Detecting Antibiotic Resistance Proteins in Clinical Samples Using Proteomics

Lead Research Organisation: University of Bristol
Department Name: Cellular and Molecular Medicine

Abstract

Approximately 40,000 people die in the UK every year as a result of Sepsis, which is a medical condition usually triggered by the body's reaction to bacteria in the blood. When bacteria are present in the blood this is called bacteraemia. The bacteria can come from all sorts of places and can be of many different species. So a diagnosis of Sepsis doesn't tell a doctor what bacterium is responsible. Antibiotics kill bacteria, and so antibiotic therapy is absolutely critical to treating Sepsis. Without removing the underlying cause - the bacteraemia - treatment of Sepsis is unlikely to succeed. The dilemma that doctors face is that because they don't know the identity of the bacterium they want to kill, they are not certain what antibiotics to use. The rise of antibiotic resistance in bacteria makes this choice even more difficult. One way of dealing with this is to use "empiric therapy": to try a particular antibiotic, wait to see if the patient improves and if they don't, try another. But in the meantime, the patient may be getting more and more ill. The alternative approach is that the doctor may start treatment with the latest, most broad acting antibiotic they can find to give them the best chance of killing the bacteria. This means that this "last resort" drug might have been used when it wasn't really needed. Inappropriate use of a last resort drug is the primary driver for antibiotic resistance and will inevitably shorten its useful life.

What we really need is to give doctors information about the identity of the bacterium infecting a patient's blood and, more importantly, what antibiotics it is susceptible to. Then they can make informed antibiotic prescribing choices. At the moment, from the time a blood sample is taken from a patient where bacteraemia is suspected it can take 48 hours just to prove there are any bacteria present. Using new MALDI-TOF machines it is possible to identify the bacterium a few hours later, but that doesn't tell you anything about antibiotic susceptibility. It may take another 24 h to find out what antibiotics can be used. This means that patients can be on the wrong antibiotic for up to 72 hours. If that's a non-effective antibiotic, the patient's life is in danger, if it is an inappropriately used last resort antibiotic, the antibiotic's useful life is being shortened.

Everyone agrees that reducing the time it takes to get antibiotic susceptibility data to doctors is the key, not just for the treatment of patients, but also to better protect our dwindling supply of useful antibiotics. We feel that it may be possible to achieve this by identifying antibiotic resistance proteins - the tools bacteria employ to resist antibiotics - directly in bacteria isolated from patients' blood. If a particular resistance protein is present, the doctor would know not to use a particular drug. To test our hypothesis we want to test whether we can identify resistance proteins in bacteria in blood samples that have been cultured and processed exactly as they would be in hospital diagnostic labs. We will find out whether it is possible to use existing MALDI-TOF machines to identify at least some antibiotic resistance proteins 24 h earlier than is currently the case. We will also test whether it is possible to use more specialised LC-MS/MS machines to reduce the time to get antibiotic sensitivity data by up to 60 hours, giving a positive indication of antibiotic susceptibility about 12-15 h after sampling. It is not necessary to provide a diagnostic test that works minutes after sampling to have real clinical benefit. For severe Sepsis, each hour without working antibiotics gives a 6% increase in patient mortality, so even shaving tens of hours off the current minimum time it takes to predict antibiotic susceptibility would transform patient care.

Technical Summary

Currently it takes 18-48 h to confirm clinical bacteraemia and 2-3 h to identify the bacterium responsible using MALDI-TOF after processing the blood culture. Determining antibiotic susceptibility requires additional plating techniques, taking another 24 h. Using MALDI-TOF to identify antimicrobial drug resistance (AMR) proteins would allow clinicians to exclude certain antibiotics up to 24 h earlier than they can now, benefiting patients and prudent antibiotic use. Preliminary data lead us to hypothesise that AMR proteins are highly abundant, and so may be identifiable using the MALDI-TOF machines currently available in diagnostic microbiology labs. Objective 1 is to move towards MALDI-TOF identification of AMR proteins in culture positive blood samples. We shall (1A) create a set of transformants of key pathogens expressing key AMR genes at levels typical of clinical strains. (1B) Quantify AMR proteins in these transformants relative to ribosomal proteins using LC-MS/MS. (1C) Identify unique MALDI-TOF peaks representing these AMR proteins using protocols minimally adapted from those currently used in diagnostic microbiology labs to identify bacteria. (1D) Test whether the MALDI-TOF methodology developed in 1C can predict antibiotic susceptibility in 50 well characterised clinical isolates. (1E) Test whether antibiotic susceptibility can be predicted in 50 processed culture positive blood samples from a clinical diagnostic lab. Objective 2 is to move towards the use of LC-MS/MS to identify AMR proteins in blood cultures. This would allow a wider range of possible AMR proteins to be identified than MALDI-TOF, and it might be performed on blood cultures before they currently flag positive. To do this we shall (2) test whether AMR proteins can be identified using LC-MS/MS in 10 bloodstream isolates following growth in human blood for 12 h (i.e. less than the expected time of culture positivity) with starting inocula representative of clinical bacteraemia.

