Propionic acid use in agriculture and food production is driving evolution of novel Escherichia coli pathotypes

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Emerging multidrug resistant strains of bacteria have forced both the agricultural and food production industries to rethink the use of antibiotics as a means of preventing both food spoilage and transmission of pathogens into the food chain. Alternative anti-microbial compounds are increasingly used such as short chain fatty acids (SCFA), commonly found in the colon but with anti-microbial properties. SCFA production and use has increased greatly in the past 40 years, particularly in the Western world, with the production of one such SCFA, propionic acid (PA), now worth approximately £1.2 billion per year and rising. PA efficacy and low cost allows farmers to treat grain and animal feed while reducing antibiotic use, resulting in PA being proposed as a solution in developing countries to antibiotic overuse.
PA has proven highly successful in poultry production where through feed and water addition it has reduced carriage of Salmonella and Campylobacter, common causes of food borne sickness in the UK. PA has also proved highly popular as it is non-toxic to humans with few, if any, side effects. PA therefore can be added to foodstuffs including cakes, bread and preserves at up to 1% total weight of the food.
The Western diet now contains increasing levels of food additives whose addition while prolonging the shelf life of food and reducing food borne disease, has also coincided with worrying increases in the prevalence of debilitating gut diseases. Inflammatory bowel diseases such as Crohn's Disease (CD) and ulcerative colitis (UC) while linked to underlying genetic conditions are exacerbated by dietary and other factors, such as bacteria in the intestine.
A definitive explanation to link these genetic, dietary and microbial factors together has to date proved elusive. In this proposal we will show that PA is facilitating the transfer of pathogenic E. coli that are exposed to PA in poultry, to the PA rich environment of the human intestine.
Adherent and invasiv E. coli (AIEC) are consistently isolated from the intestine of CD sufferers. These bacteria are unusual in that they lack toxins or other factors that would set them apart as pathogens. However unlike most other bacteria AIEC can grow and survive on the preservative PA which in time induces significant changes in the behaviour of AIEC. These PA induced changes include increased invasion of cells of the intestine, persistence within these cells and an ability to form structures called biofilms that enable the bacteria to resist antibiotics at higher concentrations and for longer.
When this adaptation to PA in the lab is looked at from an agricultural perspective it can be seen that if AIEC were to encounter PA in the environment it would prepare or adapt them to the human intestine where levels of PA are high and restrictive for most pathogens, particular in the lower intestine or colon. However, AIEC establish infection throughout the human intestine, even in places where Salmonella and Campylobacter cannot. AIEC therefore appear well adapted to PA begging the question of how and where this adaptation occurred. Significantly AIEC are thought to be related to pathogens called avian pathogenic E. coli (APEC) that are found in birds such as chickens. These APECs are increasingly being recognized as causative agents of human disease with E. coli isolated from infections throughout the body, including the urinary tract and meningitis, bearing striking similarities to APECs.
This proposal will seek to establish if high levels of PA use in the poultry industry and elsewhere is contributing to the evolution of pathogens that are now capable of causing disease in the human intestine. This proposal will for the first time show a definitive link between Crohn's Disease, the Western diet and AIEC, and identify propionic acid as being a facilitator in driving this relationship.

Technical Summary

Propionic acid (PA) is a widely used anti-microbial short chain fatty acid (SCFA) reducing spoilage of food, agricultural and plant products. PA use has continually increased for over 40 years, predominantly in Western countries, and more recently as a means of addressing antimicrobial resistance (AMR) concerns. PA concentrations in the intestine mean it is regarded as non-toxic and few microbes maintain the capability of metabolising PA.

Our recent data however raises serious concerns about the widespread use of PA as an agricultural feed and water and human food additive. We have found that opportunistic Escherichia coli termed adherent and invasive E. coli (AIEC) are dramatically altered when PA is provided as a sole carbon source. These bacteria, commonly found in sufferers of the chronic inflammatory condition Crohn's Disease (CD), were seen to increase in virulence when grown on PA, increasing their adherent and invasive phenotype and also forming increasingly structured biofilms with greater numbers of bacteria. PA, which is also known to increase the virulence of Mycobacteria was seen to induce similar outer membrane changes in AIEC where increased protein production was noted alongside altered lipopolysaccharide. Perhaps most significantly these changes in virulence and protein expression were permanent, remaining after removal of PA, with single nucleotide polymorphism (SNPs) identified in known virulence genes that promote adherence, the type 6-secretion system and stress resistance.

