Building and dismantling polymicrobial pathogen communities in cystic fibrosis.

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

People whose immune defences are weakened are easy targets for infection by bacteria: they can suffer from chronic infections that last for months or even years. For example, people with the genetic disorder cystic fibrosis (CF) pick up lung infections which they cannot clear because their airways are blocked by sticky mucus. 90% of people with CF die from lung failure as a result, 50% of them before the age of 41. People with chronic obstructive pulmonary disease, HIV/AIDS or asthma, and hospital patients on ventilators, are also very vulnerable to chronic lung infection. These infections are typically resistant to many antibiotics, and it can be hard for doctors to predict which antibiotics could help a particular patient.

We have only recently understood that chronic infections are not simple infections of one type of microbe - rather, they are complex systems where different species of bacteria, fungi and viruses live together and interact inside the infected organ. The interactions are probably what make chronic infections so unpleasant and so hard to treat with antibiotics. Bacteria are especially interactive and sociable. Cells of the same and different species can glue themselves together in sticky biofilms that protect them from stress, immune clearance and antibiotics, and they can communicate with one another using chemical signals to co-ordinate the release of molecules that damage the host tissues.

There is another layer of complexity in chronic infection: the long time periods that infections persist for, and the changing environment inside the host due to medical treatment and tissue damage, mean that pathogens evolve. They can adapt to become better at living inside the host and resisting antibiotic treatment, and a clonal founding population of any given species can become very genetically diverse as it spreads into different areas or "niches" inside the infected organ.

This complexity and constant change means that it is very difficult to treat chronic infection. Antibiotic resistance can be hard to recognise when it is a function of two or more bacterial species interacting. Also, if we try to kill one species with an antibiotic, we do not know how the other species in the community will respond: more dangerous bacteria might be able to grow better if their competitors are removed. This probably explains why standard lab tests of antibiotic susceptibility of bacteria from patients - which are conducted only on one bacterial type from a patient - are not very good at predicting which antibiotics will help when they are prescribed.

People have tried to understand how chronic infection ecology drives antibiotic resistance and infection severity in samples tested in a lab by growing the bacteria in a test tube or on an agar plate, but these are not at all like a complex organ, like a lung. Tests have also been performed on mice and insects but these are very different from human lungs and so are of limited value (and there are ethical concerns about using live animals in this way).

I have developed a more realistic and ethical way of mimicking chronic lung infection using lungs from pigs killed for meat and donated human lungs from the NHS biobank. I use culture techniques that closely simulate chronic infection in people with CF. I will use this system to study how bacterial species that commonly cause chronic lung infection (in CF and other conditions), interact with one another to produce highly antibiotic-resistant, persistent infection. I will also develop my model into a new tool for conducting better, more predictive tests of which antibiotics, or which novel therapies, might work in patients.

Technical Summary

Chronic infections are caused by diverse communities of microbes that inhabit spatially-structured host environments. Inter-microbe interactions mediate virulence, persistence and antimicrobial resistance (AMR). Thus, improved diagnostic and R&D tests for the likely clinical impact of antimicrobial compounds require testing platforms that recapitulate diverse infection communities in structured tissue. Further, future treatments for chronic infection (e.g. bacteriophage) must be tested in such models to gain reliable predictions of their clinical potential.

I have developed a clinically valid, high-throughput model of polymicrobial infections of the lungs of people with cystic fibrosis (CF): ex vivo lung tissue cultured in artificial mucus. I will characterise infection communities in this model, revealing how biodiversity mediates AMR and virulence. I will study clinical isolates of key lung pathogens (P. aeruginosa, S. aureus, B. cenocepacia, H. influenzae, S. milleri + whole CF sputum samples) in single and polymicrobial infections. I will assess how the model could be developed into a clinically predictive platform for AMR profiling and the activity of novel antimicrobial/antivirulence drugs. I will use hypothesis-driven experiments about microbial communities in structured tissue to reveal key drivers of AMR in CF infection, the likely triggers of acute pulmonary exacerbation, and how these may be neutralised.

My model uses pig lung tissue from a commercial butcher and human lung from an NHS biobank. I will define how well pig lung recapitulates human tissue infections and promote its wider adoption in academia and industry to increase the clinical validity and 3Rs compliance of basic research and R&D testing. Through consultancy work with colleagues, I will explore the potential for ex vivo lung to underpin tests of novel therapeutic agents (antibiotics and phage) on realistic infection communities in structured tissue.

Planned Impact

STRATEGIC IMPACT
The project will deliver a clinically valid, high throughput model for studying how microbial interactions drive pathogenesis and antimicrobial resistance (AMR), and for testing antimicrobial susceptibility. This meets the MRC's goal of securing impact from research by maximising the translatability of lab studies. It also supports the cross-council initiative on tackling AMR, specifically the aims of "Understanding resistant bacteria" and "Understanding real world interactions". Beneficiaries include patients; academic, clinical and industry researchers; project staff and the general public.

