Chicken or the Egg: Is AMR in the Environment Driven by Dissemination of Antibiotics or Antibiotic Resistance Genes?

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

Abstract

Antimicrobial resistance (AMR) in the environment is driven by antibiotics released in the urine of humans and animals into sewage and ultimately the receiving rivers. AMR is also released from within the gut bacteria that are shed in faeces of both humans and animals. In both cases, antibiotics and AMR-containing gut bacteria are released into the environment through sewage. Despite the continued release of both antibiotics and antibiotic-resistant bacteria into our rivers, we still don't know the relative role that they play in explaining the amount of antibiotic resistance that we see in our environment. This is a critically important knowledge gap as it prevents industry and policy makers from determining where to spend our time and resources so as to lower this 'environmental reservoir of antimicrobial resistance'.

Sewage contains thousands of chemicals, many of which are at concentrations sufficient to inhibit or kill bacteria. Microbes defend themselves from these chemicals with a range of strategies, all of which have genes that are broadly classified as 'resistance genes'. Hence, sewage is an excellent place to find bacteria rich in resistance genes. Many of these genes are known to be mobile, which allows for the genes to be shared, thereby increasing its abundance within the environment. This mobility of genes is key to why it is so difficult to know what is driving AMR in the environment-a bit like 'which came first, the chicken or the egg.' Are the concentrations of antibiotics present in sewage sufficiently high to select for resistance genes in the environment or are the genes for resistance simply spreading from the gut-derived bacteria into the native environmental microorganisms? The keys to answering this question lie in the following two questions: 1) Do genes released from sewage move into and persist in the natural microbial community without continued exposure to critical threshold concentrations of antibiotics; and 2) Are the critical threshold concentrations in the environment sufficiently high to maintain gut-derived AMR genes in the natural microbial community or select for them all on their own?

In the proposed research we aim to answer these two key questions using four innovative experimental systems: 1) a small laboratory microfluidic system for the precise control and manipulation of microbial biofilms; 2) an in situ river mesocosm and 3) ex situ macrocosm which can also control and manipulate microbial biofilms under controlled conditions with the addition of antibiotics and/or antibiotic resistance genes; and finally 4) the use of the freshwater shrimp, Gammarus pulex, as an indicator species of environments where the reservoir of antibiotic resistance is elevated. In the case of the Gammarus, we will study the microorganisms that live within this shrimp and determine if these microbes acquire similar antibiotic resistance traits as those found in identically-exposed biofilms. Modern molecular techniques (i.e, metagenomes, plasmid metagenomes, qPCR, meta-transcriptomes), will be used to quantify treatment effects within biofilms and Gammarus. The data from these studies will be used to parameterise a mathematical/statistical model that will be designed for use by regulators, industry and academia to better predict and understand the risks posed by AMR in the environment.

Planned Impact

This proposal will determine the degree to which antibiotics and antibiotic resistance genes are driving antimicrobial resistance (AMR) within the freshwater environment. There are a range of stakeholders for whom the research outputs could be valuable detailed below.

Regulators
Defra and the Environment Agency require sound science to inform their response to the European Water Framework Directive (WFD) and Urban Waste Water Treatment Directive (91/271/EEC) which has a major influence on the way chemical risks are assessed and chemical control measures might be considered. As described in the scientific case for support, antibiotic resistance gene prevalence could be mediated by antibiotics and antibiotic resistance genes, but to different degrees. Understanding the relative role of these drivers for AMR selection and maintenance in the environment, will help stakeholders and policy makers prioritise the most efficacious solution to minimising the risks to humans. CEH holds regular meetings with Defra Central Evidence group and the EA Evidence group, which represent opportunities for our science to be communicated across the agencies.

Water Industry
The central suggestion of this proposal could reasonably apply to the entire network of sewage treatment plants in the UK and is therefore potentially of great relevance to the Water Industry. If control measures for antibiotics or antibiotic resistance genes were required by the WFD it would be the Water Industry that would have to implement discharge controls to limit this risk (e.g. through enhanced waste water treatment). Thus, evidence from the proposed research will be critical for the industry's risk assessments and response. It may be that the minimum selective concentration for antibiotics are prohibitively low for the solution to lie with the water industry. We are liaising with Tony Griffiths of Southwest Water and Howard Brett of Thames Water, links that will be strengthened during the project.

