Extending bicyclomycin treatment of multi-drug resistant Gram-negative pathogens

Lead Research Organisation: John Innes Centre
Department Name: Molecular Microbiology

Abstract

Antibiotics have saved millions of lives from infectious diseases and are arguably the greatest medical discovery of the 20th century. Unfortunately, one of the biggest threats to public health in the 21st century is the rise of multi-drug resistant bacterial infections. This rise has been caused by the improper use of antibiotics in medicine and agriculture combined with a shortage in the discovery of new types of antibiotics. This has prompted the World Health Organisation to warn that "with a dearth of new antibiotics coming to market, the need for action to avert a developing global crisis in health care is increasingly urgent", and the UK's Chief Medical Officer, Prof. Dame Sally Davies, to declare that "we are also not developing new drugs fast enough". This is a global problem that requires global action, particularly for the treatment of multi-drug resistant Gram-negative bacterial infections. Gram-negative bacteria are naturally resistant to many antibiotics used clinically, and have evolved to be resistant towards most other antibiotics following years of treatment with those medicines.

There are various approaches to tackle this growing problem, including re-purposing "old" antibiotics. These are drugs that were thoroughly tested for efficacy and safety in human clinical trials, but were not then widely used for a variety of reasons. For example, better broad-spectrum alternatives may have been available at the time. The rise of multi-drug resistance means that these molecules may now be very useful, especially because their lack of clinical use means that there has not been an opportunity for resistance to develop in disease-causing bacteria. Additionally, new experiments can sometimes reveal antibacterial activities that were not identified in earlier research. This is the case for bicyclomycin, an old antibiotic that had previously been shown to have moderate bacteriostatic activity towards Gram-negative bacteria. "Bacteriostatic" means that the antibiotic stops bacterial growth, but does not actively kill the bacteria, which can lead to the persistence of an infection. Excitingly, recent work has shown that bicyclomycin can actually kill bacteria ("bactericidal") when it is used alongside another bacteriostatic antibiotic. This unexpected activity makes bicyclomycin a highly promising antibiotic for the treatment of Gram-negative bacterial infections when used in combination with another drug.

Therefore, we propose to carry out further work to determine whether bicyclomycin can be widely used in the clinic. This will include testing more accurate models of infection for this novel activity and identifying new compounds that can be used alongside bicyclomycin to stop resistance developing. Bicyclomycin is a molecule that is made naturally by non-pathogenic soil bacteria. Natural products such as this are produced by the action of a series of enzymes (proteins), which are encoded by genes (DNA) in the bacterial genome. Thus, we aim to discover the genes that are responsible for bicyclomycin production. This discovery will allow us to make modifications to the pathway to make more of the compound, which will enable its study in infection models. We can also modify the pathway to produce new versions of bicyclomycin, which might have better activity than the original compound or overcome resistance mechanisms.

Technical Summary

Our research programme aims to repurpose an old antibiotic, bicyclomycin, for treatment of multi-drug resistant (MDR) Gram-negative infections. This stems from recent work demonstrating its ability to rapidly kill Gram-negative bacteria when used in combination. Bicyclomycin is a Streptomyces natural product that was discovered in the 1970s and has a narrow bacterial spectrum, poor oral absorption, and little lethal activity. Consequently, this compound, along with many others, was set aside in favour of more active agents that were available at the time.

