Team:Sheffield/Human Practices
BrightBiotics as a Diagnostic Tool
The Global Antimicrobial Resistance Issue
When choosing an issue to tackle with our diagnostic hardware, we decided to focus on the growing issue of antimicrobial resistance (AMR). AMR infections are thought to claim as many as 700,000 lives per year, a figure that is expected to rise to 10 million by 2050 if current trends continue (Figure 1) (1). The World Health Organisation (WHO) has therefore described AMR as one of the major concerns of modern healthcare (2), and it is critical that we take action now.
The use of antibiotics is the leading cause of antimicrobial resistance around the world, and in 50% of cases, antibiotic treatment is either not needed or not optimally effective for the infection (3). Doses of antibiotic that are not lethal to bacteria leads to the development of antibiotic resistance by supporting genetic change and acting as a selection pressure (4) (Figure 2). Therefore, one way clinicians can combat AMR is to conduct antimicrobial susceptibility tests (ASTs), to identify any drug resistance in pathogens and ensure effective drugs are chosen for treatments (5).
Advice from Microbiologist Consultants
To gain a better understanding of what ASTs are most commonly used in the clinic, we spoke to microbiologist consultants in Poland and Cyprus. They informed us of the Vitek 2 System, that is commonly used for ASTs.
Vitek2 is a highly automated system, in which a bacterial suspension, grown from a patient sample, is distributed into compact cards containing antibiotics (figure 3). A sensitive optical density system then measures changes in bacterial growth in the presence of the antibiotics in the cards. If there is no bacterial growth, then the organism is sensitive to the drug and it would therefore be an appropriate treatment option.
However, the Vitek 2 system is hugely expensive, costing close to $100,000 (7). The clinicians in Poland and Cyprus expressed to us that this cost was the major drawback of Vitek 2, and means that often smaller hospitals and clinics are unable to afford this state of the art machinery, and therefore miss out on the ASTs that could be crucial to prevent the spread of AMR.
Repurposing the BrightBiotics system as an affordable, automated AST device
Following our research on the Vitek 2 system, we noticed that our device could have many of the same features. It detects bacterial growth as a measure of optical density, and so may therefore detect changes in growth at rates similar to Vitek 2. In addition, we could emulate the Vitek 2 cards by putting antibiotics in the well plate, and seeing how various antibiotics affect growth of a bacterial inoculum.
Firstly, we made sure we could detect differences in growth of antibiotic resistant and sensitive strains in the presence of the appropriate antibiotic, as Vitek 2 can. We engineered antibiotic resistant strains by transforming with biobricks encoding chloramphenicol, kanamycin, carbenicillin and ampicillin resistance. Then used a laboratory automated plate reader to compare their growth with that of of antibiotic sensitive strains, in the presence of antibiotics. We found that with the laboratory plate reader we could see a difference within just a few hours at bacterial concentrations as low as 10^3 CFU/ml (equivalent to 1x10-3 OD) (figure 4). This showed us that it would be possible to differentiate between antibiotic sensitive and insensitive strains, in a time frame that could be clinically relevant.
Next, we confirmed that our device could also differentiate between antibiotic resistant and sensitive strains. …. NEW GROWTH CURVE
Integrated Human Practices to Design an Ideal Device
We then took our Antimicrobial Susceptibility Test prototype to professionals working in clinics and diagnostic settings, the markets we see our device entering. We interviewed them for feedback on our prototype device, and asked for constructive feedback. We have attempted to incorporate as much of their feedback as possible into our current device design:
The Royal Hallamshire Hospital in Sheffield, England
We spoke to senior nurses on the wards of a major hospital in Sheffield. They were enthusiastic about the cost of our device, as it is significantly cheaper than the single use tests they currently use for infections such as MRSA screening. However, they felt that the ~8 hour test time of our device would slow down patient flow on wards. Instead, they suggested we contact diagnostic labs directly, whom the hospital uses to diagnose severe patients staying overnight on the wards.
Liverpool Diagnostics in Liverpool, England
We then turned to a major diagnostic lab in the North of the UK. They highlighted to us the importance of identifying the pathogenic organism, as this will be important in patient care, and also in choosing which antibiotics to test against the organism. For bacterial identification, we investigated several mechanisms such as….**PIOTR BACTERIAL IDENTIFICATION STUFF**
They also suggested we create a way to make our device more tailored to certain types of pathogen. To do this, we will mimic the Vitek2 cards, and design well plates, pre-filled with antibiotic appropriate to a certain group of antibiotics. This will increase the autonomy and ease of use of our device, and as 96 well plates are already a standard consumable, they should be much cheaper to produce than the custom Vitek2 cards.
