Un-ravelling the network of regulation of multidrug resistance in Salmonella enterica

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection

Abstract

Every year people are exposed to non-typhoidal salmonella bacteria, typically from contaminated foodstuffs. Some people succumb to infection and get ill. This is particularly dangerous for the very old and young, and these people often need treatment with antibiotics. Unfortunately, antibiotic resistance in salmonella is not uncommon. In this project we will perform experiments that will allow us to understand how multiple antibiotic (drug) resistance (MDR) is caused. We will identify genes and/or the proteins that control the production of the AcrAB-TolC system, which exports antibiotics from the bacterial cell and so allows it to continue to grow and cause infection. If this system is inactivated the bacterium becomes very susceptible to antibiotics, finds it difficult to colonise the host (e.g. poultry) and antibiotic resistant salmonella are hard to select. We anticipate that we will find proteins that confer MDR and that can also be used as targets for drugs that will inhibit production of AcrAB-TolC. Such new drugs would make it difficult for the salmonella to cause infection and be antibiotic resistant.

Technical Summary

The AcrAB-TolC efflux pump in Salmonella enterica serovar Typhimurium confers innate multiple drug resistance (MDR), and is important in colonisation and persistence in poultry ? the primary reservoir. In isolates from humans and animals it confers clinically relevant MDR. Genetic inactivation of AcrAB-TolC confers antibiotic hyper-susceptibility, attenuates the organism and makes it difficult to select antibiotic resistant bacteria. In salmonella RamA, a member of the AraC-XylS family of transcriptional activators, can regulate expression of acrAB and tolC. Over-production of RamA confers MDR whereas in Escherichia coli MarA plays this role. However, in salmonella there are data to indicate that expression of acrAB and tolC is regulated, and MDR also conferred, by other factors. The purpose of this project is to advance our understanding of the mechanisms by which MDR via over-production of AcrAB-TolC is conferred. Four hypotheses will be explored (1) in salmonella RamA and MarA have distinct, but over-lapping, regulons; (2) RamA is the primary regulator of expression of the AcrAB-TolC efflux pump in those Enterobacteriaeceae that possess this gene, and MarA only plays a role in those bacteria that do not possess ramA (e.g. E. coli and Shigella spp); (3) both ramA/RamA and marA/MarA are regulated by other factors, possibly another regulator, which when mutated confers MDR; (4) expression of acrAB and tolC is regulated by a factors and/or gene/s other than local repressors, ramA or marA. A systems biology approach will be taken using microbiology, molecular genetics, cell biology and in vivo models and include new and novel techniques including ChIP and DNA sampling. This research project will make new and important scientific advances in how efflux pumps are regulated and this information will be relevant to those serovars that cause enteric fever, Typhi and Paratyphi, plus other Enterobacteriaceae.

Publications

10 25 50
 
Description Advisor to CMO on antibiotics
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Antibiotic Action
Geographic Reach Multiple continents/international 
Policy Influence Type Gave evidence to a government review
 
Description Antibiotic Strategy, Department of Health
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description BSAC, The Urgent Need for New Antibiotics
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
 
Description DriveAB
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description EU Joint Programming Initiative on AMR Scientific Advisory Board
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
 
Description Longitude Prize Expert Advisory Panel
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
 
Description MRC Drug target and validation
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in a advisory committee
 
Description Medical Research Council Cross Funder AMR committee
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description O'Neil AMR Review
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Secreteriat APPG on Antibiotics
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description MRC CASE
Amount £96,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 10/2020
 
Description MRC Innovate Theme 2
Amount £163,253 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2016 
End 12/2017
 
Description Roche
Amount £460,000 (GBP)
Organisation F. Hoffmann-La Roche AG 
Sector Private
Country Global
Start 10/2015 
End 10/2017
 
Title FACS selection of mutants with altered ramA or acrB expression 
Description A transposon library containing a GFP reporter gene is screened using flow cytometry. Populations with increased fluorescence are sorted and then whole genomes of the sorted population are sequenced to identify the gene/s conferring altered production of ramA or acrB. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Proof of principle of success with this novel approach has been provided by recent experiments using a ramA reporter plasmid in a TraDIS library of S. Typhimurium (SL1344). DNA sequencing with transposon specific primers revealed five potential genes, the products of which have not been previously implicated as influencing expression of ramA. 
 