Planned Impact

In addition to the Academic Beneficiaries, outlined separately, we have identified the following groups who will benefit from the research and in the following ways.

The UK Population
Sepsis is one of the most common life-threatening medical conditions seen in the UK. Around 100,000 patients suffer from this condition each year. Many will die - around 40,000 people per year - but many more will recover and the rate and extent of their recovery will affect their futures. There has recently been significant publicity of this problem, and the advocacy group the UK Sepsis Trust, have publicised the "Sepsis Six" steps to treatment. The research project presented in this application will address steps 2 and 3 (take blood cultures, give broad spectrum antibiotics). The use of even a broad spectrum antibiotic may be inappropriate if the target organism is resistant, but it is not possible for a clinician to know this without antibiotic susceptibility data. Successful completion of this project will pave the way to use proteomics to shorten (by tens of hours) the time it takes to obtain antibiotic susceptibility data for blood stream isolates. Given the estimation that one hour without a working antibiotic increases mortality by 6% for severe sepsis, an advance such as this will potentially save hundreds of lives per year. Moreover, it will reduce time to cure, which will reduce morbidity and long term complications. All of which will directly benefit the UK population.

The UK Health Service and Wider Society
According to NHS estimates (http://www.england.nhs.uk/wp-content/uploads/2013/12/spesis-brief.pdf) the cost to the NHS of acute sepsis care is around £2.5 billion per year. The more seriously ill a patient gets and the longer they take to get better, the greater the cost. The more long term complications they have - a result of prolonged and more serious disease - the more long term cost there will be for outpatient and GP care, social care, and potential reductions in the working lifetime of the patient. Any intervention that will reduce the time it takes to prescribe a working antibiotic will shorten time to cure, reduce disease severity and will ultimately cost society less money. The step changes that might result from completion of this project could considerably reduce the cost of Sepsis to society.

Industry/UK Economic Activity
Sepsis is a global problem and the UK population is not unusually burdened by it. If the UK government, through its research councils, spearheads research such as this, then there is potential for the UK to directly benefit economically from the research, development and sale of patented diagnostic equipment and test kits to healthcare systems from other countries. There is also potential for job creation in the UK and economic flow from Europe, since at least one European company that currently makes diagnostic microbiology proteomics equipment (Bruker) already has R&D facilities in the UK.

Protection of the Global Antibiotic Resources
Antimicrobial drug resistance is a massive global problem, and a real threat to humanity. Not just because of its impact on the ability to treat infectious diseases in humans, but also because of its impact on farming. There is a dearth of new antibiotics in development, and recent initiatives to stimulate this will take many years to come to fruition. In the meantime, we need to protect our existing supply of antibiotics as a global resource. This means using narrow spectrum drugs when they will suffice to protect broad spectrum, last resort drugs from the inevitable rise of resistance that comes from their inappropriate use. Obtaining reliable antibiotic susceptibility data is the keystone to appropriate antibiotic prescribing. Completion of this project will provide a means to obtaining susceptibility data sooner, allowing a switch to appropriate therapy earlier than is currently possible. This will protect existing antibiotics.

Publications

10 25 50

 
Description Improving empiric antibiotic prescribing by applying a Bayesian decision theory approach to phenotypic and genomic resistance data.
Amount £223,521 (GBP)
Funding ID MR/T005408/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2020 
End 12/2022
 
Description AMR talk at Monkton Coombe School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Matthew Avison visited Monkton Coombe School to give a talk on AMR entitled 'Superbugs: Is Resistance Futile?' Pupils came up with imaginative questions and the school reported increased interest in the subject within the school population.
Year(s) Of Engagement Activity 2017
 
Description Access to Bristol schools event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact About 50 motivated college students came to Bristol from the region to get a taster for microbiology. Matthew gave a talked entitled Superbugs: Is Resistance Futile? Some students reported it had given them an interest in going into the field of microbiology.
Year(s) Of Engagement Activity 2016
 
Description American and Canadian High School visit to UoB 
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 Pupils from American and Canadian High Schools attended a day at UoB. Matthew gave a talk entitled Superbugs: Is Resistance Futile? Pupils had many challenging questions and some said afterwards that they had no idea you could select for an antibiotic resistance gene by using another antimicrobial like ammonium compounds.
Year(s) Of Engagement Activity 2016
 
Description BBC Radio Bristol Interview 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Matthew Avison had 30 minute interview with Steve Yabsley on BBC Radio Bristol about AMR research project. Stimulated discussion via phone in comment from members of the public
Year(s) Of Engagement Activity 2016
URL http://www.bbc.co.uk/programmes/p04dxl57
 
Description BridgeBridge AMR Schools Outreach Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 160 students from the region came to a cross-faculty schools conference recently for post-16 biology and chemistry students held within the School of Chemistry to examine why AMR is an escalating global threat. Matthew opened the conference and gave a talk entitled 'Superbugs: Is Resistance Futile?'
Year(s) Of Engagement Activity 2016
URL http://www.bristol.ac.uk/news/2016/december/schools-conference-amr.html
 
