Selection for AMR in complex microbial communities at sub-therapeutic antibiotic concentrations

Lead Research Organisation: University of Exeter
Department Name: University of Exeter Medical School

Abstract

Antimicrobial resistance (AMR) is an increasing problem in human and animal pathogens, and has been highlighted as a serious threat to public health by the Chief Medical Officer. There is increasing evidence that antibiotic usage in agriculture may be contributing to the emergence of AMR in the clinic and the government's 5 year AMR strategy highlights improved knowledge and understanding of AMR as a key priority. This project will investigate the evolution of AMR in complex microbial communities using laboratory evolution experiments. For the first time, selection for AMR in a clinically important opportunistic pathogen, E. coli, will be studied in the presence and absence of a complex community of bacteria. The community will be from pig faeces, and will be incubated in anaerobic fermenters that have previously been used as simple gut models.

Traditional approaches to studying AMR in bacteria often consider only one species in isolation. This approach can be useful in studying mutation based adaptation to antibiotics; however it does not consider the impact of horizontal gene transfer where resistance genes are acquired from the microbial community. Single species experiments also fail to consider competition between different species of bacteria that have differing intrinsic resistance to antibiotics.

Competition experiments in the presence and absence of a complex microbial community will help us understand how evolution of AMR occurs. We will expose experimental microbial communities to different concentrations of antibiotic representing the sub-therapeutic concentrations found in the animal and human gut during oral antibiotic therapy. This will give insights into the way complex interactions that occur within microbial populations affect evolution of AMR, relative to selection for AMR in single species experiments. Exposure to low concentrations of antibiotics was traditionally thought to be unimportant in selection for AMR. However recent data suggests that selection occurs at much lower concentrations than previously thought, and preliminary data in Gaze's lab suggests that selection for AMR gram-negative opportunistic pathogens can be greater at lower sub-therapeutic concentrations than at higher concentrations closer to those used to treat infections.

We will also investigate the effects of selection by a single antibiotic on relative abundance and diversity of all known resistance genes. This will provide data on indirect co-selection for AMR genes due to genetic linkages in the genomes of bacteria in the complex community and on mobile genetic elements capable of transferring multiple genes between bacteria. Cell sorting and next generation sequencing techniques will identify all known AMR genes transferred to a genetically tagged E. coli under antibiotic selection. Conversely, we will also investigate AMR gene transfer from a tagged E. coli to all other bacteria within the complex community.

Technical Summary

Evolution of AMR will be studied using a genetically tagged focal strain and AMR plasmid in the presence and absence of a complex microbial community using next generation sequencing and bioinformatic approaches.

In this instance we have chosen to use a pig derived community under selection with oxytetracycline, as selection for AMR within the pig microbiome is an important issue given the size of the global pig population and the amounts of antibiotics used in pig husbandry. However, this model system will be used to study phenomena that are likely to occur in all complex microbial communities under antibiotic selection.

We will use genetically tagged host strains and AMR plasmids, allowing quantitation using real-time PCR and separation of host strain and plasmid bearing bacteria using fluorescently activated cell sorting. The latter approach has been validated by Barth Smets who has kindly agreed to supply strains. Selection for the resistance plasmids will be quantified as a function of antibiotic concentration from the minimum inhibitory concentration of the susceptible focal strain to zero, in the presence and absence of the complex microbial community. Gene transfer between the focal strain and the community will be investigated using cell sorting to separate the focal strain and plasmid recipients from the community. The extent of gene transfer from focal strain to the community and vice versa will be assessed using metagenomic methods. Changes in community structure and AMR gene abundance and diversity during selection experiments will be assessed using amplicon and shotgun sequencing respectively followed by bioinformatic analyses using Qiime and analysis using a structured antimicrobial resistance gene database.

This project is innovative in its combination of selection experiments using genetically tagged strains and plasmids in the presence and absence of a complex microbial community, and the use of next generation sequencing approaches.

