ANIHWA call2 - A bacteriophage-based approach to reducing infections caused by antibiotic resistant Escherichia coli

Lead Research Organisation: University of Nottingham
Department Name: School of Veterinary Medicine and Sci

Abstract

Antibiotic resistance is a significant and increasing problem in many types of bacteria which cause disease (pathogens) in animals and humans. Escherichia coli is among the most important of these pathogens, because of its role in intestinal, urinary tract and respiratory disease, and septicaemia in a variety of livestock species, including poultry; and also because many strains of E. coli that are associated with septicaemic infections in animals and humans are closely related. Antibiotic resistance in E. coli strains is increasing worldwide and this resistance can be maintained even after reducing or withdrawing antibiotic use. As such, the treatment of E. coli infections in animals and humans requires a new and sustainable approach. This project will investigate the use of bacteriophage as a biological control against harmful strains of E. coli which infect chickens. Bacteriophage, often contracted to 'phage', are viruses which infect and kill bacteria. They are quite specific, only affecting the targeted bacterial species while leaving other bacterial flora unharmed. Phages do not infect animals or humans and are widely distributed in the environment. As such, the use of phages to selectively kill harmful strains of E. coli which infect animals has the potential to be a natural and sustainable alternative to antibiotics, and may also result in new treatments for antibiotic resistant bacterial infections in humans. The effective application of phage therapy will require a thorough understanding of phage-bacteria interactions in a range of environments. This project will use laboratory experiments to build a comprehensive understanding of how phages infect E. coli strains under different environmental conditions. This information will then be used to design protocols for the optimal use of phage therapy to treat infections in animals.

Technical Summary

The use of host-specific bacteriophage to target pathogenic strains of E. coli has the potential to be an effective and viable alternative to antimicrobial chemotherapy. The selection of suitable bacteriophage biocontrol candidates requires the in depth characterisation of the virus and detailed analysis of how phages interact with their hosts in vitro and in vivo. We will isolate phages from the environment, surface water, farms, drains and sewage which are able to infect a range of Avian Pathogenic Escherichia coli (APEC) serotypes. These will be characterised in vitro, and bacteriophage biocontrol candidates will be selected for evaluation in an E. coli septicaemia model in chickens. Selection of the phage will be based on the ability to infect a wide range of pathogenic E. coli strains, in vitro phage replication kinetics, and lack of/minimal host resistance. The potential issue of E. coli resistance to phage infection will be addressed by (i) targeting surface receptors which are important for virulence and/or antibiotic resistance, (ii) the use of cocktails of phages which target different receptors, and (iii) studying the CRISPR-Cas system of wild strains of E. coli to determine its role in phage resistance and its epidemiology and evolution during phage infection. This approach can synergise with the development of new drugs and has the potential to provide a sustainable platform for control of antibiotic-resistant pathogens which could easily be extrapolated to many other animal pathogens.

Planned Impact

This proposal is in response to a BBSRC-supported research call from the Animal Health and Welfare (ANIHWA) ERA-Net to address antimicrobial and anthelmintic resistance by developing alternative curative and preventative therapies. There is general agreement that colibacillosis is the most common infectious bacterial disease of poultry and is responsible for significant economic loss worldwide. The major potential areas of impact are:
(i) The successful development of bacteriophage administration for APEC strains will reduce morbidity in infected chickens and reduce antibiotic usage, the economic scale of which is large but unquantifiable.
(ii) Successful commercial exploitation could result in widespread use off this approach with financial benefits.
(iii) Use of APEC infections represents a useful model for other bacterial pathogens where resistance is increasingly a problem, including the Pasteurella group of bacteria.
(iv) The use of phage therapy to control extraintestinal Escherichia coli (ExPEC) infections in birds is a useful comparative medicine model which may directly benefit human patients suffering from ExPEC infections such as meningitis, septicaemia and urinary tract infections.

Publications

10 25 50
 
Description The main purpose of this project is to use viruses that infect bacteria (bacteriophages) to control E. coli infections in chickens. An added benefit of this work is that the strains of E. coli which cause serious infections in chickens share many similarities with E. coli strains that infect people, and this work could lead to a new type of treatment for human infections. We have isolated over 250 E. coli bacteriophages from sewage, lake, river water and poultry abattoir samples. These phages were isolated using a diverse collection of 31 E. coli strains comprising avian pathogenic E. coli (APEC), mutant E. coli strains and extraintestinal pathogenic E. coli (ExPEC) strains. A host range profile has been performed for each phage in the collection. The replication profiles of each of these phage in liquid broth cultures has been determined, and we are now determining the replication profiles of combinations of the 'best performing' phage. The genomes of these phage have been sequenced, assembled and characterised using a novel method developed in our group. We have found that the presence of certain genes within some of these phage predicts their success in phage therapy. We have now published these results and analysing the genomes of other phage to see whether these genes can predict therapeutic success more generally. The most promising bacteriophage are currently being further characterised to determine their suitability for therapeutic trials to treat E. coli infections in poultry. We are in the process of investigating the use of insect models (e.g. the wax moth, Galleria) of phage therapy as a surrogate for vertebrate models to reduce the number of chickens we use in our therapeutic trials.
Exploitation Route We anticipate that this project will result in a panel of bacteriophage which can be used as an alternative to, or adjunct to antimicrobial therapy in poultry production. Because of the similarity of extra-intestinal E. coli infections in chickens and humans, we also anticipate that our findings may be of use to develop new bacteriophage-based treatments for human E. coli infections.
Sectors Agriculture, Food and Drink,Education,Healthcare,Manufacturing, including Industrial Biotechology