Bicyclomycin has now become a promising candidate for redevelopment as members of the research team have shown that combining bicyclomycin with a bacteriostatic inhibitor of transcription (e.g. rifampicin) or translation (e.g chloramphenicol) converts bicyclomycin from a bacteriostatic agent to a highly lethal agent against multiple Gram-negative species. This discovery of bicyclomycin lethal synergy was surprising, because with most, if not all, other antimicrobial combinations, bacteriostatic inhibitors of gene-expression antagonise rather than stimulate the lethal activity of bactericidal agents. We have evidence that indicates that in the absence of a suitable partner antibiotic, a protein that protects against the lethal activity of bicyclomycin can be expressed. A small-molecule inhibitor of this protective protein combined with bicyclomycin would create a novel, highly lethal combination therapy for MDR Gram-negative infections. To achieve this we need to: (1) determine whether lethal synergy is observed in an in vivo model of infection; (2) establish a robust, high-titre platform the production of bicyclomycin by its natural Streptomyces producer, and develop methods to produce novel derivatives by both pathway engineering and synthetic chemistry; and (3) characterise the protective mechanism using proteomics and subsequent gene deletions to inform a screen for inhibitors of this process.

Planned Impact

The main beneficiaries of the proposed research will be the academic research community (see academic beneficiaries) who will benefit from the result of proposed work across multiple scientific disciplines, including infection studies, biosynthesis, medicinal chemistry and structural biology. This will result in the generation of various models we build from this work (such as a model of lethal synergy, and a model of bicyclomycin biosynthesis). This will be published in top class open access journals and will be presented at national and international conferences across disciplines. The project will establish a new collaboration between groups at the John Innes Centre (JIC) and Xiamen University, and the researchers involved in this project (UK PDRA, Chinese PhD students and research assistants) will benefit from exposure to a multidisciplinary project that encompasses a variety of research areas that are valuable for 21st century antimicrobial drug discovery.

A public consultation by JIC (coordinated by Ipsos MORI) determined that antibiotic resistance is seen by the general public as one of the major challenges facing both the UK and the developing world. It is therefore vital that we effectively communicate our research to the public so they can understand what we are doing to develop new approaches to overcome AMR. We will employ various outreach strategies that have previously proven to be highly successful. This includes our 'Antibiotic Hunters' exhibit about antibiotic discovery that was first prepared for the Great British Biosciences Festival in 2014. The bicyclomycin story will make a great addition to this exhibit, which allows the public to directly speak to members of the research team in a relaxed environment. This will be presented at local and national public outreach events and at local schools and hospitals. We will also reach the public by engaging with the media via our press office when we publish the results of this study. This will ensure there is impact from this research beyond academia.

The major mode of outreach for this project involves the SAW Trust (www.sawtrust.org, reg. charity no. 1113386), which is a science education initiative based on the Norwich Research Park that uses art and creative writing to effectively communicate a diverse range of cutting edge research topics in schools. It has already been a great success in Shanghai, where it was introduced into schools by the Xuhui Education Bureau. Since the first Xuhui SAW workshop in 2012, the programme now involves 18 schools and kindergartens, 140 teachers and around 50 social volunteers. The SAW learning style has proven to be very popular with Chinese teachers and creates opportunities for parents to work alongside teachers to deliver valuable curriculum enrichment activities in schools. This prior Chinese experience means that SAW is ideally placed to implement an antibiotic-themed project, which will be initially implemented at a middle school affiliated with Xiamen University, and would then be shared widely to other schools as an important learning resource. The project will be designed with scientific input from the research team and will be tested in a UK school before being translated and transferred to China. Both the UK PI and co-PI have been involved in multiple SAW activities in the UK relating to antibiotics.

There is also scope for the discovery and development of novel IP in the form of new versions of bicyclomycin, improved production methods, and co-administration strategies. This has the potential for economic benefits in additional to the obvious social benefits that come with developing new treatment modalities for antibiotic resistant bacterial pathogens. As appropriate we will continue to liaise closely with our technology transfer offices to protect and exploit any novel IP that arises from this work.