PreventX Integrated Diagnostics, Sheffield, England
PreventX is a major diagnostics lab based in Sheffield. They told us it was important that our device had an integrated software that can analyse the sample and produce a definitive, easy to read outcome. To do this, we would implement machine learning software, and use a large amount bacterial growth data to ‘teach’ our software difference between ‘growth’ and ‘no growth’, i.e. sensitivity or resistance to the drug tested. It would then provide the user with an easily interpreted result. We have imagined what this software might look like in figure 5.
Teaching
University
We met with Dr. Melanie Stapleton, Research Technician at University of Sheffield, Undergraduate Teaching Labs to talk about the possible applications of our device in teaching. She provided us with examples experiments for which our device could be used by undergraduates such as protein assays, enzyme assays and bacterial growth experiments.
Bradford Protein Assays
The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. Our device will be used to measure the absorbance of the sample containing Bradford reagent at 500nm.
Problems with current equipment:
- Requires the use of many cuvettes
- Requires performance of multiple experiments
How our device can solve it:
- Pipetting time reduced
- Multiple samples can be run at the same time
- Results generated immediately
Enzyme Assays
Our device could be used to measure enzyme activity in enzyme oxidation/reduction reactions. NADH is often oxidized to NAD+ in these reactions. Since NADH absorbs light at 340 nm, our device could measure the change in absorbance of 340 nm light, as a result indicating a change in the amount of NADH.
Problems with current equipment:
- Manual data collection
- Overnight readings cannot be taken
How our device can solve it:
- Automatic data collection
- Allows overnight results collection
High Schools/ Secondary Schools
Our team also explored the possible applications of our device in teaching labs at high schools/ secondary schools. Possible experiments include measuring the rate of enzyme reactions.
Propanone and iodine reaction
Our device could be used to measure the rate of a reaction that involves iodine in a solution of propanone. The solution of iodine in propanone starts off brown, and then fades through orange to yellow to colourless as the iodine is used up and the change in absorbance can be read by our device.
Research
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Cras euismod, quam eu imperdiet aliquam, felis ipsum eleifend est, volutpat malesuada lacus urna a quam. Nam in egestas odio. Curabitur malesuada et ex elementum tempor. Aliquam hendrerit ultrices molestie. Curabitur sed nulla leo. Donec eget est ornare, porttitor nisi vitae, suscipit arcu. Vivamus quis nisi sit amet purus blandit convallis ut ut risus. Mauris magna ex, volutpat sed varius id, semper at urna. Nunc libero justo, lacinia nec volutpat id, euismod eget mi. Nulla facilisi. Phasellus pharetra vulputate leo, eu sodales ante placerat eu. Cras nec fermentum mi, ut sagittis erat. Aenean fringilla magna nec hendrerit accumsan. Nulla non nisi vitae mi lacinia dapibus ut at augue. In at lorem quis nunc sodales luctus vitae vitae ante. Vivamus mattis, nibh sed consequat vestibulum, mi ante imperdiet lorem, dictum condimentum purus lectus quis metus. Aliquam feugiat porta turpis ut vulputate. Sed cursus luctus posuere. Vestibulum vel lorem sollicitudin, ultricies nisl fermentum, semper lacus. Mauris ut semper neque. Nunc ullamcorper scelerisque massa, sed vehicula tortor porttitor vel. Donec quis arcu dui. Curabitur vel laoreet erat. Interdum et malesuada fames ac ante ipsum primis in faucibus. Aliquam mauris magna, feugiat vel lorem sagittis, gravida.
Outreach
Mini-iGEM competition with high school students
We organized an outreach event with the aim of raising awareness about synthetic biology among A-Level students from schools in Sheffield. The day started off with introductory lectures about synthetic biology from Dr. Egbert Hoiczyk, senior lecturer at the University of Sheffield. We then organized a mini iGEM competition where students were divided into teams and had to brainstorm and build their own synthetic biology iGEM device. Following this, teams delivered a presentation about their device to a panel of judges (our advisors). These creative students produced exciting and original solutions to tackle issues such as hemophilia, breast cancer, and plastic recycling, using the power of synthetic biology.
