Using next generation sequencing to reveal human impact on aquatic reservoirs of antibiotic resistant bacteria at the catchment scale

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

The threat of antibiotic resistance has been compared to that posed by climate change and global terrorism by the Chief medical Officer Dame Sally Davies. Bacterial resistance to antibiotics has existed for hundreds of millions of years, as it evolved to combat antibiotics produced by bacteria and fungi. Resistance is conferred either by mutation or by uptake of DNA from other bacteria which may not even be closely related. This horizontal resistance gene transfer is one of the most important issues facing the fight against infection in the clinic. Novel resistance genes that are taken up by clinical pathogens originate in environmental bacteria, and once in human pathogens or even harmless commensal bacteria, will be selected for by clinical use of antibiotics. However, little is known about the conditions under or locations in which these genes are mobilised into human associated bacteria, or what the human exposure routes for transmission of these resistance genes are. Increasing evidence suggests that the use of antibiotics in agriculture contributes to the increase in resistance seen in the clinic, however much less research has focused on evolution of resistance in farm animals than in humans so less evidence is available. Even less is known regarding reservoirs of resistant bacteria in the natural environment, particularly locations heavily polluted by human or animal waste.

11 billion litres of waste water are discharged into UK rivers every day; critically much of this treatment does not significantly reduce numbers of resistant bacteria. Millions of tons of animal faecal wastes are spread to agricultural land every year, providing additional inputs of resistant organisms into the wider environment. Our previous work has shown that the use of a marker gene, which is predictive of levels of antibiotic resistance genes in sediments, varies by up to 1000 times between clean and dirty sediments. Our data also shows that waste water treatment plants are responsible for the majority of this effect (about 50%), and 30% is associated with diffuse pollution from land adjacent to the river. Other data generated by the consortium suggests that there are real human exposure risks to these environmental reservoirs of resistant organisms, with several million exposure events occurring each year in England and Wales through recreational use of coastal waters alone.

This project will, for the first time, use cutting edge high through put DNA sequencing technologies and computational analyses to increase our understanding of the human activities that drive increased levels of antibiotic resistant bacteria across the River Thames catchment. Abundance and identity of over 3000 different resistance genes will be determined at 40 sampling sites, in triplicate at three time points over one year, to capture impacts of seasonality and flow. We will also measure a range of antibiotic residues, metals and nutrients. We will use graphical information system data on waste water treatment plant type, size and location and land use throughout the catchment. Together this data will be used to produce a model which will reveal the main drivers of resistance gene abundance and diversity at the catchment scale. We will also identify novel molecular markers associated with different sources of pollution that can be used as source tracking targets. We aim to analyse the effects of specific mitigation strategies that are able to reduce levels of resistant bacteria, this will enable estimates of reduction in resistance levels that can inform policy and regulatory targets.

A translational tool will be developed for surveillance of the most important marker genes identified from the DNA sequence analyses and modelling work. This will be an affordable test that will help identify key factors for human health risk assessment.

Planned Impact

In addition to researchers, regulators and government bodies described previously, the wider stakeholders who will benefit will include the water industry, farmers, shellfish producers / harvesters and parts of the tourism sector who are involved with recreational freshwater and coastal water use. They will gain insights into potential hazards, and mitigation strategies for reducing exposure to AMR bacteria.

Antimicrobial resistance (AMR) has been highlighted by Dame Sally Davies (Chief Medical Officer), and crucially the government's 5 year AMR strategy highlights the need for a "One Health" approach including consideration of the agricultural and natural environments.

"Antimicrobial resistance is a global problem and we all need to take responsibility. I have already been speaking with the World Health Organization, the G8 and with countries across the world to make sure we're all working together and I am pleased to see that steps are being taken in the UK to help us take the fight to resistant bacteria".

"After her report in 2013, AMR was put on the government's national risk register of civil emergencies - which provides guidance on potential threats such as terrorist attacks, pandemic flu and major flooding".

In the long term the outputs of the proposed research are extremely important to society as it is crucial that we gain fundamental insights into evolution and dissemination of AMR, not only in the clinic, but in the wider environment. As discussed later in this proposal, there is increasing evidence that pollution introduces clinically significant AMR bacteria into river catchments where there is a real human exposure risk, particularly in receiving coastal waters. In addition, a widely agreed research priority is to investigate the potential for environmental antibiotic residues to select for AMR in polluted natural environments such as rivers.

We will work with project partners in the UK (LGC) and abroad (Austrian Institute of Technology) to develop surveillance tools for the most important resistance markers we will identify during the proposed research. We will also work with UK based Advanced Anaerobics Ltd to evaluate the impacts of treatment of animal waste on AMR dissemination.

Our project outputs will be available to academic and private sector researchers to study the ecology of AMR in a key UK catchment. This will consist of a Thames catchment metagenomic sequence database, plus a database of metadata at each sample site and time point plus replicate sediment samples from each sample site and time point stored at -80 degrees C. The significance of this resource should not be underestimated as it will facilitate a wide range of in silico analyses and future research projects that can make use of our state of the art project outputs.