Publications

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Hutchings MI (2019) Antibiotics: past, present and future. in Current opinion in microbiology

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Hutchings MI (2019) Editorial overview: Antimicrobials: Tackling AMR in the 21st century. in Current opinion in microbiology

 
Description The objectives described for the UK partners in this UK-China grant have been met. We discovered the biosynthetic gene cluster for bicyclomycin in the known producer Streptomyces cinnamoneus DSM 41675. This revealed that highly similar gene clusters are also present in the genomes of in multiple bacteria, including hundreds of isolates of Pseudomonas aeruginosa, a human pathogen, which was a truly surprising result (https://aem.asm.org/content/84/9/e02828-17.abstract). We could not detect bicyclomycin production in Pseudomonas aeruginosa, but heterologous expression of this gene cluster in the non-pathogenic bacterium Pseudomonas fluorescens SBW25 proved that the P. aeruginosa gene cluster produces authentic bicyclomycin. This represents a quicker and cleaner route to producing bicyclomycin compared to using the original Streptomyces producer.

We therefore developed this Pseudomonas-based fermentation system to produce and purify sufficient bicyclomycin for our Chinese partners (Prof. Xianming Deng and Prof. Xilin Zhao at Xiamen University) in this MRC Newton Fund project. This material is currently undergoing the biological testing in China as detailed in the grant proposal. These tests involve in vivo studies of lethal synergy, testing against multi-drug resistant clinical isolates of bacteria and studies on the mechanistic basis for lethal synergy (these results will be reported in the future). Additionally, we have used this Pseudomonas system to generate bicyclomycin and diketopiperazine derivatives using a mixture of genetic engineering and synthetic chemistry (manuscript in preparation). Given the promising results of this research and the potential importance to antibiotic drug development, we are making plans to take this project forward with further grant applications and we have shared the Pseudomonas production system with our Chinese partners to ensure that they have the local capacity to make bicyclomycin in the future.
Exploitation Route There are multiple aspects of this project that could be taken forward by others. Firstly, the development of an easy-to-engineer Pseudomonas production system for bicyclomycin has potential for future production of bicyclomycin and related derivatives. This may be carried out by our Chinese collaborators, a company, a different research group or potentially even a charity. The results of the biological testing in China are very likely to impact the future research of other research groups. Currently, bicyclomycin is not in use, but encouraging results from biological testing by our Chinese partners would highlight the promise of this novel and safe antibiotic, especially given its activity towards Gram-negative bacteria. We have also shared material from the project (with an appropriate MTA) with a USA-based research group.

On a very fundamental level, our discovery of the widespread nature of the bicyclomycin gene cluster raises questions about gene cluster evolution and horizontal transfer within nature - a number of research groups will be interested to know why and how this gene cluster become so widespread - for example, understanding the role of bicyclomycin in nature.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Dr Andy Truman attended the China-UK AMR Workshop (Beijing, China) in December 2019, where this research was presented alongside Xianming Deng and Xilin Zhao. Here, we were also involved in discussions around strategies for future research into AMR in the UK and China. Dr Andy Truman also participated in the International Research Alliance for Antibiotic Discovery and Development (IRAADD) Workshop Meeting, (Saarbruecken, Germany), where a European strategy for Future AMR research and funding concepts was discussed. A white paper resulting from this network meeting is currently under review for publication in Nature Reviews Drug Discovery. Based on the results of the project, we received a grant from the John Innes Centre knowledge exchange and commercialisation (KEC) Innovation Fund to further develop our bicyclomycin production system to enable interactions with industrial partners. be Discussions are ongoing with fermentation facilities for larger-scale fermentation work that could provide longer-term support for pre-clinical studies of bicyclomycin efficacy and safety.
First Year Of Impact 2017
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Policy & public services

 
Description Contribution to SAW Antibiotics book
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
URL http://www.sawtrust.org/buy-the-books/saw-antibiotics/
 
Description Establishing a new production platform for the antibiotic bicyclomycin (JIC Innovation Fund)
Amount £19,583 (GBP)
Organisation John Innes Centre 
Sector Academic/University
Country United Kingdom
Start 02/2018 
End 06/2018
 
