Team:Sheffield/Human Practices


Different Applications of our Device



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). These identify any drug resistance in pathogens and ensure effective drugs are chosen for treatments (5).





Phase I: Learning from Microbiology labs


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.

Vitek 2 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 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.




Phase I: Integration of feedback


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 cells 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.


The next step will be to test our device for its ability to distinguish between an antibiotic sensitive and resistant strain, and ensure our results are comparable to the laboratory plate reader.





Phase II: Taking our device to healthcare professionals


We then took our Antimicrobial Susceptibility Test prototype to professionals working in clinics and diagnostic settings, the markets we imagine our device entering. We interviewed them about 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 microcalorimetric methods. This idea comes from research by Chang-Li et al. 1988, who showed that growing bacteria leave a very characteristic thermal finger print. In their study Chang-Li et al. were able to distinguish among Escherichia coli, Staphylococcus aureus, Salmonella typhosa, Salmonella chaleraeuis and Shigella flexneri using micro temperature sensors. Given eenough time, we would like to try and employ similar technology in our device. However this technology is largely eexperimental and we may therefore need to explore other options.


They also suggested we create a way to make our device more tailored to certain types of pathogen. To do this, we would mimic the Vitek 2 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 Vitek 2 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 of 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 designed what this software might look like in figure 5.



PreventX were also concerned with patient confidentiality, as our device would be sending patient data over the cloud. To combat this issue, we spoke to various members of research ethics staff at the University of Sheffield, and decided that the best course of action would be to design our own encryption software.

The software uses a discrete mathematical model (based on cellular automata) similar to conway's game of life to generate a string of pseudo random numbers. It uses a JPEG image as an input, then performs image processing to convert it to a binary array and then performs the encryption algorithms on this data.This is then applied to data like a shift cypher and sent to the server. To decrypt the data the shift cypher is removed and the original data is obtained.

They also advised on best practices such as assigning patients anonymous identifier and then not sending information linking identifiers to their actual name through the same medium.




A Teaching Tool




In Universities


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 of 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 a sample containing Bradford reagent at 500nm.

Problems with current cuvette spectrophotometers :

  • 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 enzymatic activity during 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 cuvette spectrophotometers :

  • Manual data collection
  • Overnight readings cannot be taken

How our device can solve it:

  • Automatic data collection
  • Allows overnight results collection

In High/Secondary Schools


Our team also explored the possible applications of our device in teaching labs at high/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.




A Research Tool


We began our project with an idea for a device that would allow for cheap and quick detection of microbial growth. As our engineers worked on the device the team of biologists started by testing out several concepts that we had in mind. We wanted to see what is the minimal concentration of microbiological sample to be able to say with high confidence that we are seeing growth or lack thereof. We decided to use a microplate reader to start our experiments. We used the only device that was available to us - VICTOR X Multilabel Plate Reader. Quite soon after starting our first experiments we found several obstacles on our way. The experimental regime looked as follows:


1- Microtiter plate preparation.

2- Taking first measurement.

3- Incubating microtiter plate at 37°C on a shaker.

4- Taking measurements every hour.


Our experience when using Viktor: Due to the nature of our experiment we had to take microtiter plate out of incubator every hour, and take the measurement. This resulted in microtiter plate not being incubated for fifteen minutes during each hour, which definitely had a negative impact on the timeline of our experiment. What is more, we couldn’t take more than seven measurements due to working hours restrictions. This resulted in us not being able to determine how growth curves look like for lower dilutions of microbiological samples.


How would our experience look like using BrightBiotics: The frustration arising from described obstacles led us on to idea of what would the features of a perfect microplate reader be. We started creating a device that would allow us to take measurements continuously and remotely, without a need to move microtiter plates. Using our device we could set up an overnight experiment and collect all the data in the morning which would had a positive effect on our laboratory workflow, costs and data quality. We firmly believe that device like our would be an exceptional research tool for all scientists that are interested in continuous, uninterrupted and remote workflow.




Outreach

Mini-iGEM competition with high school students


Inspired by the 2016 Sheffield iGEM teams manual for organizing miniaturized iGEM competitions, we decided to hold our own outreach event which was aimed at introducing the topic of synthetic biology among 17-18-year-olds. We wanted to give these individuals the chance to use synthetic biology to propose solutions to real-world problems.





We organized an outreach event with the aim of raising awareness of 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 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.




References:

http://www.nature.com/articles/srep23375
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3625966/
https://drive.google.com/drive/folders/0Bz2Fq_I7H9J5aV80c2FQOGl1djg
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378521/
https://academic.oup.com/cid/article/49/11/1749/344384/Antimicrobial-Susceptibility-Testing-A-Review-of
http://www.biomerieux-usa.com/clinical/vitek-2-healthcare
https://procure.ohio.gov/PriceList/800014pricelist.pdf