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

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.

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.