Planned Impact

This proposal would impact the following stakeholders as outlined below.

Research community:
Food security research:
Food security concerns have led to dramatic changes in production of food, grains and animal feed. Antibiotic use has reduced significantly due to concerns over increasingly antibiotic resistant pathogens emerging across the world. Propionic acid (PA) is being used increasingly across the world, spreading more recently to developing countries where antibiotic overuse is still a considerable problem. This proposal is timely in that it highlights concerns about the role of PA in driving evolution of novel human adapted opportunistic pathogens, in this case adherent and invasive E. coli (AIEC). Many alternatives to PA are available and likely do not induce similar issues as regards pathogen selection. This proposal would impact food security research by highlighting a to-date unrecognized issue in the use of specific preservatives driving research to identify and ensure the safety of alternatives.
Antimicrobial resistance research:
Reducing worldwide antibiotic use is a primary concern presently and will remain so for many years to come. PA use in farming has helped greatly in this regard. However debilitating diseases such as Crohn's Disease (CD) are increasing as food processing changes and the role of PA and other organic acid preservatives must be addressed. It is essential to retain trust in the goal of reducing AMR, and to do so safe and economical alternatives must be offered to farmers. Acknowledging and addressing safety issues with preservatives is essential to finding alternatives. Those researching AMR including the pharmaceutical industry would benefit through increased knowledge of issues of AMR pertaining to preservatives, something not previously described.
Crohn's Disease research and the medical community:
The reason as to why AIEC and Mycobacterium avium paratuberculosis, as opposed to other bacterial species, are found in greater numbers in CD sufferers is unknown. This proposal puts forward a unifying theory that links both pathogens; both are unique PA utilizing pathogens that trace their origins back to agricultural backgrounds as either avian or bovine pathogens. This finding would lead to new ways of examining CD as a disease, and potentially new ways of modulating the microbiome to help control the disease impacting the way the medical community treated CD.

Outside of scientific research the following groups would be impacted by this research:
The Department for Environment, Food and Rural Affairs (DEFRA) and the Food Standards Agency (FSA):
Both would be impacted as both play significant roles in deciding policy as regards preservatives that are suitable for use in food in the UK. Previously they have moved to ban certain additives based on adverse effects.
Farming industry/Agribusiness:
The farming industry would not be significantly impacted as alternatives to PA are already available. Changes in farming practice to reduce PA use would be the most obvious impact. Agribusiness which relies heavily on compounds such as PA would be impacted as an alternative approach to agricultural produce being preserved would be needed.

Public/consumers:
This proposal would impact on public perceptions of food preservation by educating the public as to how their food is treated and preserved long term. For sufferers of CD and other inflammatory bowel conditions being aware of how their diet may exacerbate their condition would have a significant impact on their treatment.
Pharmaceutical industry:
PA production is worth over £1 billion per year worldwide and a number of companies within the UK produce the compound. However these companies are not limited to PA production for the most part, and any impact would most likely be limited to producing a safer alternative to PA. Alternatives are already manufactured in the UK for the agriculture industry.
 
Description We have discovered that a specific short chain fatty acid (SCFA) called propionic acid (PA) can act as a source of nutrition for pathogenic bacteria in the human intestine. This is highly unusual as PA normally is antimicrobial in the intestine and can kill bacteria. Due to this it is now used in agricultural and environmental settings to reduce the presence of pathogens such as Salmonella and Campylobacter (in chickens for example it is added to feed to reduce these bacteria in the chicken intestine and reduce transmission to humans).

The bacterium we study, adherent and invasive Escherichia coli (AIEC), not only grows on PA but it becomes increasingly virulent in its presence. By altering the PA concentration in a mouse model of infection AIEC increases 10-fold in the intestine over 3 days. Further long term work indicates that over the course of 3 weeks this increased microbial load remains far higher after supplementation with PA. This leads to increased inflammation both in the intestine and systemically. We are presently looking into translating these findings clinically by using biopsies sourced from the NHS to check the clinical relevance of our findings.

