Understanding the relationship between bacteriophages and pathogenicity in the gut pathogen Clostridium difficile

Lead Research Organisation: University of Leicester
Department Name: Infection Immunity and Inflammation

Abstract

Clostridium difficile associated diarrhoea (CDAD) is one of the most worrying emerging bacterial pathogens and over 2000 people in the UK were killed last year (Kuijper, 2006). Only two antibiotics can be used to treat C. difficile and resistance has developed towards them both. However, bacteria have viral enemies, or bacteriophages which specifically target them. All bacteria have bacteriophages and they have been engaged in an arms race for as long as they have existed. Bacteriophages represent a natural, specific and extensive resource of novel antimicrobial agents. They could be either used directly as therapeutic agents, or products derived from their genomes (e.g. lysins which degrade bacterial walls) could be harnessed to lyse them (Fischetti, 2004). Bacteriophages are used extensively to control other bacterial pathogens, they are used widely in Eastern Block countries, and in USA last year the FDA recently released approval to use them in hot dogs to control Listeria.

Whilst other bacterial pathogens have well characterised bacteriophages, those which infect C. difficile are very poorly known. This is in part due to the difficulties in working with the appropriately named C. difficile. To understand how bacteriophages could be used to control C. difficile first a comprehensive collection of them has to be isolated and characterised and an appropriate selection of them sequenced to characterise what components of their genomes could be exploited to control C. difficile.

A different set of bacteriophages integrate into the genomes of C. difficile and alter their virulence. Whilst hiding in the bacterial genomes, they can encode a range of toxins which increase their ability to proliferate in the gut and hospital environment. An understanding of the way in which bacteriophage achieve this can be related to hospital practices and may result in strategies to minimise the spread of virulence factors.

I will provide the first comparative study of the bacteriophages that are associated with patients who have C. difficile associated diarrhoea and compare them to those who have C. difficile but not diarrhoea. I will characterise both the lytic phages and those that reside in the C. difficile genomes. I will establish if diarrhoea free patients have a more effective natural bacteriophage population than those who have diarrhoea. Sequence information will allow me to characterise how bacteriophages kill C. difficile. It will also allow tests to be developed that can see how environmental stimuli can trigger toxin release in the ?hidden? bacteriophages.

Technical Summary

Clostridium difficile associated diarrhoea (CDAD) is a major problem in UK hospitals and was responsible for more than 2000 deaths across the UK last year. It is thought to cost the NHS more money than any other bacterial pathogen. Only two antibiotics are available to treat C. difficile and resistance is developing to both of them. Like all bacteria, C. difficile is infected by a wide range of viruses (bacteriophages) which specifically target it. Bacteriophages are engaged in an arms race with their hosts and their genomes offer a rich source of novel antimicrobials. Lytic bacteriophages control the population structure and evolution of bacteria by selectively killing certain strains. The lysins and holins used for this purpose have enormous therapeutic potential. A different subset of bacteriophages integrate into C. difficile host genomes where they encode toxins or virulence factors which influence host pathogenicity. The bacteriophage community associated with C. difficile is poorly characterised and it is not known if specific phages infect either CDAD associated or non-CDAD associated C. difficile strains. My preliminary studies have shown that there is a diverse bacteriophage community associated with just seven C. difficile strains isolated from patients who had tested positive for the clostridial toxin tcdA. I propose to take an ecological approach to understanding the role of bacteriophages in CDAD. To determine their prevalence and diversity in C. difficile, I will isolate them from C. difficile colonised patients at the Leicester Royal Infirmary over a one year time period. I will characterise them according to their morphology, genome size and ecological attributes. These studies will direct the sequencing of 12 bacteriophage genomes, 6 associated with patients with CDAD, and 6 from C. difficile colonised but non-CDAD presenting patients. This will reveal novel lysins and holins and other pathogenicity factors. Genome information will be used to design molecular probes which will establish the genetic diversity of the C. difficile bacteriophage community. Differences in the composition of bacteriophage genomes can then be related to hospital practices and other environmental factors. A collection of well characterised bacteriophages will provide an essential resource which will underpin future biochemical and genetic analysis of novel therapeutic agents and establish how bacteriophages influence C. difficile virulence.

