The evolutionary emergence of multidrug resistant bacterial pathogens

Increasing antibiotic resistance in bacterial infections is a serious threat to modern medicine, so understanding why some bacteria become resistant to multiple antibiotics whereas others do not is an important challenge for microbiologists, doctors and vets. In pathogenic E. coli - the most common cause of blood and urine infections worldwide and recognised by the World Health Organisation as a top global health threat - antibiotic resistance is associated with gaining extra loops of DNA called plasmids that carry several different antibiotic resistance genes. Not all E. coli strains seem to be able to maintain such plasmids, which is probably because gaining plasmids is typically very costly because the genes the plasmids carry interfere with the workings of the bacterial cell. Our work has led us to hypothesise that this variation between strains of E. coli is due to differences in how the various strains regulate the expression of their genes allowing some to mitigate for the disruption caused to the cell by the plasmid.
In this project we will test this idea in the laboratory using evolution experiments and cutting-edge sequencing technology. We have gathered together a collection of plasmid-free E. coli strains whose close relatives do typically carry antibiotic resistance plasmids in nature. First, we will compare the costs of acquiring a plasmid and the level of antibiotic resistance provided in both types of strain. We predict that costs will be higher in strains that don't typically carry plasmids. Second, we will compare the disruption caused to the cell by acquiring a plasmid in both types of strain by measuring patterns of how they express their genes. We predict that disruption to the cell caused by gaining the plasmid will be greater in strains that don't typically carry plasmids, and that this will scale with the costs we measure of carrying the plasmid. Third, we will experimentally evolve bacteria carrying plasmids in the lab to observe in real time how natural selection compensates for the cost of acquiring a plasmid. We predict that evolved bacteria will gain new mutations in regulators that turn genes on or off, to mitigate the disruption to the cell caused by carrying a plasmid, and that this process will be faster and more efficient in strains that typically carry plasmids. Fourth, we will test how the mutations observed during experimental evolution affect the expression of genes in cells with and without plasmids, to understand the molecular mechanisms by which evolution allows bacteria to maintain plasmids.
This project will advance our fundamental understanding of how and why pathogenic strains of E. coli that are highly resistant to antibiotics evolve. In future this insight could help us to identify potential superbugs before they emerge, or suggest novel targets for drugs that force bacteria to lose their antibiotic resistance plasmids making them susceptible to conventional treatments.

A major risk factor in the emergence of bacterial pathogens from commensal or environmental reservoir populations is the evolution of drug resistance, either pre- or post-emergence, which then makes treatment of the infection more challenging and increases the likelihood of invasive disease. This process is facilitated in bacteria by acquisition of multidrug resistance (MDR) plasmids carrying multiple resistance determinants. Understanding the evolutionary processes and mechanisms by which emerging pathogens acquire and maintain MDR plasmids is an important area of research in the fight against rising levels of antibiotic resistance. At present our understanding of this process is limited by a paucity of studies where the evolutionary hypotheses derived from observational data are interrogated with experimental tests. The large amount of observational population genomic data available for E. coli makes it an ideal model system in which to investigate the emergence of pathogenic MDR lineages that are the major cause of blood and urinary infections worldwide. This project combines experimental evolution, high-throughput genome sequencing, and transcriptomics to understand the evolutionary and molecular mechanisms by which pathogenic lineages of MDR E. coli emerge via horizontal gene transfer.

Who will benefit and how?
This is fundamental blue-skies research but results from this programme of research, and its practical application in clinical and environmental management of antibiotic use, has the potential to deliver far-reaching benefits across a wide range of sectors, from individuals and organizations, to societal benefits from long-term potential gains in the overall lifespan of existing and new antibiotics. The mechanistic understanding of the evolution of antibiotic resistance gained from this project will enable informed clinical decisions of antibiotic use, identify potential biomarkers of high-risk emerging pathogenic strains, and improve advice to the veterinary and farming community on the use of antibiotics. Although banned now in the EU, use of antibiotics as growth promoters is still widespread elsewhere - up to 40% of antibiotics used are as livestock growth promoters. The beneficiaries of this project can be divided in to 2 groups:
1) General Public: AMR is high on the news agenda, with recent reports of colistin resistance bacteria in China widely reported in the UK and beyond. Despite this, in August 2015, a report from NICE (National Institute for Health and Care Excellence) claimed that 10 million doses of antibiotics are inappropriately prescribed in the UK each year. We envisage that one key strand of impact activities associated with our project will be directed towards improving public understanding of the impending societal crisis surrounding rising antimicrobial resistance. There exists widespread misunderstanding of antimicrobial resistance (AMR) among the general public as shown by recent Wellcome Trust research ("Exploring the consumer perspective on antimicrobial resistance" report). The report identifies 3 key challenges:

1. Public attitudes to antibiotics and treatment are a barrier to improving antibiotic stewardship.
2. Poor knowledge of how antibiotics work and what resistance means lead to misconceptions about the nature of the AMR problem.
3. "Doomsday" scenarios and the huge numbers surrounding the AMR crisis in the media make individuals feel powerless to effect change.

Public engagement events designed to reach different age groups in schools and the wider community will raise awareness of the science underpinning the evolution of antibiotic resistance, and what we can all do to minimise the risk. We will exploit the high-profile of Antibiotic Action, based at UoB, to promote the events and key findings among the general public.

2) Government, Clinical, Agriculture and Veterinary Policymakers: AMR is also high on the policy agenda, with antibiotics of last resort increasingly at risk. Many reports across all these areas are highlighting AMR as a concern, yet there is a dearth of knowledge about the evolutionary mechanisms of AMR emergence particularly in pathogens affecting both humans and animals and where AMR arises through horizontal gene transfer. Our findings will be communicated to key stakeholders that can impact the use of antibiotics and their dissemination in clinical and agri-food environments, including: medical professionals, veterinarians, farmers and the agri-food industry.