Molecular basis of foodborne disease risk of variants of Salmonella Typhimurium DT193 and U288

Lead Research Organisation: Quadram Institute Bioscience
Department Name: Gut Microbes and Health

Abstract

The bacterium Salmonella accounts for about 125 million incidents of disease worldwide each year, and nearly a million deaths. The morbidity and mortality caused by this pathogen has a significant impact on the economies of both resource rich and resource poor countries. Most cases of non-typhoidal Salmonella are thought to result from fecal contamination of food and food products, either directly in the food chain or by cross contamination in the home or restaurants. A common and therefore critical step for this is the entry of the bacterium into the food chain from livestock and poultry in which this pathogen is commonly found. However, even though virtually all types of Salmonella have the potential to cause disease in man, not all are commonly associated with disease in man. Understanding how these processes work is critical to the detection of high risk types of Salmonella in livestock and the food chain, and efforts to decrease the likelihood of Salmonella entering these environments. We propose to study two common types of Salmonella that are both present in pig herds butter present distinct risk to food safety. We will study these bacteria at a genetic and behavioural level to understand how the different types circulate in pig populations in the UK and how they enter and survive in our food. First a collection of pig and food chain isolates of Salmonella Typhimurium will be whole genome sequenced and the variation in their genome used to define the how they spread into the food chain and into the human population. Then we will study important behavioural variations that may impact the threat posed by the variants in food. As the types of Salmonella to be studied are genetically closely related, the number of genetic differences are small, which makes it possible to identify candidate differences associated with altered behaviours of the variants. Genetic differences in types of Salmonella are potential candidates targets for surveillance to identify types more likely to represent a risk to food safety or for other intervention strategies aimed at decreasing the likelihood that they will enter the food chain.

Technical Summary

The considerable economic and health impact of pathogens of the genus Salmonella is the result of their presence in livestock and poultry, entry into and survival within the food chain, and their ability to cause intestinal or systemic disease. However, not all variants of Salmonella are equally likely to cause disease in man and an understanding of the molecular basis of the likelihood that a particular variant will enter the food chain and cause disease in man is critical to food safety. Some variants of Salmonella are prevalent in food animals yet are not a predominant cause of human clinical infections. This research proposal seeks to address these questions using a molecular epidemiology and comparative whole genome sequence approach combined with classical molecular biology and infection models to compare two highly related variants of S. Typhimurium (DT193 and U288) that exhibit distinct risk profiles for foodborne disease in man, despite similar epidemiology in livestock. Little is known about the variation in genotype and phenotype of closely related variants of bacterial pathogens circulating in zoonotic reservoirs and the environment. Crucially, genotypic polymorphisms are not only potential candidate targets for intervention strategies aimed at decreasing the likelihood that these pathogens enter the food chain but also targets for distinguishing variants of pathogens that differ in their risk to food safety, useful for surveillance. This study will define the molecular epidemiology of common variants of Salmonella Typhimurium in the UK pig herds and those entering the food chain via abattoirs by analysis of whole genome sequence variation. Genotypic, transcriptomic and phenotypic variation will be determined and important genotypic differences understood in the context of their associated phenotype by whole genome recombination and mutagenesis approaches.

Planned Impact

The beneficiaries of this research include research scientists, government agencies involved in surveillance activities, the food industry and the general public.
1. Research scientists will benefit from a greater understanding of the diversity of genotype and phenotype of the Salmonella pathogen. Salmonella is used as a model pathogen organism in thousands of research labs around the world. These labs tend to use a single or a limited number of strains in their studies. However, it is not known how generally applicable the conclusions are from experiments from a single strain. Our study will provide a baseline data for the diversity of genotype and phenotypes of closely related isolates of S. Typhimurium. These data may also provide insight for other bacterial pathogens in addition to Salmonella. Our work will also generate sequence data, phylogenetic information and genetically modified microorganisms that will be made freely available to the research community. As part of this project we will develop methodologies to construct chimeric strains of S. Typhimurium using high frequency recombination. The strains used for this methodology will be broadly applicable for many molecular genetic studies using S. Typhimurium and these will also be made freely available.
2. Government agencies (eg AHVLA and PHE) involved in surveillance of bacterial pathogens will benefit from the data and data analysis generated in this project. The Salmonella strains to be analysed are part of an on-going epidemic in the UK and new diagnostic methods to detect and differentiate these isolates are needed. The Dr Liljana Petrovska (AHVLA) and Dr Elizabeth de Pinna (PHE) are collaborators on this application and projects run internally within these organisations will benefit directly from the output of this project. The Principal Investigator is also an active member of the Global Microbial Identifier task force that is actively engaged in using the type of data generated in this project to identify pathogens and genes of interest in these pathogens for diagnostics and surveillance. The project will therefore have a potential global impact.
3. The food industry and in particular the pig rearing industry is interested in decreasing the incidence of Salmonella in the food chain. This is in part from the EC. The output from this project will provide the knowledge required to discriminate between pathogens of high and low risk to human disease.
4. In the longer term the general public will benefit from potential advances in diagnostic and surveillance methodologies that will become available with the knowledge generated from the proposed work. The network of scientists involved as collaborators on the research project have the expertise to deliver these improvements in parallel and as a direct result of the proposed work.
STRATEGIC RELEVANCE: The proposal addresses gaps in the fundamental knowledge impacting the ability of the UK food industry to deliver safe food with a reduced risk of contamination with food borne pathogens and in particular Salmonella. It is therefore directly relevant to the BBSRC strategic priorities 'Food, nutrition and Health'. The generation of a large amount of whole genome sequence data of Salmonella from food sources and the food chain is the starting point of this project. Integration of these data with existing whole genome sequence data to study the phylogenetics and molecular epidemiology of Salmonella in livestock and the food chain is an example of 'Exploiting New Ways of Working'. In addition the project benefits from working from this point back to mechanisms and classical molecular biology approaches. This work therefore in part fulfils the major responsive mode priorities 'Data driven biology'.
 