 
Title Novel method of phage genome analysis 
Description As part of the genomic characterization of bacteriophages, we have devised a novel method to confirm completeness of the phage genomes. Briefly, each assembled genome was re-ordered by arbitrarily splitting the assembly at half the length and the ends of assembly were joined with a string of 'Ns'. A custom python pipeline was used, mapping the raw sequence reads from the quality filtered read files against the re-ordered assembled genome as the reference. 
Type Of Material Data analysis technique 
Year Produced 2016 
Provided To Others? Yes  
Impact None yet, but a manuscript has been prepared for peer-review where this method has been described. This manuscript will be submitted to a peer-reviewed journal in March 2017. 
 
Description ANIHWA AntibioPhage 
Organisation AmpliPhi Biosciences Corporation
Country United States 
Sector Private 
PI Contribution Co-ordinator of project (contracts, IP, meetings, research decision-making). Isolation and in vitro characterisation of bacteriophage candidates for phage therapy trials. Genome analysis of bacteriophage. Hosting and training postgraduate research students and staff.
Collaborator Contribution Access to key equipment and facilities (e.g. for preparation of commercial-grade bacteriophage suspensions for in vivo trials). Analysis of bacteriophage genomes and the characterisation of CRISPR spacer regions and their role in phage resistance. Expertise in development of in vivo models of septicaemia and salpingitis in chickens.
Impact Outcomes: So far, the collaboration has resulted in the pooling and sharing of bacteriophage and E. coli isolates, and associated biological and genetic characterisation data between the partners. Some of this data has been used to prepare a joint manuscript which will be submitted to a peer-reviewed journal in March 2017. A consortium agreement and material transfer agreements have been signed by all partners.
Start Year 2016
 
Description ANIHWA AntibioPhage 
Organisation French National Institute of Agricultural Research
Department INRA Loire Valley Centre
Country France 
Sector Public 
PI Contribution Co-ordinator of project (contracts, IP, meetings, research decision-making). Isolation and in vitro characterisation of bacteriophage candidates for phage therapy trials. Genome analysis of bacteriophage. Hosting and training postgraduate research students and staff.
Collaborator Contribution Access to key equipment and facilities (e.g. for preparation of commercial-grade bacteriophage suspensions for in vivo trials). Analysis of bacteriophage genomes and the characterisation of CRISPR spacer regions and their role in phage resistance. Expertise in development of in vivo models of septicaemia and salpingitis in chickens.
Impact Outcomes: So far, the collaboration has resulted in the pooling and sharing of bacteriophage and E. coli isolates, and associated biological and genetic characterisation data between the partners. Some of this data has been used to prepare a joint manuscript which will be submitted to a peer-reviewed journal in March 2017. A consortium agreement and material transfer agreements have been signed by all partners.
Start Year 2016
 
Description ANIHWA AntibioPhage 
Organisation University of Copenhagen
Country Denmark 
Sector Academic/University 
PI Contribution Co-ordinator of project (contracts, IP, meetings, research decision-making). Isolation and in vitro characterisation of bacteriophage candidates for phage therapy trials. Genome analysis of bacteriophage. Hosting and training postgraduate research students and staff.
Collaborator Contribution Access to key equipment and facilities (e.g. for preparation of commercial-grade bacteriophage suspensions for in vivo trials). Analysis of bacteriophage genomes and the characterisation of CRISPR spacer regions and their role in phage resistance. Expertise in development of in vivo models of septicaemia and salpingitis in chickens.
Impact Outcomes: So far, the collaboration has resulted in the pooling and sharing of bacteriophage and E. coli isolates, and associated biological and genetic characterisation data between the partners. Some of this data has been used to prepare a joint manuscript which will be submitted to a peer-reviewed journal in March 2017. A consortium agreement and material transfer agreements have been signed by all partners.
Start Year 2016
 