Title High throughput screening of compound libraries 
Description A GFP reporter is used to screen a compound library using the Birmingham Drug Discovery Facility, which contains a whole cell high throughput screening (HTS) platform. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Following a preliminary study the data was used as the basis of a successful research grant application with Roche. 
 
Title High througput GFP induction assay 
Description Promoters of interest were fused to GFP on a plasmid, these constructs were transformed into various backgrounds and induction of GFP was performed with a variety of compounds. High throughput was attained by using a 96 well format and utilising a FluoSTAR OPTIMA to measure fluoresence. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact We have been able to evaluate numerous compounds on their ability to induce our genes of interest. This tool gives the ability to screen for inhibitors of efflux in bacteria. 
 
Description Dirk Bumann 
Organisation University of Basel
Department Biozentrum Basel
Country Switzerland 
Sector Academic/University 
PI Contribution Use of promoterless GFP plasmid to construct various GFP reporter fusions
Collaborator Contribution Provided us with promoterless GFP plasmid (pMW82)
Impact We have exploited GFP reporter fusions to measure RamR-dependent regulation of the ramA promoter, which confirmed the identification of the DNA target for RamR. We also utilised the GFP reporter fusion to perform error prone PCR and create a mutant library in order to investigate which region(s) of the RamR protein interacts with the DNA target. A superrepressor mutant was identified. Published 2012, PMID:22948865 We have utilised a transcriptional GFP reporter system in order to identify compounds which increase the transcription of ramA. We found that in the the absence of a functional AcrAB-TolC efflux pump and with compounds that inhibit efflux ramA expression was induced. Published 2013, PMID:23493314
Start Year 2009
 
Description Gerry Wright 
Organisation McMaster University
Department Institute for Infectious Disease Research
Country Canada 
Sector Academic/University 
PI Contribution Intellectual input, experimental design and experiments
Collaborator Contribution Intellectual input, experimental design and experiments
Impact None so far
Start Year 2013
 
Description Luke Alderwick 
Organisation University of Birmingham
Department School of Sport, Exercise and Rehabilitation Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provide GFP reporter constructs for use in high throughput screening of compound libraries
Collaborator Contribution High throughput screening facilities
Impact None as yet
Start Year 2015
 
Description Paul Williams 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Construction of in-vivo reporter plasmids and reporter chromosomal mutants.
Collaborator Contribution To develop the in vivo model of infection to measure efflux status, measurement of efflux inhibition in vivo
Impact None as yet
Start Year 2015
 
Description Peter Warn (Evotec) 
Organisation Evotec
Country Germany 
Sector Private 
PI Contribution Academic supervisor on iCASE studentship and provision of research facilities for iCASE student
Collaborator Contribution Industrial supervisor on an iCASE studentship (2016-2020). Will support student and provide experience in assays and diagnostic development .
Impact NA
Start Year 2016
 
Description Simon Gibbons 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental design, Intellectual input and experiments
Collaborator Contribution Experimental design, Intellectual input and experiments
Impact None so far
Start Year 2010
 
Description Vass Bavro 
Organisation University of Birmingham
Department Institute of Microbiology and Infection
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental design, Intellectual input and experiments
Collaborator Contribution Intellectual input and computer modelling
Impact None so far
Start Year 2013
 
Description Conference Talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact 08/09/16 Microbiology Society AGM Microbiology Society, London Prize winner lecture From outreach to basic research - addressing the crisis of AMR
28/09/16 Resistance and You! PHE, University of Manchester Speaker All about antibiotics - how they have changed our world
28/09/16 Resistance and You! PHE, University of Manchester Panel Chair Antibiotics and You!
07/11/16 Chevening leadership Foundation: R&D policy and strategies for innovation Chevening Foundation, London Speaker An overview of AMR, the scale of the problem and possible solutions
16/11/16 Plugging the Antibiotics Gap: A Medicinal Chemist's Perspective Royal Pharmaceutical Society, Alderley park, Cheshire Plenary speaker The global challenge of antibacterial drug resistance: what can be done?
13/01/17 Early Discovery of new antibiotics EU JPI AMR, Paris Panel Chair Analysis of the current European level portfolio: how to synergize the approaches, how to progress for new strategies / optimize funding around discovery
07/02/17 SHAMROCK annual lecture University of Sheffield Speaker Multi drug efflux systems: from basic research to discovering efflux inhibitors
23/03/17 Post Graduate Symposium Key note speaker University of Warwick MDR efflux 08/06/17 Daresbury IBM laboratory, Warrington 21-23/06/17 Hannover key note lecture Molecular basis of antibiotic permeability in Gram Negative bacteria. 4-5/07/17 AMR Award Holders Meeting Poster Presentation. 17/05/17 Pint of Science, Birmingham. 08/04/17 Roche Seminar MDR efflux systems basic research and discovery of new inhibitors. 24/10/17 UBM society , University of Birmingham, Loss of function of MDR efflux systems confers loss of virulence. 24/11/17 University of Upsalla, Sweden, Antibiotic resistance and why it matters to you. 21-22nd March 2019 - Garrod Lecture at the BSAC Spring Conference 2019, International Convention Centre, Birmingham.
Year(s) Of Engagement Activity 2016,2017,2019
 