AMR has huge costs to society both economically and from a human health perspective. Our research will provide critical data on AMR, allowing mitigation of transmission of AMR bacteria and fundamental research on drivers of resistance in the environment and in the clinic. The latter will stem from our work on selection for resistance at low antibiotic concentrations which are present in the environment and also in the human body.

We anticipate that we will generate data that will inform government policy, and that of key regulators, within the lifetime of the grant. This will impact human health if steps are put in place to reduce environmental dissemination, selection and human exposure.

Publications

10 25 50
 
Description Current major concerns have focused on the recycling of antibiotic resistance genes (ARGs) as these may move independently of their pathogen hosts. Human sewage contains both resistant bacteria and may also contain sub lethal concentrations of antibiotic residues and biocides, which can enter the environment, unchanged and exert selective pressures on resistance gene carriage. Thus we identified two major challenges in understanding risk in environmental exposure to AMRP which relates to their viability and ARG acquisition. We have hypothesised that ARG have probably originated in environmental resistomes but due to selection pressures in the clinic have become upregulated and carried on highly mobile genetic elements (MGEs). We focused our efforts on the more sensitive culture independent methods of analysing environments for AMRP and ARG and have developed novel bioinformatics tools which do not rely on a priori design of probes and primers but allow flexibility and the use of Hidden Markov Models to find emerging resistance genes. This defined our third objective to enable discovery of ARG diversity in addition to establishing possible location in association with MGEs and attempt extraction of AMRP genomes form metagenomes to investigate survival and selective effects of environmental exposure to antibiotics, biocides, ARG and MGEs. Mathematical modelling has the ability to connect multiple disciples towards a better understanding of complex biological, chemical, physical, geographical, environmental and social interactions of ecological systems. Our group has recently produced the first multi-factorial risk model for antibiotic resistance gene exposure in river catchments based on observations made using culture independent methods, coupled with geographical, chemical and environmental measurements and we have refined and updated this model. The model was adapted to investigate the effects AMRP persistence in the presence and absence of antibiotics and possible mitigation effects of waste water treatment plant (WWTP) efficacy. We proved definitively that distance from WWTP was a key factor in affecting prevalence of ARG and was positively correlated with ARG levels measured both by qPCR and by relative prevalence in reads within the annotated metagenome.
A further key finding was that in the metagenome recovered genomes (MAGS) clinically relevant ARG were found in indigenous river associated bacteria rather than human associated groups. This result has significant implications for the impact of antibiotics on the environmental resistome and prevalence and dissemination of ARG.
Exploitation Route Our results are being submitted for publication in learned journals and a full report is being prepared for NERC, Defra and EA, we were delayed in these activities due to the large amount of bioinformatics analysis and modeling work that had to be done towards the end of the project as it was governed by seasons and sampling had to be done into the final year so that all the analysis was only completed when the last sampling was done. A large database of results containing all the metadata will be deposited within the next month with EBI.
Sectors Agriculture, Food and Drink,Environment,Leisure Activities, including Sports, Recreation and Tourism

 
Description Provided data on the prevalence of antibiotic resistance genes (ARG) to stakeholders at a large meeting in London on 14th March 2018. Gave prevalence data across the entire Thames catchment so that Thames Water could also evaluate where hot spots for pollution occurred. In addition a model has been developed to help identify drivers for pollution and this can also be used for evaluating mitigation impacts which will be very useful for Defra, EA, Rivers Trust and others.
First Year Of Impact 2015
Sector Environment,Healthcare,Leisure Activities, including Sports, Recreation and Tourism,Pharmaceuticals and Medical Biotechnology
Impact Types Policy & public services

 
Description Advice given to Defra regarding the incidence of antimicrobial resistance in rivers
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
 
Description Contribution to extensive discussions on the concerns over environmental reservoirs of antimicrobial resistance culminating in the publication of a technical white paper "Initiatives for Addressing Antimicrobial Resistance in the Environment: Current Situation and Challenges report available on the wellcome website under antimcirobial-resistance-environment.
Geographic Reach Multiple continents/international 
Policy Influence Type Gave evidence to a government review
URL https://wellcome.ac.uk/sites/default/files/antimicrobial-resistance-environment-report.pdf
 
Description Chicken or the Egg: Is AMR in the Environment Driven by Dissemination of Antibiotics or Antibiotic Resistance Genes?
Amount £1,500,000 (GBP)
Funding ID NE/N019717/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 06/2016 
End 03/2020
 
Title Novel use of minION for diagnostics 
Description Novel approach to diagnostics for typing pathogens in faecal samples directly 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact New approach for direct typing of a pathovar or spoligotype directly from cells in a faecal or other samples for example soil, blood, tissue. This will allow rapid identification of pathogens in the environment and help track dissemination pathways. 
 