Description Xiamen University Bicyclomycin Project 
Organisation Xiamen University
Country China 
Sector Academic/University 
PI Contribution In the process of fermenting a natural product antibiotic from a Streptomyces strain that will then be purified for use by collaborators in Xiamen University.
Collaborator Contribution Assessing the biological activity and mechanism of action of the Streptomyces antibiotic.
Impact Collaboration started recently so no outputs so far.
Start Year 2016
 
Description Dubrovnik Summer School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I provided a seminar and led multiple small group discussions as part of the Dubrovnik Summer School in Applied Molecular Microbiology. This was attended by 45 postgraduate and post-doctoral researchers from around the world to learn about concepts and methods in natural product biosynthesis. Following the summer school, I have been contacted by multiple attendees regarding various aspects of the work I discussed.
Year(s) Of Engagement Activity 2018
URL https://www.jic.ac.uk/training-careers/summer-schools/applied-molecular-microbiology/
 
Description International Research Alliance for Antibiotic Discovery and Development (IRAADD) Workshop Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attendance and presentation as part of the International Research Alliance for Antibiotic Discovery and Development (IRAADD) Workshop Meeting (Saarbruecken, Germany), which is a Europe-wide network of researchers and industry partners involved in antibiotic discovery and development.
Year(s) Of Engagement Activity 2019
 
Description Interview with science writer (Kat Arney) about antibiotic discovery and combating antimicrobial resistance. 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview with science writer (Kat Arney) about antibiotic discovery and combating antimicrobial resistance. In particular a focus on the re-purposing of old antibiotics for treating multi-drug resistant infections. This was in relation to an in-depth article she is preparing on AMR and antibiotic discovery, which was later published in the Daily Mail.
Year(s) Of Engagement Activity 2018
URL https://www.dailymail.co.uk/health/article-5623417/Could-antibiotics-mens-beards-soil-weapons-battle...
 
Description Invited talk at China-UK AMR Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A UKRI-funded workshop held in Beijing to discuss ongoing and recent UK-China research projects on AMR (antimicrobial resistance), as well as future directions and prospects for this research area.
Year(s) Of Engagement Activity 2019
 
Description Invited talk at the University of Ghent 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited talk on natural product discovery at the University of Ghent (Belgium).
Year(s) Of Engagement Activity 2019
 
Description Invited talk at the University of Leeds 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited talk for the seminar series in the School of Molecular and Cellular Biology, University of Leeds. Participation in a discussion about my research with a group of MSc students.
Year(s) Of Engagement Activity 2018
 
Description JIC 50 Open Day 
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 Public/other audiences
Results and Impact Outreach stand for Department of Molecular Microbiology as part of an Open Day in relation to the 50th anniversary of the John Innes Centre in Norwich.
Year(s) Of Engagement Activity 2017
URL https://www.jic.ac.uk/news-and-events/blog-copy/2017/09/open-day/
 
Description Presentation at Science for Innovation Showcase Event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Provided presentation entitled "Discovery and biosynthesis of bacterial peptides with antibacterial and anticancer activities" to an audience of industry and research council representatives invited to the John Innes Centre. Participated in a follow-up panel discussion.
Year(s) Of Engagement Activity 2018
 
Description Presentation at Xiamen University (China) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited presentation given to faculty, post-docs and students at Xiamen University in China.
Year(s) Of Engagement Activity 2019
 
Description Science Museum Superbugs Late Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact "Antibiotic Hunters" exhibit at a Science Museum Superbug Late Event. This was an adult-only event held in the evening at the Science Museum in London and attended by thousands of people. The exhibit was organised jointly between members of my research group and members of the Wilkinson group (also at JIC).
Year(s) Of Engagement Activity 2018
 
Description Year 10 Science Camp Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A talk on natural product discovery to attendees of the year 10 Science Camp at the John Innes Centre. This was followed by questions on antibiotic and anticancer compound discovery.
Year(s) Of Engagement Activity 2018
URL https://www.jic.ac.uk/training-careers/work-experience/year-10-science-camp/