Description Media actitivies 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Professor Laura Piddock gave interviews to local, national and international media (TV, Radio and print). In the last 12 months this has included:
BBC Newsnight
BBC Radio 4
Live interview with Channel 4
Sky News live interviews
Numerous Interviews about teixobactin including The Jeremy Vine Show, BBC Radio 2 (Jan 2015)
• Provided quote to The Independent (March 2015)
• Provided quotes to The Times, The Telegraph and The Independent on 'AncientBiotics' (March 2015).
• Letter to The Times,' Britain faces an 'antibiotic Armageddon' (April 2015)
• Filming for Panorama (April 2015)
• Interview with BBC Radio 4 'The Report' (April 2015)
BBC R4 Today x3

Various TV interviews syndicated worldwide including to CNN.
Global media coverage (quoted >200 times) following the WHO's report on 30 April 2014.
Federation of Infection Societies annual meeting: interview for short film: What is the cost of not developing another antibacterial drug
Numerous print and online media interviews including The Times, The Telegraph, The Daily Mail, The Daily Express, The Metro. Quotations have often been repeated in worldwide media including in USA print media.
BBC Horizon (broadcast 22 May 2014)
Swiss National Science Foundation (SNSF) National Research Programme (NRP) Steering Committee with the title "Antimicrobial Resistance: a one-health approach"
Plenary lecture at the 'Stimulating Antimicrobial Innovation' one day meeting at the NHLI, Imperial College London on October 22nd 2015. This is being organised by the Academy of Pharmaceutical Sciences GB, through the focus group on Microbiology and Anti-infectives.
Board of Examiners in Biochemistry and Biotechnology at Imperial College London, I am writing to invite you to join us as an External Examiner for our BSc courses commencing with the 2015/16 academic year
Invitation to be a panellist at BioInfect 2015
Guest lecture on antibiotic resistance for a new course we are developing as part of the University of Glasgow online learning course.
Plenary Lecture Invitation: Research Day-St George's University of London 2 Dec talk on the subject of 'The challenges of AMR' at the launch of our new EPSRC funded AMR 'Bridging the Gaps' launch event University of Loughborough 7 Dec
Swedish Research Council review panel to evaluate applications for a Networking call to bring together researchers from Sweden and India on e-Science for Life Science and Antibiotic resistance in a 'One Health' perspective
Science Foundation Ireland review panel, Dublin SFI Investigators Programme 25th and 26th of February 2016.
European Science Foundation Invitation to join Review Panel on AXA postdoctoral fellowships 2016 meetings in Feb and May 2016
ZIBI Interdisciplinary Center for Infection Biology and Immunity
Symposium Therapeutic Resistances in Infectious Disease - Status Quo and Future Challenges, Berlin (June 27/28, 2016
Science Representative of the Buckinghamshire Federation of Women's Institutes to warmly invite you to speak at our science Investigation and Discovery Day on 7 March, in Aylesbury
Invitation to Convene and speak in symposium: 2016 ASM Microbe plenary session
Plenary lecture on AMR at the IPS conference September 2016
'Turn your nose up' campaign with the Farms not Factories organisation
Took part in a written panel interview which will appear in Future Medicinal Chemistry's upcoming New Frontiers in Antimicrobial Drug Discovery special issue
• Interviewed by Bloomberg News, Australia
• Invited to interview with The Naked Scientist
Flix Interactive about a Wellcome Trust funded project on an antimicrobial resistance computer game
• Release of BSAC policies on antimicrobial use in animals and humans (May).
• Interviewed by The Atlantic on the O'Neill Report, LJVP, (May).
• Interviewed by BBC Radio 4 Today programme (1:22), LJVP (May).
• Interviewed by BBC2 Victoria Derbyshire programme (May).
• Interviewed by BBC Radio 4 Today programme (2:25) LJVP, (May).
• Interviewed by BBC World on superbugs, LJVP, (May).
• Invited to interview by Good Morning Britain about the O'Neill Report, LJVP, (May)
