A quantitative method to evaluate AMR distribution in complex communities based on methylome profiling

Lead Research Organisation: University of Surrey
Department Name: Microbial & Cellular Sciences

Abstract

Antimicrobial resistance (AMR) inhibits our ability to deal with once easy-to-treat bacterial infections. AMR can be acquired by disease-causing bacteria from other organisms living in the same environment that do not pose a threat to our health e.g. in our intestines, which is home to trillions of bacteria. The acquisition of AMR by some harmful bacteria will enable these organisms to survive antibiotic exposure whereas as sensitive organisms, both beneficial and harmful to us, are killed. This means that the use of antibiotics, even at low levels, can promote the expansion of resistant populations in niches where there is little competition from other microorganisms.

Understanding the chain of events taking place during transmission of AMR is essential to inform more effective treatments and to rationalise use of our current repertoire of antibiotics. Unfortunately, methods for studying transmission are largely based on being able to grow the organism in the laboratory, something that is only possible for a relatively small number of species. As a consequence, all the events in which non-culturable species - those that cannot be grown in the lab - are involved are missing from our studies. Only recently, methods based on the production of fluorescent proteins have been used to understand transmission events in the environment and they have already given insights into previously unknown bacterial interactions. These methods are however limited to those conditions in which the fluorescent proteins work, which are largely dependent on the presence of oxygen. Specific anaerobic niches crucial in antibiotic treatments such as the intestine cannot be analysed using this methodology.

We propose an alternative approach to monitor transmission of AMR in complex bacterial populations like the gut microbiota. This method is based on the analysis of changes in the DNA of resistant populations in response to changes in environmental conditions such as during treatment with antibiotics. We will take advantage of the presence of other genes that can be transferred together with AMR to do this. These genes encode for enzymes called methyltransferases that produce permanent modifications in the DNA of the recipient cell. Monitoring the presence or absence of those modifications by a new sequencing technology will be used as proof of the acquisition of AMR. Since this method works in a high-throughput fashion, we can monitor thousands of species in a single experiment. In that way we will generate a complete dynamic picture of the main AMR interactions in the community and the rate at which these occur.

If successful, this new technique will help us to determine the flow of AMR in the natural environment, identifying potential reservoirs that favour the development of resistance. These results could be used to design tailored-made treatments to optimise the way that we use antibiotics to minimise the spread of AMR.

Planned Impact

This proposal aims to produce a method for the high-throughput analysis of the spread of antimicrobial resistance (AMR). We envision that, if we understand the rate and chain of transmission of this process, we will be able to interfere with it, optimizing the use of our current or future antibiotics. The driver of this project is the development of a new technology, and as such, the potential application will not be only limited to the study of AMR but will benefit other sectors of society.

Commercial exploitation and industrial partnership
The method proposed relies on a particular deep-sequencing technique developed by Pacific Biosciences (CA, USA). If successful, our work will be a 'technology push' that will clearly benefit this company over other competitors. One of the partners in this project (Dr. Gang), acts as a regular consultant of PacBio and we will seek commercial opportunities for further development based in the UK. For instance, another of the collaborators, the Centre for Integrative Biology (NERC-hub Liverpool), would be in an excellent position to become the reference institution to provide this kind of DNA analysis. Besides the DNA sequencing technological development, we believe that the applications of the method will be of interest for other sectors of industry, such as those related to environmental monitoring in agriculture, food, water, etc. At a later stage and once the proof of concept has been established, we will identify companies in the UK with an interest in the topic for collaborative endeavours. In addition, we will increase the impact of this work in industry under the umbrella of the EU program Horizon2020.

Improvements to the One Health Initiative
The impact of this technology in human health will be highly relevant. There are no current methods to understand the chain of events taking place during AMR transmission in the human microbiota, especially in microaerophilic conditions. This pump-priming bid is focused on mice but we will look for future opportunities to test the method under controlled conditions with human samples. Depending on the final coverage obtained by sequencing and thanks to the stability of the modifications, it could be possible to investigate methylation patterns in samples obtained in previous studies, alleviating the requirements of human sample collection.
As stated before, we think it is feasible to inform better clinical practice based on the results of this proposal. Establishing the rate and structure of the network of microbial interactions would be an important milestone towards a more rational use of antibiotics (i.e. specific combination therapies) slowing the spread of AMR.