Public Health
Public Health England are responsible to Government for clearly identifying and reporting on challenges to public health. AMR is a significant issue for PHE as evidenced by their 5 year Antimicrobial Resistance strategy, published in September 2013. Clarification on the potential for human exposure to a wide range of different antimicrobial resistance genes through wide-spread environmental prevalence will be important for their future review of the topic. As part of this project, we aim to engage with PHE, with particular reference to the further development of our statistical model that aims to predict high AMR exposure areas and the implications of our Gammarus 'sentinel' research for rapid monitoring of the environment.

We aim to conduct three seminar/workshops with an open panel discussion about the implications to policy, regulators, academics and industry of our research in light of changing national and international priorities on antibiotic use and regulation. The PI is very active on twitter (>1100 followers) and will use this to engage with the wider scientific community, along with the CEH twitter account. We will post blogs to the CEH website, which will be disseminated via Twitter and Press Releases. Our science outputs will be prepared for publication in high impact, open access journals. Our policy relevant messages will be communicated both through blogs and policy briefings and disseminated to the many relevant stakeholders already identified as well as CEH's wider stakeholder contacts. The PI will draw upon the expertise of the CEH Business Development and Engagement Section for advice on social media, events and workshops, public relations, media activities and science writing.
 
Description That antibiotic resistance proteins can be excreted in quite large quantities and proteins are very stable leading to protection of sensitive bacteria and some E. coli can be active below 30o C in environmental conditions. Faecal bacteria survive well in flume systems which mimics river and natural sediment and can remain active to transfer genes. We have developed analytical methods to detect potential antibiotic exposure in situ and determine degradation rates. Few studies have considered both pathogens and ARGs together with comparative analysis of waste treatment procedures, and no model has looked at mitigation strategies such as anaerobic digestion to reduce AMRP load with an integrated modelling approach. The main achievements can be summarised as follows:
1. Development of new bioinformatic tools to examine association of ARG with MGEs and AMRPs in environmental metagenomes.
2. Gained an improved understanding of antibiotic decay and degradation rates in different environments and measured exposure.
3. Developed an improved understanding of antibiotic degradation pathways especially in riverine microbiomes and model systems as we have now produced a key model for sulfonamide impacts at sublethal doses in model river systems using flumes in Department of Engineering. These flumes provide first clear evidence for enrichment of resistance genes in indigenous environmental bacteria as sublethal levels of antibiotic. This result has wide reaching implications for the impact of antibiotic residues continuously be pumped out of waste water treatment plants (WWTP) as both faecal bacteria and pharmaceutical are readily detected in rivers downstream of WWTPs.
Exploitation Route Currently there is no real evidence that sublethal levels of antibiotics present in WWTP effluent has an impact on the environment, nor the levels observed in land runoff resulting from the use of antibiotics in agriculture. Our study which was repeated now provides the first evidence that there is a measurable impact on the prevalence of resistance genes after exposure to sublethal doses of antibiotic and thus the Water Utility Companies need to consider controlling the output of antibiotics in WWTP effluent. Defra, the EA and other agencies such as the Rivers Trust were briefed about our results and we are writing a position paper for Government and in the process of submitting a manuscript to learned journal showcasing our results.
Sectors Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology

 
Description 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. This model can be adapted to investigate the effects AMRP persistence in the presence and absence of antibiotics and possible mitigation effects of antibiotic degradation. We have developed analytical methods to detect potential antibiotic exposure in situ and determine degradation rates. Few studies have considered both pathogens and ARGs together with comparative analysis of waste treatment procedures, and no model has looked at mitigation strategies such as anaerobic digestion to reduce AMRP load with an integrated modelling approach. The main achievements can be summarised as follows: 1. Development of new bioinformatic tools to examine association of ARG with MGEs and AMRPs in environmental metagenomes. 2. Gained an improved understanding of antibiotic decay and degradation rates in different environments and measured exposure. 3. Developed an improved understanding of antibiotic degradation pathways especially in riverine microbiomes and model systems as we have now produced a key model for sulfonamide impacts at sublethal doses in model river systems using flumes in Department of Engineering. These flumes provide first clear evidence for enrichment of resistance genes in indigenous environmental bacteria as sublethal levels of antibiotic. This result has wide reaching implications for the impact of antibiotic residues continuously be pumped out of waste water treatment plants (WWTP) as both faecal bacteria and pharmaceutical are readily detected in rivers downstream of WWTPs.
First Year Of Impact 2019
Sector Environment
Impact Types Societal,Policy & public services