Planned Impact

Beneficiaries of the research will include academics (see academic beneficiaries) and key stakeholders from government agencies including Defra, the Food Standards Agency (FSA), the Veterinary Medicines Directorate (VMD) and members of the Defra Antimicrobial Resistance Committee (DARC) and Advisory Committee on Antimicrobial Resistance and Healthcare Associated Infections (ARHAI). Individuals working in areas of veterinary and clinical medicine concerned with antibiotic therapy and AMR will also benefit including vets, pharmacists, infection control practitioners and clinical microbiologists. Private sector stakeholders within the animal and human biopharma industry would also benefit from a deeper understanding of the way AMR evolves and the role sub-therapeutic concentrations of antibiotics play in selection for resistance.

There is a fundamental lack of knowledge regarding the relationship between antibiotic exposure and selection for resistance in complex communities such as those within the human and animal microbiomes. Whilst patterns of resistance in different microbial communities under different selective regimens will vary the proposed research represents one of the first attempts to investigate selection for AMR in complex microbial communities under controlled replicated conditions using cutting edge molecular tools. Previous research using animal models is subject to lack of true controls and adequate replication to account for confounding variables. Working with genetically tagged strains in animals is also logistically difficult and costly. In vitro work to date largely relies on single or dual species experiments which do not account for horizontal transfer of resistance genes or competition between taxa with differing intrinsic levels of resistance. Where mixed communities are studied, the system is usually regarded as a "black box" with limited ability to disentangle changes within the community.

Our innovative approach combining molecular microbial ecology and experimental evolutionary biology utilising next generation sequencing and bioinformatic approaches is well suited to tackling this complex problem.

Increased understanding of how antibiotics select for resistance in complex microbial communities is crucial in developing best practice in terms of prescribing and prophylaxis in animals. Interventions that reduce the rate at which AMR evolves have the potential to improve the nation's health and reduce social and economic costs associated with AMR. Our data will strengthen the evidence base relating to the impacts on microbial communities of exposure to antibiotics at a range of concentrations encompassing those likely to be present in the human and animal gut.

It is likely that novel insights into the evolution of AMR will be made within the lifetime of the grant, potentially informing policy on antibiotic usage and identifying key questions for further research.

THE RF and technician will develop understanding of the evolution of AMR and will receive training in the use of molecular tools and experimental approaches required to investigate the problem. This expertise will be valuable within the academic, public and private sectors.
 
Description ? Invited to meeting in South America (Argentina or Uruguay in June 2018) to advise the Interagency Coordination Group on Antimicrobial Resistance (IACG) co-chaired by the UN Deputy Secretary-General and the Director General of the World Health Organization.
Geographic Reach Multiple continents/international 
Policy Influence Type Gave evidence to a government review
 
Description Co-authored a box in Chief Medical Officers annual report
Geographic Reach National 
Policy Influence Type Citation in other policy documents
URL https://www.gov.uk/government/publications/chief-medical-officer-annual-report-2017-health-impacts-o...
 
Description Invited by Lee Slater (Senior Policy Advisor on AMR in the environment at Defra) to sit on a panel of academic experts in a series of workshops between the US, UK, China and India on antimicrobial resistance and antibiotic pollution in the environment. It's being co-ordinated by the UK Science and Innovation Network and the USA Centers for Disease Control and Prevention.
Geographic Reach Multiple continents/international 
Policy Influence Type Gave evidence to a government review
 
Description Invited to speak to Defra Antimicrobial Resistance Coordination (DARC) Group Autumn 2017 on AMR in the environment.
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
Impact Talk to Defra and veterinary medicines directorate about antibiotics in the environment
 
Description Invited to speak to Defra Hazardous Substances Advisory committee about AMR in the environment
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Report for United Nations Env Programme
Geographic Reach Multiple continents/international 
Policy Influence Type Gave evidence to a government review
Impact Report was published alongside UNEA commitment to tackle AMR form an environment and human health perspective
 
Description Strategies for Addressing Antimicrobial Resistance - Future challenges for UK policy
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description Mapping the evidence for the risks of human exposure and transmission of AMR in the natural environment
Amount £80,590 (GBP)
Funding ID NE/S015965/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 01/2019 
End 09/2019
 