Description Bristol Grammar School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Matthew Avison gave a talk entitled 'Superbugs: Is Resistance Futile?' to Bristol Grammar Senior School pupils. Pupils gave feedback such as "I used to think antibiotics were normal when you have a bad cold but I now know it's more likely to be a virus so will only use them if I have to."
Year(s) Of Engagement Activity 2017
 
Description Bristol Health Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Matthew gave a talk to over 100 members of the general public during a session called Bristol Love Bugs. His talk was about the Impact of Antibiotic Use on the Microbiome.
Year(s) Of Engagement Activity 2016
URL http://www.bristolhealthpartners.org.uk/events/view/2016/10/22/bristol-loves-bugs-the-human-microbio...
 
Description Bristol Tackles Global Challenges World Café 
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 Matthew Avison and Kristen Reyher gave presentations on their work and discussed potential areas of collaboration in the future with researchers from the UK as well as abroad, including many delegates from Thailand. Many interesting ideas came from this week of talks and workshops.
Year(s) Of Engagement Activity 2017
URL http://www.bristol.ac.uk/bristolbridge/events/2017/bristol-tackles-global-challenges.html
 
Description Cheltenham Ladies' College - talk on AMR 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Matthew Avison gave a talk called "Superbugs: Is Resistance Futile?" to 90 students at Cheltenham Ladies College. This talk provoked questions and debate about AMR and Matthew was also approached by students afterwards with further queries.
Year(s) Of Engagement Activity 2017
 
Description Filton College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Matthew gave a talk entitled 'Superbugs: Is Resistance Futile?'. Response was good and some students asked about careers in microbiology.
Year(s) Of Engagement Activity 2016
 
Description Gave talk at Sidmouth Science Festival 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact About 100 members of the general public attended my talk, "Superbugs: Is Resistance Futile?". Feedback included surprise at "cross resistance caused by biocides," that "antibiotics occur in soils" and "how long E.coli can live in the body". The audience reported changed views such as "I used to think basically that the more antibiotics you used, the healthier and better prepared you were. Now I'll be more cautious."
Year(s) Of Engagement Activity 2016
 
Description Langford Vets Antibiotic Awareness Evening 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact CPD evening for vets, although was also attended by a medical microbiologist and a medical pharmacist. There was much discussion between the speakers and audience, and feedback included: "As a medical microbiologist working in human health in a local hospital, I found it refreshing and very interesting to see the extent, commitment and drive to work to combat antimicrobial resistance in the animal sector - really for the greater good of population health." One vet enquired about consultancy for their practice.
Year(s) Of Engagement Activity 2016
 
Description Presentation to Chief Scientific Advisor Chris Whitty 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Professor Chris Whitty, Chief Scientific Advisor for the Department of Health visited University of Bristol to hear of our work and latest findings. Matthew Avison and Ashley Bryce gave presentations, and Professor Whitty sent feedback later in the week saying he'd taken our findings on board and had found the visit useful.
Year(s) Of Engagement Activity 2017
 
Description Sheldon School - AMR talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Matthew Avison gave a talk entitled 'Superbugs: Is Resistance Futile?' to 80 secondary school pupils. Students asked questions about antibiotics and resistant bacteria and the school sent good feedback about the talk.
Year(s) Of Engagement Activity 2017
 
Description St Lawrence School talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Matthew gave a talk entitled 'Superbugs: Is Resistance Futile?' There were many interesting questions from pupils and teachers reported the kids felt they had learnt a lot.
Year(s) Of Engagement Activity 2016
 
Description Surgeon X Comic 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Matthew Avison was asked to advise and contribute to the Surgeon X comic (Wellcome Trust Funded), which imagines a world beyond the antibiotic age, which was published via Wowbagger Productions. The comic is sold in print form and via an app. There has been significant international impact.
Year(s) Of Engagement Activity 2016
URL http://surgeonx.co.uk/our-team/
 
Description Swindon College AMR talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Matthew Avison gave a talk entitled 'Superbugs: Is Resistance Futile?' This was to inspire a new generation of microbiologists and raise awareness of AMR. Students fed back change in perceptions and increased interest in microbiology.
Year(s) Of Engagement Activity 2017
 
Description Talk at Warwick SWON Alliance Industry day. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Talk at the SWAN industry day to industry and academic representatives. Significant interest generated in methods for determining antibiotic action via proteomics
Year(s) Of Engagement Activity 2016
URL https://www2.warwick.ac.uk/fac/cross_fac/wamic/swon/industry/
 
Description Winterbourne International Academy - school talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Parents and pupils of Winterbourne Academy attended their Festival of Science lectures, one of which was a talk by Matthew entitled "No more drugs for superbugs"
Year(s) Of Engagement Activity 2016
URL http://www.myyate.co.uk/yate/e/18972/festival-of-science-lectures