We have now moved our observations into the clinical setting trying to take our research from 'bench to bedside'. We have applied our basic laboratory science findings and shown that they are applicable in paediatric Crohn's disease patients. The changes we see in PA treated bacteria in the lab are reflected within this patient population and for the first time we have identified a specific carbon source these bacteria associated with Crohn's disease respond to. We are presently looking into ways we can turn this finding to our advantage and use this signal as a means to alter the behaviour of these bacteria so they reduce rather than increase inflammation in these patients.
Exploitation Route Our findings could likely lead to a change in the way antimicrobials are used in agriculture. Our findings indicate that in the same way bacteria develop antibiotic resistance they are also developing resistance to less common antimicrobials such as SCFAs. Given our body's natural defences rely heavily on such substances their excessive use in agriculture etc. needs to be addressed.

We are also attempting to translate our findings into the clinical setting. We know what these bacteria respond to now so in disease such as Crohn's disease where they are found we are attempting to alter their phenotype so they are a positive presence rather than a negative one as they are now.
Sectors Agriculture, Food and Drink,Environment,Healthcare

 
Description Appointment to BBSRC Pool of Experts
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description BBSRC Committee B member May 2018 and February 2019
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
URL https://bbsrc.ukri.org/about/governance-structure/committees/committee-b/
 
Description Cystic Fibrosis Trust Scientific Advisory Board
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Horizon2020 Panel
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
URL https://ec.europa.eu/programmes/horizon2020/en
 
Description BBSRC-AstraZeneca 4 year PhD
Amount £140,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 09/2023
 
Title Crohn's disease animal model based on SCFA supplementation 
Description We have generated a new mouse model of Crohn's disease using bacterial strains isolated from CD patients. Previously these bacteria were not infective in animal models casting doubt on their relevance to disease. However we have shown that by altering SCFA levels in the murine gut these bacteria cause a productive and long term infection with both local and systemic inflammatory consequences. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact We believe this highlights an important difference between humans and animal models of disease. SCFAs are far lower in mice resulting in entirely different outcomes in infection. 
 
Description Clinical application of findings from BBSRC grant 
Organisation National Health Service
PI Contribution Looking at patient samples to translate our findings into the clinical setting. We use biopsies to detect metabolic signatures of bacterial growth as an indication of how that may contribute to gastrointestinal disease such as IBD.
Collaborator Contribution Provide the clinical samples/
Impact No outputs as yet as collaboration is new. Multi-disciplinary: bacteriology, immunology and clinical departments from NHS involved.
Start Year 2018
 
Description Phylogenetics of adherent and invasive Escherichia coli 
Organisation Heriot-Watt University
Department Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Working on collaborative publications exploring the origin of adherent and invasive E. coli strains associated with inflammatory bowel disease. My laboratory has been leading the research with our colleagues at Heriot-Watt providing bioinformatic input. Our work has primarily focused on classifying differences between the isolated strains from IBD patients with regular commensal or non-pathogenic E. coli. This has all been through in vitro analysis in immune and intestinal epithelial cells.
Collaborator Contribution Our partners have attempted to phylogenetically translate our laboratory findings bioinformatically to see if our data shows a greater overall trend in these bacterial strains.
Impact Presently working towards publication
Start Year 2015
 
Description SCFA detection 
Organisation Glasgow Royal Infirmary
Country United Kingdom 
Sector Hospitals 
PI Contribution Providing samples from animal models of Crohn's disease for comparison to those of humans as regards their short chain fatty acid content. We believe this may be indicative of perturbations in gastrointestinal metabolism and disease.
Collaborator Contribution Short chain fatty acid detection by LC-MS.
Impact Paper under review with respect to this work.
Start Year 2017
 
Description Glasgow Microbiology Collective 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact A meeting of microbiologists from across Glasgow with a view to disseminating ideas and forming the basis for future collaborations. The meeting ran over two days and will continue on an annual basis. A number of collaborations have arisen from the meeting already resulting in joint PhD students, joint grants and further get togethers to share ideas.
Year(s) Of Engagement Activity 2017,2018
 
Description School visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presented microbiology based work to P6 and P7 students. Have been asked to return to help set up a basic laboratory experiment showcasing bacteriology.
Year(s) Of Engagement Activity 2018