Description We have investigated the molecular epidemiology of two types of Salmonella commonly found in UK pig herds (DT193 and U288) that differ in their risk to food safety and distribution in animal hosts. DT193 Salmonella account for over half, while U288 Salmonella account for less than 1% of all human clinical infections by Salmonella Typhimurium. We have characterised the phylogenetic relationship of these bacteria and identified distinct microevolution leading to functional diversification consistent with their distinct epidemiology by whole genome sequencing over 500 Salmonella isolates and in vitro and in vivo .

DT193 and U288 form two distinct groups in the phylogenetic tree of Salmonella, but share a common ancestor that existed at an as yet unknown time but that is likely to be at least several hundred years in the past. DT193 evolved by acquisition of genes conferring resistance to antibiotics and copper and these may have led to their rapid spread into pig populations where antibiotics and copper are used to treat infections or as growth promotors, respectively. U288 acquired genes involved in resistance to antibiotics as well, but also lost coding capacity in their genome, a common signature of host adaptation. In both cases these variants evolved over a 20-30 year period, but gain or loss of genetic material that resulted in their ability to enter the pig population and spread rapidly.

The evolution of U288 was largely characterised by a decrease in coding capacity in the core genome as a result of small insertions and deletions predicted to affect the expression of a functional protein (hypothetically attenuated coding sequences - HACs). HACs were in genes involved in metabolic functions including those linked to pathways involved in survival of desiccation, and genes previously implicated in interactions of Salmonella with the host. The change in coding capacity is therefore consistent with changes that affect survival in the food chain and potentially adaptation to circulation specifically in pig populations. In contrast the microevolution of the DT193 genome was characterised by the acquisition of novel phage and genomic island that is a member of a previously unknown family of integrative conjugative elements (ICE).

The decrease in coding capacity of the U288 genome was associated with phenotypic changes in characteristics that have the potential to negatively impact their ability to enter the food chain, and survive or replicate. The two variants exhibited distinct interactions with the pig on oral infection. DT193 colonised the intestine more efficiently than U288 strains, but was recovered in lower numbers from lymphoid tissue associated with the intestine. Such differences in tropism may have a significant impact on the likelihood of these two variants entering the food processed at the abattoir. Furthermore, we also observed less severe disease symptoms in pigs infected with U288 compared with DT193 Salmonella, a finding that may have important considerations for the detection and treatment of Salmonella on farms. U288 also grew more slowly in rich broth in aerobic and anaerobic conditions and is more sensitive to desiccation compared to DT193. These distinct physiological characteristics may contribute to differences in risk to food safety due to survival in associated stresses.

Our findings therefore suggest that U288 may be less able to both enter the food at the abattoir, but also less able to survive and replicate in the food.
Exploitation Route Our analysis has identified candidate genetic markers associated with risk to food safety of two closely related types of Salmonella. We have identified key differences in the way that variants of Salmonella with distinct risk to food safety colonise pigs, the principal zoonotic reservoir. This information is most relevant to the surveillance of Salmonella by the Animal and Plant Health Agency and the agriculture industry involved in pig farming. These stake holders would benefit from an understanding of how different types of Salmonella colonise pigs and the molecular markers that may be used to predict the relative risk each type poses to human health.
Sectors Agriculture, Food and Drink

 
Description Molecular epidemiology and genomics of Salmonella Typhimurium in livestock and wild animals 
Organisation Animal and Plant Health Agency
Country United Kingdom 
Sector Public 
PI Contribution My research group designs research programs, processes samples for genome sequencing, analyses data and carries out molecular microbiology studies to test hypotheses generated from sequence analysis. We write manuscripts for publication in peer reviewed journals. We work with our partners to identify potential use of genome sequence data to improve surveillance and diagnostics of foodborne pathogens.
Collaborator Contribution A partners at APHA help to design research programs with advice and epidemiological data from national surveillance programs. They prepare genomic DNA for whole genome sequence and help interpret data and analysis.
Impact Collaboration is multi-disciplinary IFR scientists provide sequence analysis and molecular biology expertise and APHA scientists provide epidemiological expertise
Start Year 2015
 
