Applying New Tools to Identify Inhibitors of Antimicrobial Resistance Plasmid Transmission or Stability in Gram Negative Bacteria

Antibiotics and antimicrobials underpin modern human and veterinary medicine. They are commonly used not only to treat infections, but also prior to surgery, and are extremely important for vulnerable patients with reduced immune function, including the young, elderly, transplant, cancer, and HIV/AIDS patients. However, the spread of multidrug resistant bacteria is rendering these treatments ineffective. The number of infections in people and animals caused by antimicrobial resistant bacteria are increasing to alarming proportions around the world, and new treatments are urgently needed. However, it has proven very difficult to find antibiotics that get inside bacteria such as E. coli, Salmonella and Pseudomonas aeruginosa (called Gram-negative bacteria). Furthermore, over the last few decades very few new antibiotics have made it to the market. Therefore, finding ways to make resistant bacteria susceptible to already existing antimicrobials is a very attractive strategy.

One of the reasons antimicrobial resistance is such a growing problem is that bacteria are able to easily share their genetic information through a number of ways, including plasmid transmission. Plasmids are pieces of genetic material that can be moved from one bacterial cell to another. These plasmids often contain genes that allow bacteria to become resistant to antimicrobials (termed antimicrobial resistance genes). Therefore, bacteria are able to share antimicrobial resistance. This occurs on a global scale, with antimicrobial resistance plasmids rapidly traversing the globe, causing serious and difficult to treat infections worldwide.

In this project, we will use a novel system we have developed to discover compounds that get rid of these antimicrobial resistance plasmids from bacterial populations. This could be used in a number of settings including animals, patients, farming, and waste water treatment. In animals, large quantities of antibiotics are used in a number of countries including high income countries such as the USA, which provides a platform for resistance to develop. This resistance can then spread to other nations. Our work would reduce the number of antimicrobial resistance genes in bacterial populations, therefore reducing the chances that these genes would get into human pathogens, where they could cause untreatable infections. Compounds identified in this study could also be used to clean surfaces, especially in hospitals, where antibiotic resistance is prevalent. They could also be used to eliminate antibiotic resistance genes from waste water, soil, compost, and farms. Our work could also be applied in hospitals where patients are routinely treated with antibiotics. Drugs developed from our work could be given to patients prior to antibiotic therapy, thus increasing bacteria susceptibility to antibiotics.

In order to complete this work we have assembled a team of scientists with expertise in a wide range of different techniques. We will work together to provide new scientific information and knowledge crucial to combating antimicrobial resistance, by making bacteria sensitive to the antibiotics we already have available. Ultimately, this research will have a health benefit on the treatment of patients with life-threatening infections caused by antibiotic resistant bacteria.

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. Therefore, innovative strategies to address the growing problem of AMR are needed.

This project uses a new fluorescence based reporter system constructed using three clinically relevant Gram-negative associated global AMR plasmids (pCTblaCTX-M-14, pNDM-HKblaNDM1, and pKpQILblaKPC) which encode genes conferring resistance to some of our 'last line of defence' antibiotics. These tools will be used to screen for novel compounds that inhibit AMR plasmid transmission and/or stability. This will be done in a high throughput fashion at the University of Birmingham Drug Discovery Facility, where we will screen approximately 40,000 compounds, from the Diversity Library and the FDA approved Prestwick Library. Flow cytometry will be used to analyze compound effects on AMR plasmid transmission and stability. Identified target compounds will then be verified, and the specificity of their effect will be determined. Our preliminary work shows that the fluorescent reporters and affiliated flow cytometry assays are an effective way to determine plasmid stability and transmission.

The work proposed in this project has significant implications for global levels of AMR. Identified compounds could be used in a number of applications including but not limited to: animals feed, farming, veterinary medicine, waste water treatment and possibly human medicine. Our group has established collaborations that will allow these applications to be examined in future work. Using compounds that reduce the prevalence of AMR genes in people and the environment will sensitize bacteria to already existing antimicrobials.

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 by providing new scientific information about the plasmid inhibitors. Knowledge exchange will be delivered through 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.

- The Post-doctoral research fellow (PDRF, Dr. Buckner), will be provided with an excellent platform for pursuing a successful career, which in the long-term will contribute to the scientific advancement and economic prosperity of the UK. This project presents an excellent opportunity for the PDRF to develop her career within a strong research team located within an institution where PDRFs have been able, and mentored, to progress to independence, transitioning into PIs in their own right. The project will also increase the number of scientists with expertise in antibacterial drug discovery.

- The MRC and the BBSRC, by adding to the UK's capacity and research strength in antibiotic action, resistance and antimicrobial discovery research.

- Industry (small medium enterprises, agricultural industries and the pharmaceutical industry), who will have the opportunity to build upon novel data about compounds that remove AMR plasmids 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. Information about antibiotics has appeal and interest to a broad lay audience. This will also generate impact by improving societies understanding of AMR and how individuals can make a difference.

- Low and medium income countries through established collaborations of the applicants with researchers in Africa, India, Brazil 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 plasmid inhibitors to help formulate evidence based policy in relation to national and global infection challenges.

There is an urgent need for new antimicrobials to treat Gram-negative bacterial infections. We anticipate that this project will uncover compounds that can be used to remove AMR plasmids from bacterial populations. This will increase the susceptibility of bacteria to already existing antibiotics. Thus our results will be applicable to the development of novel treatment strategies providing societal and economic benefit for (1) Agriculture - animal feed and compost treatment, (2) Hospital cleaning - maintaining clean AMR free environments, (3) Waste water treatment - removal of AMR genes from waste water bacterial pools, (4) possibly even new patient treatments - compounds identified may lead to drugs to increase susceptibility of bacteria to existing antibiotics. Knowledge gained from this project will be exchanged with other commercial stakeholders by publication and conference presentations. Assisting the development of novel antibiotics will increase the health and wealth of the UK population and economy.