Description Advgisory group member for Eden Project Wellcome Invisible Worlds exhibition 
Organisation The Eden Project
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Advisory group to Invisible Worlds (£1 million Wellcome funded exhibition that includes microbiology)
Collaborator Contribution Advisory group to Invisible Worlds (£1 million Wellcome funded exhibition that includes microbiology)
Impact The Eden Project will be hosting the Programme Integration Grant (NERC Environmental Microbiology and Human Health) final meeting and we will develop a display of the NERC programme data
Start Year 2017
 
Description Environmental reservoirs of microbial pathogens 
Organisation University College of Islamabad
PI Contribution Working on developing bids for GCRF and other funding agencies to halt the spread of microbial pathogens and AMR in the environment
Collaborator Contribution Hosted a workshop and paid our team's expenses to visit and work in a collaborative pilot study in addition to talks and visits to field sites
Impact The collaboration resulted in the analysis of field sites to consider the environmental reservoirs of microbial pathogens and understanding transmission pathways through water and food chain routes.
Start Year 2019
 
Description Korean partnering on metagenomics and microbiome analysis 
Organisation Chung-Ang University
Country Korea, Republic of 
Sector Academic/University 
PI Contribution Understanding the risk posed by the widespread dissemination of antimicrobial resistant bacteria (AMRB) and pathogenic variants (AMRP) in the environment depends on persistence of the bacteria and the resistance genes (ARGs). ARGs can move into indigenous environmental bacteria but risks of infection will be reduced or removed. What has been observed is the high diversity of resistance genotypes in waste water polluted environments coupled with the expansive diversity of the environmental resistome and so the extensive mixing of human and animal wastes with environmental bacteria particularly in the presence of antibiotics could produce AMRP capable of extensive resistance phenotypes. Uncertainty exists over the longevity and activity of AMRP in the environment outside of their hosts and to what extent they may participate in gene exchange so current work in both the Cha and Wellington groups is focused on this aspect and finding new ways of monitoring AMRP and their activities. Thus we identified two major challenges in understanding risk in environmental exposure to AMRP which relates to viability and ARG acquisition. In the form of two workshops and exchange of personnel the Wellington group members Dr Chiara Borsetto and Dr Robert James demonstrated their methods for using long read sequencing to gain an improved analysis of resistance genes in the environmental resistome, Chiara Borsetto talked about using mesocosms to establish if sublethal levels of antibiotics in the environment select for resistance phenotypes or are simply collocated with already resistant bacteria in waste water effluent. By characterizing the host genome it is possible to establish if the resistant bacteria are human adapted or environmental bacteria. From our work on river flumes used to model impact of waste water effluent in UK rivers it appears that sublethal antibiotic has a distinct impact on the prevalence of the relevant resistance genes.
Collaborator Contribution Professor Chang-Jun Cha's group have expertise in bioinformatics and hold a resistance gene database in addition to having a WaferGen SmartChip machine and the arrays which can detect very sensitively over 300 types of resistance genes using PCR conducted in hundreds of minicells.We were given access to this machine and were able to compare results and also establish diversity and new combinations of various mobile genetic elements. We aim to write a position paper comparing our results on anthropogenic impacts on Thames riverine microbiome compared with the study they are conducting on the Han river in South Korea. They provided access to databases and SmartChips plus shared sequences and databases.
Impact Two papers are in progress and one submitted currently: Submission no: ENVINT_2019_218, Submission title: A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria Corresponding author: Professor Chang-Jun Cha, Listed co-author(s): Dr Kihyun Lee, Dr Cung Nawl Thawng, Professor Elizabeth Wellington, Dr Dae-Wi Kim.
Start Year 2018
 
Description Presentation to MRC sponsored scientists and panel members 
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 Gave a presentation entitled "AMR in Soil" MRC workshop Antimicrobial Resistance Cross Council Initiative, London July 4-5th.
Year(s) Of Engagement Activity 2017
 
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. 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 ? 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 MRC Steering group for AMR 
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 Sit on the MRC AMR Steering Panel to plan further activities and review existing programmes of funding
Year(s) Of Engagement Activity 2017,2018
 
Description Presentation at International meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave presentation entitled "Metagenomics and culturomics: The environmental resistome" FEMS 2017 7th Congress of European Microbiologists Valencia Spain July 9-13th.
Year(s) Of Engagement Activity 2017
 
Description Presentation at a workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Two workshops the first initially to engage with policy makers and practitioners to understand the issues surrounding the spread of resistant bacteria in Pakistan.
Year(s) Of Engagement Activity 2017,2018
 
Description Public Science Evening 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presentation given at an evening event on the Microbiome event to inform the public about the importance of bacteria in the human gut, in the soil and in the bioremediation of polluted environments.
Year(s) Of Engagement Activity 2017
 
Description Working group activity 
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. On the 27-28th of September 2017, ca 20 scientists with core expertise on the Environmental Dimensions of Antibiotic Resistance, invited and/or selected by the member states, met in Gothenburg, Sweden, to discuss potential steps in a roadmap for the Environment pillar within JPIAMR (see appendix for participant list). The steps discussed primarily covered the need for an update of the Environment part of the current Strategic Research Agenda (SRA), but also more specifically the potential content of a research call with focus on the environmental dimensions.,
Year(s) Of Engagement Activity 2017
 
Description Workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Participated in an international workshop with Indian scientists across a range of disciplines to understand teh issues of AMR in India
Year(s) Of Engagement Activity 2017