Description The environmental dimension of antimicrobial resistance: informing policy, regulation and practice.
Amount £72,525 (GBP)
Funding ID NE/S006257/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 01/2019 
End 12/2020
 
Description Towards Developing an International Environmental AMR Surveilance Strategy
Amount £44,356 (GBP)
Funding ID MR/S037713/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2019 
End 12/2019
 
Description ? Attended a Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) workshop in Gothenburg 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Attended a Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) workshop in Gothenburg in September 2017 to explore and identify critical research needs that relate to the environmental dimensions of AMR, both in the longer term for providing input to an updated JPIAMR Strategic Research Agenda, but also in the shorter term to provide guidance for specific calls.
Year(s) Of Engagement Activity 2017
URL https://www.jpiamr.eu/wp-content/uploads/2017/11/Report-_JPIAMR-workshop-on-Environmental-dimensions...
 
Description ? Gaze WH. Co-selection for AMR by quaternary ammonium compounds. Royal Academy of Science. Stockholm. March 2016. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk at the Royal Academy of Science in Stockholm on AMR in the environment
Year(s) Of Engagement Activity 2016
 
Description ? Gaze WH. The environmental aspects of antibiotic resistance. Learned Societies AMR meeting. June 25th, 2015 London. 
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 Antimicrobial resistance: environments,evolution and transmission Networking workshops for researchers
Year(s) Of Engagement Activity 2015
URL https://www.responsibleantibioticuse.org/#xl_xr_page_index
 
Description ? Gaze WH. The environmental aspects of antibiotic resistance. Learned Societies AMR scoping meeting. Society for Applied Microbiology symposium on AMR, Royal Society of medicine. December 2015. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk to diverse audience on AMR in the environment
Year(s) Of Engagement Activity 2015
 
Description ? Gaze, W.H. Selection for, dissemination of and exposure to antibiotic resistant bacteria in the natural environment. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact 4th International Conference on Responsible Use of Antibiotics in Animals, The Hague, September 2016.
Year(s) Of Engagement Activity 2016
URL https://www.responsibleantibioticuse.org/#xl_xr_page_index
 
Description ? Gaze, W.H. Taking action to improve healthcare by addressing the links with environment chemistry. Scotland (NHS), 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk to NHS Scotland on AMR in the environment
Year(s) Of Engagement Activity 2016
 
Description ? Invited by MRC to work with Viadynamics to develop an AMR framework to map out research challenges and ways in which we are addressing those challenges. 
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 Advise RCUK policy on research priorities on AMR
Year(s) Of Engagement Activity 2017
 
Description ? Invited to EA meeting on AMR in waste water to plan EA programme of investigation. March 2017 
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 Help inform water industry AMR research programme
Year(s) Of Engagement Activity 2017
 
Description ? NERC Planet Earth article "Have we opened the floodgates on antimicrobial resistance?" Winter 2016/17. 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact ?Article for Planet earth detailing role of flooding and climate change on environmental transmission of Amr
Year(s) Of Engagement Activity 2017
 
Description Filming for BBC4 program on AMR 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Filmed with freelance program makers for a Michael Mosley BBC4 documentary on AMR
Year(s) Of Engagement Activity 2017
 
Description GW4/BristolBridge: Systems approaches to AMR in Different Environments (16 Mar 2017) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Presented outline of grant to researchers working on AMR in the environment
Year(s) Of Engagement Activity 2017
 
Description NERC KE Fellowship working with EA, Defra, Water and pharmaceutical industries 
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 This is in its early stages but it has already resulted in a JPIAMR (MRC) funded network on environmental AMR surveillance which has resulted in engagement with gov stakeholders and national / international partners
Year(s) Of Engagement Activity 2019
 
Description Speaking at House of Commons UKRI AMR research strategy refresh 
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 Speaking at House of Commons UKRI AMR research strategy refresh focusing on existing UKRI funded work in the area of AMR and the environment
Year(s) Of Engagement Activity 2019