form robust biofilms that can enhance their survival under stress conditions. In this multidisciplinary proposal we will isolate and determine the whole genome sequence of 500 Listeria spp. from in-house culture collections or isolated from chilled food factory environments. We will compare the epidemiology of the isolates and their persistence on specific factory surfaces. These diverse genomics data will be used to track the pathogen through different food factory areas and enable us to identify genotypes and candidate genetic determinants associated with persistence. In parallel we will perform 16S rDNA based metagenomics of the Listeria spp.-associated and non-associated microbiomes of food factory origin. We will establish correlations with specific commensal bacteria and isolate relevant bacteria. We will establish a lab 'model factory' at IFR to examine the ability of these Listeria spp. to form biofilms, either alone or as mixed species biofilm on factory relevant surfaces and under associated environmental parameters. The in-depth knowledge gained from this study will underpin our understanding of the survival and growth of Listeria spp. and will directly inform the industrial development of new intervention strategies to minimise the presence of this pathogen in food processing environments and thereby increase consumer safety and confidence.

This SPFI proposal presents an outstanding opportunity to address key health issues related to the understanding of the growth and survival of Listeria in the food processing environment. As such, outputs of this project will directly contribute to BBSRC's 'Food, nutrition and health' priority which spans both the 'Agriculture and food security' and Bioscience for health' key strategic priorities. The in-depth knowledge generated by the project has substantial strategic value in the long term for informing novel strategies for intervention and biocontrol of this microorganism. This will assist the food industry to overcome the regulatory challenges currently threatening it through its use of quaternary ammonium biocides as disinfectants in factory environments. In addition to academic beneficiaries, the outputs of the research will deliver high-quality impacts to the following wide-ranging stakeholder groups:

Food processing and Food Retail industries: This project will have a major impact on food manufacturing industries both in the UK and worldwide, particularly those involved in production of minimally processed ready-to-eat foods, demand for which is ever increasing, both in developed and developing countries. The project will inform the industries on the genotyping of the Listeria spp. isolates as well as the types of factory surfaces that are conducive to Listeria spp. growth. In the long term, this project's outputs may lead to improved strategies for preventing and inhibiting the growth of Listeria on these surfaces. The generic principles involved in this research are expected to provide additional opportunities for the food industries to develop innovative knowledge-led biocontrol measures as alternatives to biocides, to minimise levels of environmental Listeria spp. and the potential for consequential food contamination. For food retail industries, the presence of Listeria in their food products requires them to instigate product withdrawal with concomitant risks to brand equity and reputational damage. Hence, greater confidence in the improved cleanliness of food processing environments to produce safer food products will benefit both industrial sectors.

Societal impacts: Several segments of the population are at increased risk of infection by L. monocytogenes, including pregnant women, new-borns, immunocompromised people, persons with cancer, diabetes and kidney disorders and the elderly. Overall reduction of L. monocytogenes in the food chain will have positive impacts on the healthcare setting and the vulnerable groups within our society.

Economic impact: L. monocytogenes contamination and infection has a significant economic burden, both for health costs as well as to the food industry. In USA alone, the annual cost of listeriosis is estimated to be between $2.3 to $22 billion per annum, and to the food industry the cost of implementation of safety measures against L. monocytogenes represents 0.2% of product sales. Long term benefits of this proposal leading to improved control measures in food processing industries to provide the public with safe food will have economic benefits both for national health as well as industry.

Engagement with policymakers: This project contributes to the FSA's strategic plan 2015-2020 on the outcome that 'Food is safe'. Listeria reduction is cited alongside Campylobacter as a key food pathogen of concern. The outputs from this project will inform the FSA on improved control measures in the food processing setting.

The prime routes for dissemination and knowledge exchange will be via: 1) the industrial partners and project management team members to the chilled food market sector; and 2) by Ian Charles and the project management team to other stakeholder groups, including the wider scientific community, through giving presentations at international conferences and stakeholder events, as well as through publication in high impact, open access journals.