Determination of the dynamics of antimicrobial resistance genes in the human and animal gut microbiome.

This research aims to develop a much deeper understanding of how antimicrobial resistance (AMR) resistance genes survive and spread within people and animals and to develop an understanding of how we might change the way we use antibiotics to reduce the amount and spread of AMR. AMR does not operate in an ecological vacuum and to understand the process properly, we need to obtain a much clearer appreciation of the underlying bacterial population and community dynamics of bacteria possessing AMR genes.

The aims of this research are to measure the numbers and AMR genes in bacteria found in patients, farm animals and the environment and the way they change over time (the dynamic changes), and in response to the use of antibiotics with a particular emphasis on the clinical outcomes in cases of human and animal disease. The research will use state-of-the-art DNA sequencing technology and analytical techniques to look at mixed populations of bacteria on samples of bacteria obtained from experiments performed in the laboratory and samples taken from hospitals and farms. Many of the bacteria in the gut are impossible or very difficult to grow in the laboratory and are considered to be 'unculturable'. Previous research has ignored these.

The overarching hypothesis to be tested is that the unculturable microbiome provides a reservoir of AMR genes that may both receive and donate AMR genes, in particular when the microbiome is under antimicrobial selective pressure.

The specific objectives are:

1. To establish the accuracy and reproducibility of two novel DNA sequencing methods that enable the DNA from individual bacterial species containing AMR genes to be identified from within faecal microbiome samples.

2. To use these novel genomic techniques in experimental conditions (in mice and pigs) to investigate AMR gene transfer from E. coli expressing Extended Spectrum Beta-Lactamase (ESBL) and Salmonella enterica serovar Typhimurium with fluoroquinolone resistance within the gut microbiome both following the treatment with antibiotics that are effective and when an antibiotic is used to which the bacteria are resistant.

3. To perform studies of the gut bacteria over time in farmed pigs and humans receiving antibiotics for treatment of clinical disease.

4. To develop mathematical models that capture the flux of AMR genes in bacterial populations to identify factors that lead to the build-up or transmission of resistance.

Research on Antimicrobial resistance (AMR) in host people and animals has focused on pathogenic bacterial species which are readily cultured in the laboratory. Recent advances in chromosome conformation methodology such as enable the metagenomic DNA sequencing of complex microbiomes with the assembly of genomic data from individual constituent bacterial species. Using these techniques it is possible to track the fate of AMR genes when they move between species of bacteria, or move between separate lineages of the same species. The aim of the proposed research is to define the nature and frequency of transfer of antimicrobial resistance (AMR) genes between pathogenic and commensal bacteria within their hosts under varying selection pressures.
The proposed research consists of 6 work packages (WP) with the following objectives:
WP1. To establish the accuracy and reproducibility of two novel metagenome sequencing methodologies that enable the individual genomic backgrounds of bacteria containing AMR genes to be identified from within faecal microbiome samples.
WP2 & WP3. To use these novel genomic techniques in experimental conditions (in mice and pigs) to investigate horizontal AMR gene transfer from E. coli expressing Extended Spectrum Beta-Lactamase (ESBL) and Salmonella enterica serovar Typhimurium with plasmid-mediated fluoroquinolone resistance within the gut microbiome both following the therapeutic use of antimicrobials that lead to bacterial cure, and following the use of antimicrobials to which these strains are resistant.
WP4 & WP5. To perform longitudinal studies of the gut microbiome in farmed pigs and humans receiving antibiotics for treatment of clinical disease to test the external validity of observations made from experimental animal infections.
WP6. To develop mathematical models that capture the flux of AMR genes in bacterial populations to identify key parameters in the build-up or transmission of resistance.

Increasing frequency of antimicrobial resistance (AMR) resistance genes in bacterial pathogens is a major problem in human and animal medicine. This research will provide important information about the impact of antimicrobial drug use in populations of bacteria found in agricultural and hospital environments. This knowledge is essential for the development of strategies concerning the responsible use of antimicrobial drug in both veterinary and human medicine. Animal agriculture and particularly farmed species such as pigs, poultry and dairy cows currently have a high dependence on blanket treatments of antibiotics in the face of infectious disease. Changes in these practices would have profound effects on the economics of food production and rational decisions need to be based on high quality evidence from appropriately conducted scientific studies. The results from this research would have impacts in the welfare of farm animals at the individual and group level; at the farm level; and for the whole industry. It would enable informed discussion to precede the adoption of industry-wide strategies to reduce levels and further induction of AMR arising from agricultural use of antibiotics. The dynamics of AMR gene flux in both animals and people will enable public and animal health policies to be developed that maximise the clinical effectiveness of narrow spectrum antimicrobial treatments, and help inform decisions on the most judicious use of broader spectrum antibiotics and those of greater importance to human health.

The proposed research will provide the groundwork to inform future antimicrobial stewardship in animal and human health. It is an ambitious and fundamental study of the way antimicrobial resistance (AMR) genes behave in the context of a host's microbiome.

One of the outcomes of the research is the generation of data about the previously unknown species of bacteria that contribute to human and animal microbiota. The application and further development of novel metagenomic sequencing techniques will identify the considerable proportion of the gut microbiome that is 'unculturable'. The proposed research will generate genome data from these occult species of bacteria that will not only be of value in the study of AMR but also provide data that will improve our understanding of the microbiology of the gut in healthy and diseased individuals.