MICA: Understanding how bacteria respond to efflux inhibition

The number of infections in people and animals caused by antibiotic resistant bacteria are increasing to alarming proportions around the world, and new treatments are urgently needed. However, historically, it has proven very difficult to find antibiotics that can get into bacteria such as E. coli (called Gram-negative bacteria). Even when they penetrate into the cell, most get pumped out/exported (called 'effluxed'). This export is via a biological 'vacuum cleaner' that sucks substances out of the inside to the outside of the bacterial cell. A single system can transport many different types of antibiotics. Without such systems Gram-negative bacteria are killed by very low concentrations of antibiotics. The combination of restricted entry of drugs into the bacterial cell (called outer membrane permeability) and export by multidrug resistance (MDR) efflux systems are why so many antibiotics that are effective treatments for infections caused by Gram positive bacteria, such as Staphylococcus aureus, do not work against Gram negative bacteria. Efflux systems that transport antibiotics out of the cell are not only required to give antibiotic resistance but without them bacteria are unable to cause an infection or form a biofilm - a community of bacteria that live on surfaces in the host and environment. Efflux inhibitors make the bacterium very sensitive to antibiotics, make them less able to colonise or infect their hosts and less able to form a biofilm. Therefore, efflux inhibitors offer the potential to increase the effectiveness of currently available and new drugs.

Our aim is to investigate the hypothesis that drug-resistant bacteria can emerge even in the presence of an efflux inhibitor. In this project, we will build upon our preliminary data by determining when E. coli becomes resistant to a combination of an antibiotic and efflux inhibitor. We will identify how the bacteria become drug resistant and find out if this type of resistance already exists in bacteria isolated from people and animals. Using a model that reproduces drug levels over time in people, we will identify ways to suppress the emergence of antibiotic and efflux inhibitor resistance.

To achieve the project aim, there are two applicants, a scientist and a doctor, plus three expert collaborators from the pharmaceutical industry and two universities. We will work together to provide new scientific information and knowledge crucial to the drug discovery of efflux inhibitors. Ultimately, this research will have a health benefit on the treatment of patients with life-threatening infections caused by antibiotic resistant bacteria.

Antibiotics are a cornerstone of modern medicine and used extensively by healthcare professionals. Therefore, antimicrobial resistance (AMR), especially by Gram-negative bacteria, poses a threat to human health. Furthermore, developing new antimicrobials has proved increasingly difficult over the past few decades. For Gram-negative bacteria this is due to combination of poor drug permeability coupled with active efflux. Therefore, one strategy to address the growing problem of AMR is to develop inhibitors of multidrug resistance (MDR) efflux pumps thereby sensitizing bacteria to already existing antimicrobials. Using efflux inhibitors in combination with antibiotics will increase and prolong their effectiveness.

This project will build upon our preliminary data that indicates that bacteria can become resistant to an efflux inhibitor alone and in combination with a fluoroquinolone antibiotic. In this project we will (1) investigate the bacterial response to efflux inhibition, (2) the conditions by which bacteria can evolve resistance, and (3) identify clinically relevant strategies to suppress resistance. We will do this using fluorescence based reporter systems that will detect transcription of RND MDR efflux pump and stress response genes in populations and single cells. We will use methods established in our laboratory including flow cytometry methods to (1) measure efflux activity in bacterial cells, and (2) sort populations of bacterial cells after exposure to drug +/- efflux inhibitor followed by whole genome sequencing. The mechanisms of resistance will be elucidated. We will also use established pharmacokinetic pharmacodynamic models to identify when resistance emerges and how it can be suppressed.

The work proposed in this project has significant implications for the treatment of bacterial infections.

There will be numerous beneficiaries of the programme of research, which will lead to academic, economic and social impact. These include
- The academic research community through research outputs and scientific publications generated by the applicants, researchers and project partners.
- The researchers, who will be provided with an excellent platform for pursuing successful careers, which in the long-term will contribute to the scientific advancement and economic prosperity of the UK. They will also be provided with a unique opportunity to work with project partners in their laboratories.
- The MRC and RCUK in general, by adding to the UK's capacity and research strength in antibiotic resistance and drug discovery research.
- Industry (small medium enterprises and the pharmaceutical industry), who will have the opportunity to build upon new and novel data about efflux inhibitors for antibacterial drug discovery as a basis for new treatments to address serious infection challenges.
- The public, though a programme of dissemination and engagement activities, will be told about our discoveries, which could impact on their health and wellbeing. Information about antibiotics has appeal and interest to a broad lay audience.
- Developing countries through established collaborations of the applicants with researchers in Africa and China in relation to mechanisms of antibiotic resistance and impact of resistance upon infection and treatment.
- Government departments including the Department of Health and Public Health England as well as NHS Trusts, who can use the knowledge gained about multi-drug efflux to help formulate evidence based policy in relation to national and global infection challenges.
This project will benefit the academic community by providing new scientific information about efflux inhibitors as well as resistance development and mechanisms. Therefore, data arising from our research will be of interest to several active academic research communities. They will be informed by presentations at national and international conferences, publication of data in international peer reviewed journals, and by researcher exchange. All staff engaged within the project will contribute to the generation and dissemination of academic outputs. This project presents an excellent opportunity for PDRFs to develop their careers within strong research teams located within institutions where PDRFs have been able, and mentored, to progress to independence, transitioning into PIs in their own right.
There is an urgent need for new antibiotics to treat Gram-negative bacterial infections. This project will provide new scientific data about antibiotic-efflux inhibitors. Thus our results will be directly applicable to the development of novel therapies providing commercial benefit; (1) resistance data will be crucial in helping to understand the mechanism of efflux and inform the design of inhibitors; (2) new understanding of the pharmacokinetics and pharmacodynamics of drug-inhibitor combinations will be obtained; and (3) strategies to suppress emergence of resistance to combinations will be proposed. Results will be shared with other commercial stakeholders by publication and conference presentations. Assisting the development of antibiotic-inhibitor combinations will increase the health and wealth of the UK population and economy.