Description Molecular epidemiology and genomics of Salmonella Typhimurium in livestock and wild animals 
Organisation Public Health England
Country United Kingdom 
Sector Public 
PI Contribution My research group provides expertise in sequencing and analysis of sequence data of foodborne bacterial pathogens and molecular biology for the characterisation of pathogens. My team analyses data and disseminates findings to the scientific community and other groups of interested parties by the preparation of manuscripts or oral presentations. The activity aims to contribute underpinning scientific evidence for bacterial pathogen surveillance and development of intervention strategies aimed at decreasing the likelihood that pathogens will enter the food chain.
Collaborator Contribution Collaborators at PHE contribute surveillance, samples and molecular epidemiology expertise. They contribute to preparation of publication material and monitor potential use of scientific output for translation into surveillance or intervention.
Impact none as yet
Start Year 2015
 
Description AB Agri Ltd introductory meeting to discuss potential areas of collaboration 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Discussion on how work in the Kingsley lab could be used by AB Agri (leading animal feed production company) to increase productivity and food safety in the pig rearing industry.
Year(s) Of Engagement Activity 2017
 
Description Discussion with Nestle food safety technologists 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Exploratory talks about how scientists at the Quadram Institute could collaborate with Nestle in studies aimed at investigating the impact of genetic diversity on risk to food safety
Year(s) Of Engagement Activity 2018
 
Description Discussions with 'Nature Menu' on potential collaborations aimed at investigating the diversity of Salmonella in raw dog food and implementation of intervention strategies 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Discussion of previous collaboration outcomes and planning for future collaborations and investment from Natures menus in collaborative research
Year(s) Of Engagement Activity 2018
 
Description Discussions with Mike Salter and Steven Jagger from ABAgri 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Discussions about how work on pathogen variation of Salmonella and transferable copper resistance in the Kingsley lab could be used to increase productivity and food safety in the pig rearing industry.
Year(s) Of Engagement Activity 2016
 
Description Food production site visit 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Visit to a ready to eat food production site to discuss bacterial pathogens in the food processing environment and interaction with bacterial communities.
Year(s) Of Engagement Activity 2017
 
Description Norwich Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Dr Mark Kirkwood, PDRA working on a BBSRC funded project, met with the general public to explain the work going on at the Institute of Food Research, with specific reference to his work. He explained the importance of understanding the risks to food safety posed by Salmonella and how we are using advanced molecular biological techniques and genome sequencing to innovate new surveillance, risk assessment and interventions.
Year(s) Of Engagement Activity 2016
URL http://www.theforumnorwich.co.uk/norwichsciencefestival
 
Description Oral presentation and three posters presented at 5th ASM Salmonella Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dr Rob Kingsley presented a talk 'Genotypic and phenotypic diversity arising during the clonal expansion of the current MDR monophasic Salmonella Typhimurium epidemic', and Dr Priscilla Branchu, Dr Matt Bawn and Dr Mark Kirkwood presented posters entitled 'Altered copper homeostasis in the multidrug resistant monophasic S. Typhimurium epidemic clone'. 'Signatures of microevolution in Salmonella Typhimurium revealed through whole genome sequencing', and 'Molecular Basis of Foodborne Disease Risk of Variants of Salmonella Typhimurium DT193 and U288' to a large group of scientists from around the world with the objective of sharing ideas and research outputs.
Year(s) Of Engagement Activity 2016
URL http://conferences.asm.org/index.php/component/content/article/140-conferences/5th-conference-on-sal...
 
Description Poster presentation at 'Microbes in Norwich' symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact A meeting to exchange ideas and research findings for scientist with an interest in microbes in Norwich. Dr Matt Bawn presentation title 'UK Salmonella Typhimurium Epidemic Associated with Acquisition of Novel Genomic Island'
Year(s) Of Engagement Activity 2017
URL https://www.facebook.com/events/221060494967981/
 
Description Seminar presented at the Roslin Institute, Edinburgh 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited seminar entitled 'Genome variation of Salmonella: epidemics, evolution and pathogenesis'. Dissemination of research findings and plans for several projects. Discussion with research leaders.
Year(s) Of Engagement Activity 2016
 
Description Third year BSc Animal Science students and 2 lecturers from Writtle college visit to Earlham Institute 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact Dr Matt Bawn met with visiting students from Writtle College and described his work with food borne pathogens including work funded by three BBSRC funded projects. The visit from 19 third year BSc Animal Science students and 2 lecturers from Writtle college in 2016 enabled us to promote our science and highlight colleague career paths in genomics and bioinformatics. It provided a forum for two-way dialogue where staff could engage directly with the students and lecturers, bringing application and relevance to key areas of study. A 'three-horizons' task also prompted the students to consider the potential limitations of current research themes and to consider future possibilities and outcomes. The event also provided Matt with the experience of working with students and the practice of tailoring the communication of their work to varied audiences. 100% of student respondents thought the event was 'great' and would recommend future visits.
Year(s) Of Engagement Activity 2016