• Interviewed by Kevin Fong, National Geographic, LJVP (May).
• Interviewed by Bang! Oxford University's Science Magazine, LJVP (May).
• Interviewed by a 7th grader for a project on antibiotic resistance, LJVP (May).
• Interviewed by the Daily Mail Good Health, LJVP (May).
• Provided comment to Science Media Centre on colistin-resistant bacteria in US woman, LJVP (May).
• Invited to attend the UK premier of the documentary "Clean Hands", 28th June, London, LJVP (May).
• Invited to interview by The Los Angeles Times, LJVP (June).
• BBC Radio 4 'In our Time' programme, Penicillin, 9th June 2016, LJVP (June).
• Invited to interview by "Sputnik - Orbiting the World with George Galloway", LJVP (June)
• Invited to write an article for The Conversation, LJVP (June).
• Reviewed and edited the script for a cartoon series generated by the Dundee Design School, LJVP (June).
• Invited to write an article for the Spanish newspaper 'El País, LJVP (June).
• Provided a quote to the British Medical Journal for a feature on the impact of NICE sepsis guidance on antibiotic subscribing, LJVP (June).
• Provided NICHE with Antibiotic Action posters.
• Invited to join expert panel for Innovate UK and Department of Health Funding competition: antimicrobial resistance, 29th November (Oct)
• Invited by the Food Standards Agency and the Universities of Southampton and Newcastle, to participate in a joint antimicrobial resistance workshop. (Oct)
• Invited to give a plenary lecture on antimicrobial resistance at the St George's Annual Research Day. (Oct)
• Interviewed by the Sunday Express. (Oct)
• Interview with Strategy& for a 'State of the Nation report' into AMR research & development in the UK. (Oct)
• Invited to provide quotes to the Bureau of Investigative Journalism on the topic of antimicrobial resistance. (Oct)
• Attended the 75th anniversary of penicillin, 7 November, University of Oxford. (LJVP)
• Invited to appear and provide expertise in a BBC4 documentary with Michael Moseley
• Supplement in the Guardian (Media Planet) under the One Health Agenda
• Twitter: True Stories on video joint release with PHE (stories embargoed by PHE plus BBC Radio 4 stories and Tony Maxwell videos
• Interview with Good Housekeeping magazine for WAAW
• Invited to provide a comment on 'everyday germs in the home, their effects and how you can reduce the risk of illness' for a campaign run by Domestic Innovations. (July)
• Working with BBC Five Live on concerns in relation to availability of antibiotics via online pharmacies.
• Invited to provide comment for a story the Guardian are covering that looks at an antibiotic that kills Staphylococcus aureus. (July)
• Invited to interview with TIME magazine about the anthrax outbreak in Russia
• To provide expertise on a script for a feature film under development by Neon Films
• To provide a quote for the cover of a forthcoming science book by the Royal Society of Chemistry 'The Microbes Fight Back: Antibiotic Resistance'
• Interviewed by James Gallagher, BBC Health website - UN Resolution. (Sept)
• Interview with BBC Radio 5 Live Drive - UN Resolution. (Sept)
• Interviewed with Deutsche Welle Television, Berlin - UN Resolution. (Sept)
• Interviewed by The Lancet Infectious Disease on UN Resolution to be published in November. (Sept)
• Interview with BBC World Service - The Inquiry to discuss the antibiotics crisis, following the recent UN meeting.
• Interview with BBC News - UN Resolution. (Sept).
• BBC News filming for Antibiotic Awareness Week (Oct 2018).




Promotion of Antibiotic Action (AA) being led by Prof. Laura Piddock. Antibiotic Action is a global initiative by the British Society for Antimicrobial Chemotherapy (BSAC) to highlight the threat to world health from multi-drug resistant bacteria and lobby for urgent action to develop new antimicrobial treatments
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description School Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact 460 students attended the lecture which sparked questions and discussions afterwards

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Year(s) Of Engagement Activity 2013