Description ANIHWA AntibioPhage 
Organisation University of Leuven
Department Health Psychology
Country Belgium 
Sector Academic/University 
PI Contribution Co-ordinator of project (contracts, IP, meetings, research decision-making). Isolation and in vitro characterisation of bacteriophage candidates for phage therapy trials. Genome analysis of bacteriophage. Hosting and training postgraduate research students and staff.
Collaborator Contribution Access to key equipment and facilities (e.g. for preparation of commercial-grade bacteriophage suspensions for in vivo trials). Analysis of bacteriophage genomes and the characterisation of CRISPR spacer regions and their role in phage resistance. Expertise in development of in vivo models of septicaemia and salpingitis in chickens.
Impact Outcomes: So far, the collaboration has resulted in the pooling and sharing of bacteriophage and E. coli isolates, and associated biological and genetic characterisation data between the partners. Some of this data has been used to prepare a joint manuscript which will be submitted to a peer-reviewed journal in March 2017. A consortium agreement and material transfer agreements have been signed by all partners.
Start Year 2016
 
Description Bacteriophage symposium at INRA, Tours, France.. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact A bacteriophage research symposium attended by researchers and students and industry representatives from the UK, France, Belgium and Denmark. Some of the participants agreed to student and staff exchanges during the lifetime of the current project.
Year(s) Of Engagement Activity 2016
 
Description Bacteriophage symposium at KU Leuven, Belgium. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact A bacteriophage research symposium attended by researchers and students and industry representatives from the UK, France, Belgium and Denmark. Some of the participants agreed to student and staff exchanges during the lifetime of the current project.
Year(s) Of Engagement Activity 2015
URL http://www.inra.fr/Entreprises-Monde-agricole/Nos-partenariats-nos-projets/Toutes-les-actualites/pro...
 
Description Bacteriophage symposium at the University of Copenhagen, Denmark 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact A bacteriophage research symposium attended by researchers and students and industry representatives from the UK, France, Belgium and Denmark. Some of the participants agreed to student and staff exchanges during the lifetime of the current project.
Year(s) Of Engagement Activity 2017
 
Description Contributed to UK parliment briefing on using bacteriophage to reduce antibiotic use in domestic animals 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact RA invited to contribute to a PostNote briefing for the Houses of Parliament on approaches to reduce UK antibiotic use in farm animals. Bacteriophage were highlighted as a key innovation which will allow us to reach this objective. This briefing is available freely online as well as published for MPs and others in Parliament.
Year(s) Of Engagement Activity 2018
URL https://researchbriefings.parliament.uk/ResearchBriefing/Summary/POST-PN-0588#fullreport
 
Description Evergreen international phage meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact International bacteriophage conference at Evergreen State College, Olympia, WA, USA. Post-doctoral scientists associated with our project presented their work, and initiated discussion groups with phage researchers from (primarily) developing countries with the aim of applying for joint funding and projects.
Year(s) Of Engagement Activity 2017
URL http://blogs.evergreen.edu/phage/2017-evergreen-meeting/
 
Description Massive Open Online Course - Antimicrobial Resistance in the Food Chain 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We produced a MOOC on antimicrobial resistance in the food chain which discussed the research we are undertaking as part of the present study. The MOOC was hosted on FutureLearn in November 2016, with over 3,000 people enrolling on the course. The discussion forum, which ran in parallel to the course, attracted comments from medics, veterinarians, food workers, hygiene inspectors, patients and laypeople and this prompted some excellent discussions on how to tackle antimicrobial resistance.
Year(s) Of Engagement Activity 2016
URL https://www.futurelearn.com/courses/antimicrobial-resistance-food-chain
 
Description Presentation at EMBO international conference (Viruses of Microbes), 9th-13th July 2018, Wroclaw, Poland. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of poster at "EMBO VIruses of Microbes" meeting in Wroclaw, Poland. Title: "Efficacy of coliphages to prevent chicken embryo mortality". Authors: Angélina Trotereau, Julie Kern, Angèle Thiriet, Arshnee Moodley, Rob Lavigne, Robert Atterbury and Catherine Schouler (AntiBioPhage consortium).
Year(s) Of Engagement Activity 2018
URL http://meetings.embo.org/event/18-virus-microbe
 
Description Society for Applied Microbiology Science Policy Expert Group Discussion on Food Safety and Microbiology 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact RA was invited onto the panel specifically to discuss recent developments in bacteriophage reserach in tackling bacterial pathogens which may be resistant to antimicrobials. The work for AntibioPhage was discussed and will be included in a policy document which will be circulated to all members of SfAM and then used in discussions with the UK Parliament to recommended changes necessary.
Year(s) Of Engagement Activity 2018
 
Description Visit to poultry company in Nigeria 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact The purpose of this activity was to visit a large poultry producer in a rapidly-growing developing country (Nigeria) with the aim of develping links for future research collaborations in the area of phage therapy applications in agriculture. I presented our results from the current project to the CEO and directors, as well as the company Laboratory Manager and their team. We identified a number of requirements before further progress could be made. This mainly involved organising training for the Nigerian laboratory staff to work with phage, and upgrading some of their facilities. Once this has been arranged, we will look for suitable funding opportunties for this project e.g. Newton.
Year(s) Of Engagement Activity 2017