BENEFITTING PATIENTS BY PROVIDING A NEW RESEARCH TOOL
The ultimate beneficiaries (beyond the lifetime of the grant) will be people with chronic infections. During the grant, I will liaise with the James Lind Alliance to align current and future work with their recommended priorities for cystic fibrosis research. By the end of the grant, we will have determined whether antibiotic resistance profiling of infection isolates could be made more predictive by using ex vivo lung to test polymicrobial samples, and assessed the potential of lung models for testing phage therapy. We will work with Warwick Ventures to identify routes to future commericialisation, validation and regulation of lung models for use in routine clinical testing (health service labs) and in testing pipelines for new infection treatments (academia & industry).

3RS IMPACT
Ex vivo lung models will reduce the number of animals used in infection research, saving time and money and helping researchers meet national aims to Reduce, Refine and Replace animals. Studies of basic in-host microbiology can be carried out in ex vivo lung instead of live rodents, and new clinical interventions can be trialed to rule out cytotoxic/ineffective treatments and narrow down experimental variables (e.g. dose) before moving in vivo. Of 25 P. aeruginosa mouse lung infection papers, 36% dealt with questions that we can investigate in ex vivo lung. These used a median of 36 mice, implying that annually, c.1300 mice are used in experiments that may instead be carried out in ex vivo lung. This number will be larger if unpublished pilot work and work on other bacteria are considered. This aspect of the project is an important societal impact, because the British public feel strongly about this area. A recent poll found that support for animal research is conditional on the lack of alternatives, that 78% of respondents wanted more research into alternatives and that people are interested in hearing about such research (http://bit.ly/1SWdtXN). We will track & publish the uptake and 3Rs impact of the project over the lifetime of the grant (and reassess these at intervals beyond the lifetime of the grant).

STAFF
The post-doc and technician will gain technical (molecular microbiology, genomics, imaging) and general professional (presentation, writing, statistics, communication, management of students or junior staff) skills that are transferrable to careers in academia, industry or policy, benefitting themselves and the economy. They will benefit immediately after the grant by moving to new employment.

PUBLIC
Besides the general benefits of providing dialogue with researchers and higher education outreach, talking about this project will help show that scientists are working to solve AMR and chronic infection - problems that touch many people's lives. It will also show that scientists are actively researching alternatives to animal experiments. During the grant, we will take part in various activities as outlined in the Pathways to Impact, including working in science centres/museums, science cafes and schools. We will engage with national news media by working with MRC/university press officers and using contacts from my previous media experience (e.g. work with BBC Radio 4). We will specifically engage the cystic fibrosis community by liaising with CFUnite (cfunite.org)

Publications

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Brockhurst MA (2019) Assessing evolutionary risks of resistance for new antimicrobial therapies. in Nature ecology & evolution

 
Description Led development of new module for MSc in Medical Biotechnology and Business Management. Topic: discovery and translation of treatments for infectious disease.
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
 
Title Ex vivo lung model 
Description In line with the aims of the grant, we are optimising and sharing our ex vivo lung model, the first version of which was published in 2014. The current grant is allowing significant improvements and flexible re-optimisation of the model to make it more useful and tractable for colleagues, especially those in industry. At this stage, please refer to the sections of Collaborations & Partnerships and Other Outputs for details of ongoing uptake and use, and the dedicated website at https://warwick.ac.uk/fac/sci/lifesci/people/fharrison/exvivolung 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact At this stage, please refer to the sections of Collaborations & Partnerships and Other Outputs for details of ongoing uptake and use. We will continually monitor uptake of the model through the lifetime of the grant and beyond, an in particular record any concomitant reduction in animal usage by users of the model, and report on this at a later stage. 
URL https://warwick.ac.uk/fac/sci/lifesci/people/fharrison/exvivolung
 
Description Dr Jo Purves / Dr Julie Morrissey 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution Discussion of transferrability of our ex vivo lung infection model to the study of chronic obstructive pulmonary disease, training of Dr Joanna Purves & Dr Julie Morrissey in use of the model, provision of written support for Dr Purves's successful application for fellowship funding from the Wellcome Institutional Strategic Support Fund.
Collaborator Contribution Knowledge transfer on adaptability of the model for future use by either party.
Impact None as yet.
Start Year 2018
 