Publications


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Alleyne SA (2009) Stethoscopes: potential vectors of Clostridium difficile. in The Journal of hospital infection
Hargreaves KR (2014) Clostridium difficile phages: still difficult? in Frontiers in microbiology
Malik DJ (2013) On the difficulties of isolating Clostridium difficile from hospital environments. in The Journal of hospital infection
 
Title A sculpture of a Clostridium difficile cell being infected with bacteriophages phages 
Description I worked with the artist Aimee Lax to make a sculpture of a C. difficile cell and phages, it was displayed at the Eden project in Cornwall as part of a WT funded 'Invisible You' exhibition. 
Type Of Art Artwork 
Year Produced 2016 
Impact The sculpture was widely visited 
URL https://wellcome.ac.uk/press-release/invisible-you-human-microbiome-exhibition-open-eden-project
 
Description Continued development of C. difficile phages as a therapeutic
Amount £135,154 (GBP)
Organisation AmpliPhi Biosciences Corporation 
Sector Private
Country United States of America
Start 11/2014 
End 10/2015
 
Description Developement of C. difficile phages as a therapeutic
Amount £193,145 (GBP)
Organisation AmpliPhi Biosciences Corporation 
Sector Private
Country United States of America
Start 04/2013 
End 10/2014
 
Description Midlands Innovation Fund
Amount £14,000 (GBP)
Organisation NHS Innovations East Midlands 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 05/2011 
End 08/2011
 
Description Wellcome Trust Institutional Award
Amount £31,000 (GBP)
Organisation The Wellcome Trust Ltd 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 05/2012 
End 12/2012
 
Title Phages, strains, genomes 
Description ~500 C. difficile stains have been isolated in this study, ~ 20 phages have also been isolated 
Type Of Material Biological samples 
Provided To Others? No  
Impact The strains are of interest for different biological purposes. Several are currently being sequenced by the Sanger Centre who are interested in them. This will motivate further research and be of use to the research community. The phages also have the potential to be of significant use to the research community. 
 
Description C. difficile collaborations 
Organisation Faculty of Medicine
Department Department of Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Provided samples, phages, driven research
Collaborator Contribution Providing protocols and technical helpProviding expertise in strain identification and epidemiologySequenced several genomes
Impact Outputs to follow in 2012
Start Year 2009
 
Description C. difficile collaborations 
Organisation Leeds Teaching Hospitals NHS Trust
Department Microbiology, Leeds General Infirmary, Leeds
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Hospitals 
PI Contribution Provided samples, phages, driven research
Collaborator Contribution Providing protocols and technical helpProviding expertise in strain identification and epidemiologySequenced several genomes
Impact Outputs to follow in 2012
Start Year 2009
 
Description C. difficile collaborations 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Charity/Non Profit 
PI Contribution Provided samples, phages, driven research
Collaborator Contribution Providing protocols and technical helpProviding expertise in strain identification and epidemiologySequenced several genomes
Impact Outputs to follow in 2012
Start Year 2009
 
Description Clostridium difficile cell walls 
Organisation Imperial College London (ICL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Isolation of suitable bacteriophages
Collaborator Contribution Provided protocols for removing S-Layers
Impact We are still working on this project but expect outputs in 2013
Start Year 2007
 
Title Therapeutic phages for Clostridium difficile 
Description We have patented seven phages specific for use against Clostridium difficile infections and seeks to protect therapeutic uses of any one phage individually for therapeutic uses or any combination of the seven bacteriophage strains. 
IP Reference GB1215184.1 
Protection Patent application published
Year Protection Granted 2012
Licensed Commercial In Confidence
Impact N/A
 
Description School visit, Lutterworth 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 3 groups of 20 pupils attended talk this motivated a discussion after all sessions

School teacher has fed back positively that the students really enjoyed it and has asked me to go back next year
Year(s) Of Engagement Activity 2011