 
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 LinkPI: Linking Phenotype function with Identity: a novel integrated single-cell technology and metagenomics approach
Amount £83,396 (GBP)
Funding ID NE/S008721/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 12/2018 
End 11/2020
 
Description Strain resolved metagenomics for medical microbiology
Amount £497,161 (GBP)
Funding ID MR/S037195/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2019 
End 09/2021
 
Title Metagenomes: Impact of sulfamethoxazole on the microbial community of a riverine environment 
Description BioProject PRJNA693684: Bacterial community amplicon sequencing (16S rRNA gene) and metagenomes of in vitro study to mimic riverine microbiome exposure to low concentrations of sulfamethoxazole. Flumes systems set with river water and sediment were used to mimic river conditions in the laboratory. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? No  
Impact New insight on the effects of sub-lethal concentrations of sulfamethoxazole on the riverine microbiome and related Antimicrobial Resistant Genes (ARGs) spread. 
 
Title Metaproteome: Impact of sulfamethoxazole on the microbial community of a riverine environment 
Description In vitro study to mimic riverine microbiome exposure to low concentrations of sulfamethoxazole (SMX). Flumes systems set with river water and sediment were used to mimic river conditions in the laboratory. Metaproteomic datasets of the water fraction was generated to monitor antimicrobial resistant proteins (ARPs) expression under low levels of SMX exposure. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact First attempt at monitoring active expression of Antimicrobial Resistant Proteins (ARPs) recovered from riverine microbial communities. New insights were obtained on environmental responses to antimicrobials exposure. The dataset will also be used to test customised bioinformatic pipelines for metaproteomic analyses. 
 
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 AMR integrated programme 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact I gave a talk entitled "Investigating environmental antibiotic resistance using flumes to mimic the river environment" to experts of the sector resulting in discussion of the problem of AMR in the environment and the spreading of these genes.
Year(s) Of Engagement Activity 2019
 
Description ISPB 2018 conference 
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 the poster "Investigating environmental antibiotic resistance using flumes to mimic the river environment" to experts of the sector resulting in valuable discussion of the problem of AMR in the environment and the role of potential pollutants in promoting the spreading of these genes.
Year(s) Of Engagement Activity 2018
 
Description MMEG 2018 conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation of the poster "Investigating environmental antibiotic resistance using flumes to mimic the river environment" to experts of the sector resulting in valuable discussion of the problem of AMR in the environment and the role of potential pollutants in promoting the spreading of these genes.
Poster prize awarded.
Year(s) Of Engagement Activity 2018
 
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 NERC-EMHH Environmental Microbiological Risk Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation of a poster entitled "Investigating environmental antibiotic resistance using flumes to mimic the river environment" and discussion over the topic with expert of the sector.
Year(s) Of Engagement Activity 2018
 
Description Operation Earth 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact General public attended the event and through simple and short activities (mainly for children) they were engaged in learning more about the soil and the microbial diversity in the environment around them. The majority of them also took part to a national study collecting soil from their gardens, testing the pH and reporting the results into a public database.
Year(s) Of Engagement Activity 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 Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact At the public science evening "The microbes on us and around us: We can't see them but can't live without them" hold at the university, the general public was introduced to the issue of AMR dissemination in the environment through oral and poster presentations followed by discussion in small groups with member of the team working on AMR. The Department reported an increased interest and understanding in the related subject area.
Year(s) Of Engagement Activity 2017
 
Description Superbugs Lates 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact General public attended the event (only adults) and soil and microbial diversity in the environment was shown through simple activities and discussed. Interest was also raised on superbugs and antibiotic resistance.
Year(s) Of Engagement Activity 2018
 
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