Description Perfectus Biomed Ltd 
Organisation Perfectus Biomed Ltd.
PI Contribution Following an initial meeting with the CEO of Perfectus, Dr Samantha Westgate, at a Warwick-organised conference, we initiated a collaboration with the aim of developing a certified version of our ex vivo lung infection model which could be used for R&D testing of novel antibacterial/antibiofilm drugs/adjuvants by clients of Perfectus. I first discussed this with Dr Westgate before winnign the MRC NIRG, but it is only since the NIRG started that we have been able to move beyond discussions and actually build a functioning collaboration. We have contributed background IP, consultancy time by F Harrison, training of two Perfectus scientists in setting up the model and intellectual input into two applications for translational, collaborative funding. We applied for an Innovate UK grant which unfortunately was not successful (but which will be submitted), and a proof of concept grant from the National Biofilms Innovation Centre (successful details given below). We are sharing data generated by each partner during the NBIC grant.
Collaborator Contribution Dr Westgate has provided expert industry-led consultancy on the translational capacity of our model, the market need and the steps needed to move towards an accredited model for use in commercial R&D testing. Contributions include meeting time, writing funding applications and conducting some pilot experimental work at no cost to us. The time contributed by Dr Westgate and senior scientists at Perfectus represented an appreciable in-kind contribution, which I have costed above as equivalent to the FEC of one senior scientist working full-time for one week plus associated consumables costs.
Impact Proof of Concept Grant from NBIC: Biofilm Fluorescent Antibiotics AssaY. Total award £17,233 January-June 2019. Not multidisciplinary but is an academic-industry collaboration.
Start Year 2017
 
Description Curating Australian Society for Microbiology twitter feed 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The project post-doc, Dr Marwa Hassan, is a society ambassador for the Australian Microbiology Society and curated their twitter account for two weeks. This involved tweeting interesting microbiology research articles and discussing them with followers, promoting the society's work and events, and general discussions with followers related to microbiology and research,
Year(s) Of Engagement Activity 2019
URL https://twitter.com/aussocmic
 
Description IAS Christmas Lectures Zone 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact I was invited to take part in the special Christmas Lectures-themed iteration of "I'm a Scientist, get Me out of Here!" This is an online event in which schools and colleges may sign up for their students to take part in 30-minute live chats with scientists. I took part in ten live online chats with pupils ranging from primary school age to A-level students, from around the UK and two international schools. I talked the students and answered their questions on topics related to bacterial infections, antibiotic resistance and the microbiome. I estimate that I interacted with almost 100 students during these Q&A sessions. Formal impact feedback form the vent has not yet been supplied, but past events have consistently reported that students felt more engaged with science, more confident in their science lessons and that STEM careers could be accessible to them.
Year(s) Of Engagement Activity 2019
URL https://identity.imascientist.org.uk
 
Description Microbiology Society ECM Forum roadshow, Birmingham, UK, 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact This was a forum for postgraduate and post-doctoral members of the Microbiology Society to network with each other and with members further on in their careers. I was asked to talk about my career path and any problems I had encountered along the way, and to engage in Q&A with ECM Forum members to discuss academic career development and make links for informal mentoring. I spoke to ECM forum members informally that evening and via email afterwards, one later came to visit me in my lab to talk about career plans and potential fellowship applications. Dr Maria Fernandes (Professional Development Manager for the Microbiology Society) gave feedback based on participants' comments that my session was "particularly well received."
Year(s) Of Engagement Activity 2017
URL https://microbiologysociety.org/about/early-career-microbiologists-forum.html
 
Description Skeptics in the Pub, Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I was invited to give a talk about my lab's MRC-funded research into ex vivo models of chronic lung infection for the Oxford branch of Skeptics in the Pub. I focussed on translational science (antibiotic susceptibility testing) and impact on reducing the use of animals in infection research. There was an extensive and conversational Q&A with the audience, covering topics ranging from fundamental microbiology and AMR through to 3Rs concerns and the ethics of research.
Year(s) Of Engagement Activity 2018
URL http://oxford.skepticsinthepub.org/Event.aspx/16582/From-butchers-block-to-biology-lab-using-offal-t...
 
Description Warwick Science Gala 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The project post-doc, Dr Marwa Hassan, led an activity at the University annual family-friendly Science Gala in which local children and teenagers (from primary school age to pre-University) talked about antimicrobial resistance and spread of infection. To demonstrate good hygiene and infection control the attendees could take part in "the sneeze zone" in which they sprayed drawings of their friends and family with aerosolised food dye to demonstrate microbial transmission and the power of simple preventive approaches like using a tissue!
Year(s) Of Engagement Activity 2019
URL https://warwick.ac.uk/fac/cross_fac/xmas/impact/xmas_scientist_experienceb/science_gala_2019/
 
Description Work experience for R Woodford 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I hosted a work experience placement for an adult aiming for career re-entry into science. My entire lab team helped to host the visitor and let him shadow their work, as did Dr John Moat from the School's antimicrobial testing facility. This experience confirmed the person's resolve to retrain and re-enter a STEM career.
Year(s) Of Engagement Activity 2018