Public engagement in scientific issues
The general public is aware of the problem of AMR thanks to a broad information campaign. They are not as aware, however, of the challenges and the steps taken to overcome them. Our results will contribute to the public debate in a positive way and will help to highlight the relevance of the problem and the efforts carried out by other groups. Since this is a sensitive topic, we will avoid sensationalisms that could hinder current public health protocols but, as we are committed to public engagement, we hope to have an impact in the perception of benefits derived from the technology.

Evidenced based policy making
This project will also contribute to the current debate on the need of new regulations that could speed-up the implementation of new molecules and/or alternative treatments to meet the demands of society. We also envision the need of new policies for controlling AMR transmission at a global level. For example, by modifying animal housing in the farming industry and fisheries, and also in the response to outbreaks. With this contribution we also hope to foster the investment in the field from both public and private sources.

Publications

10 25 50
 
Description This grant focused on developing and testing a method to track the transfer of antibiotic resistance plasmids between bacteria in complex microbial communities. The method relied on using natural enzymes to introduce modifications on bacterial DNA that can be detected using sequencing. So far, we have optimised the type and expression level of the enzyme and demonstrated that it modifies DNA in the manner that was predicted. We have inserted the enzyme sequence into some clinically-relevant antimicrobial resistant plasmids and have seen the transfer of plasmids between bacteria of a single species as well as those in a multi-species consortium grown under controlled laboratory conditions. Part of this has also involved optimising and exploring plasmid transfer rates under aerobic and anaerobic conditions as oxygen availability affects this process. We have applied this method to study plasmid transfer in the intestine, which is home to a wider diversity of bacteria numbering into the trillions. We have now sequenced all the samples from the murine model. Our data confirm that the DNA modifications caused by the enzyme could be detected in the original bacterial host but that thus far, no signal could be detected in the wider bacterial community. This suggests that plasmid transfer has not occurred as expected, at least for the one plasmid we have examined. We are still analysing the sequence data, which has been delayed due to a change in instrumentation, and so outputs from the project are still possible.
Exploitation Route If we are able to demonstrate experimentally the value of our approach to studying plasmid transfer, then our work might be taken forward or used by others. Within the scientific community, we are already discussing using our methodology to allow study of plasmid dynamics in other models systems, where complex microbial communities exist, specifically in the chicken gut and in a hospital sink drain trap. This latter work provides a link to Public Health England, and potentially into the area of infection control, something that could evolve into a longer term benefit for wider society. Outwith the scientific community, due to the delay in completing the final stages of the grant, we have not yet showcased our work to the public. However, this is something we will consider at the appropriate point.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology

 
Description International expert workshop on AMR, Canada
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
 
Description NERC Theme 3 Integration Award 'Environmental AMR bacteria as potential microbiome colonisers'
Amount £25,000 (GBP)
Organisation University of Bristol 
Sector Academic/University
Country United Kingdom
Start 10/2019 
End 05/2020
 
Title Methyltransferase gene delivery system 
Description A key step in this project is the generation of a methyltransferase gene delivery system to tag antimicrobial resistant plasmids (or strains) and enable their movement in complex microbial populations to be tracked. Five months into this project, we have achieved this and created a transposon-based delivery system that will randomly insert specific methyltransferase genes in the bacterial genome. In addition we have developed complementary non-sequencing based assays to assess the extent of methylation that occurs in cells receiving the genes, thus allowing investigators to optimise reaction conditions prior to investing in the (more expensive) sequencing-based methods. 
Type Of Material Biological samples 
Provided To Others? No  
Impact A methyltransferase gene delivery system should enable us to start to probe the spread of antibiotic resistant bacteria (or plasmids) in complex communities, but it is too early to state the success or otherwise of this approach. 
 
Title Murine intestinal bacterial methylation profile 
Description The database contains raw DNA sequencing reads. The sequences map to part of the 16S rRNA gene found in all bacteria. The DNA was extracted from the faeces of mice orally infected with antibiotic resistance strains of Citrobacter rodentium modified to express a unique methyltransferase gene. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
Impact None as yet. 
 
Description Opening event of new research facility 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Representatives from local biotechnology companies, other educational establishments, national funding agencies, animal-focused groups (e.g. NC3Rs), University post-graduate researchers and interested staff attended 3 hour informational session on the University's new animal facility. Here we showcased the technology we were developing in a manner that was accessible to all.
Year(s) Of Engagement Activity 2017