Team:UiOslo Norway/Description

Background

The UiOslo team of 2016 started to assemble in early February. By then we were four team members, and we started the long and time-consuming process of choosing an idea for our project. Medicine and health was themes that we were all concerned with, and several of the team members are doing their master thesis in that area as well. We all agreed that public health is important and engaging. We searched through the iGEM library for inspiration, and there were many good ideas being thrown around. We soon narrowed it down to doing something concerning antibiotic resistance, one of the biggest, global health problems that exist today. After discussing with our supervisors, we settled the issue; our project was to be about antibiotic resistance. For the molecular biologists in the group, this was a well-known topic; antibiotic resistance is a topic addressed early on in the bachelor program at UiO. It is also a common tool in research, especially in synthetic biology.

Investigating todays problems with antibiotic resistance
We wanted to get more insight of this problem and look into how a small group of seven students could contribute in solving, or at least improving the antibiotic resistance situation both in Norway and globally. Our supervisors got us in contact with the Tone Tønjum, the head of the microbiology department at Rikshospitalet (a part of Oslo University Hospital). Tone Tønjum is also a professor and has a research group of her own. The whole team met with her and she showed us around in her lab and gave us some insights into the current situation of antibiotic resistance in Norway.

Today’s detection methods
Following this meeting, there was one particular aspect of the problem that stood out to us; diagnostics. According to Tone, there are several issues with the diagnostic methods that are currently being employed to identify complex antibiotic resistance, the main ones being that they are too slow and/or expensive for regular use.

Usually, a patient sample is grown on agar plates that may contain 12 different antibiotics zones. Following plating, the colonies need time to grow, usually 24 hours, and then one can analyze the growth pattern to see what kind of antibiotics the bacteria are able to endure. This is called the disk diffusion method.

There are also rapid methods, Tone explained, but they all have one huge limitation in common; they rely on expensive machines that usually require a certain level of expertise to operate. PCR is one such method. PCR may be used for genotypic methods, to identify the presence of the DNA sequence conveying antibiotic resistance. One drawback here is that the sequence, or at least parts of it, must be known, and even though a resistance gene is present, there are differences in expression level and modes that may complicate the treatment of the infection. Because of convenience, efficiency and cost the disk diffusion method is possibly the most widely used method in the world. Even though this is a convenient method to test for antibiotic resistance, it is time consuming and generates problematic antibiotic waste. What Tone meant the marked and health institutions needs today is a fast, easy and cheap test to detect antibiotic resistance, something that could be readily used in all health institutions.

Extended spectrum beta lactamase
We also discussed what kind of infections that are of a high prevalence, and what kind of bacteria one should be concerned about. Tone talked about different resistant bacteria, and there was one type that particularly concerned Tone. The bacteria in question produce an enzyme called extended spectrum β – lactamase. This enzyme has the potential to inactivate all beta-lactam antibiotics, which is a group including a high percentage of the antibiotics that are most commonly used today. She talked about development and spread of these bacteria over the last couple of years, and it was clear that its prevalence increased both in Norway and globally. ESBL producing bacteria was a new topic to us, so we started doing some serious researching.

We soon understood that we needed more expertise in this area, so we contacted our team member Camilla’s previous professor Ole Andreas Løchen Økstad. He works at the department of pharmaceutical biosciences, and has a lot of experience in working with pathogenic bacteria. He kindly agreed to meet with us. Regarding the ESBL issue, he also seemed to think that this was an important topic, and that a solution to this was indeed a necessity. He gave us a lot of tips and provided us with several contacts with expertise within the field of ESBL-producing bacterial infections.

Urinary tract infections
To start our project, we had to settle on some sort of biological sample to focus on. There were many candidates, including blood, urine, saliva and so on. We discussed this topic at length and eventually decided to focus on urine samples. There were many reasons for this. Urine is easy to work with, the samples can be easily collected, and the nature of the sample is relatively consistent, as opposed to for example saliva, where the sample depends on how much/little saliva or mucus the patient is able to cough up.

We did a search in the prescription registry in Norway which is an online database containing an overview of all medicinal prescriptions. Phenomethylpenicillin was the most prescribed antibiotic in Norway in 2014. Phenoxymethylpenicillin is a beta-lactam antibiotic and its actions is inhibited by ESBL producing bacteria. The decision of focusing on urine was followed by an extensive investigation of urinary tract infections (UTIs) and its occurrence both in Norway and in the rest of the world. All of this research was necessary to figure out whether or not a project focusing on detecting ESBL producing bacteria in UTIs was something the society could benefit from before making a final decision. We went through a lot of research material and managed to get a good overview of urinary tract infections. UTI in general is the second most common bacterial infection worldwide, surpassed only by respiratory tract infections. UTI is usually caused by bacteria derived from the colon, but can also be caused by several other kinds of bacteria. It affects both genders, but because of physiological differences, women are more exposed to such infections than men.

The diagnostic tool
Thus, it came to pass that the UiOslo iGEM team would work on developing a diagnostic test for ESBL producing bacteria in urinary tract infections. Several experts in the field claimed that this is of high necessity and importance for the modern health sector, and that such a test will contribute to reducing the use of beta-lactam antibiotics in situations where they are futile.

To set out for such a big task, we had a lot of work in front of us. One of the contacts Ole Andreas gave us, Ørjan Samuelsen, resides in Tromsø in the far North of Norway, works at the National Center of Expertise on Antibiotic Resistance and is also a professor at the University of Tromsø. He provided us with a lot of information on the ESBL situation in Norway, which they have researched for over a decade; he gave us a report clearly stating that the prevalence of ESBL in urinary tract infections had gradually increased over the years, especially from 2013 to 2014 where an exponential increase was seen (see graph below). He also provided us with different ESBL isolates they had collected from institutions from all over Norway, which later on became the basis for our biobricks and proved our final product to be of a huge clinical relevance.

We are very excited about our project, and we feel confident that our work may benefit both patients and health institutions all over the world. Our diagnostic test is rapid, efficient and cheap, and it does not require any special training or expertise. It interfaces with an app of our own design, which contains an information page with the official Norwegian guidelines regarding UTIs and is able to interpret the test results and generate useful data. This is a truly cross-disciplinary synthetic biology project, containing elements of 3D modeling and printing, informatics and classic molecular biology. We have taken advantage of our multidisciplinary background, and it is our belief that we have not only created a product that is sorely needed, but we have also increased the public knowledge and awareness of antibiotic resistance. We believe that with PhoneLab, we are truly a part of the solution of the global problem antibiotic resistance constitutes.

Description

This year’s iGEM team from UiO will address one of the increasing world problems and health treat, antibiotic resistance. For many years humans have evolved together with bacteria in an evolutionary race, for each new antibiotic produced, a simple bacteria will eventually be able to resist the medicine, survive the treatment and continue to grow.. Without functional antibiotics modern medicine will fall back many centuries. It is postulated that antibiotic resistance may become a greater threat to our health than cancer, and by 2050, a frightening number of 10 million people is estimated to die due to infections caused by antibiotic resistant bacteria.

In short: The way in which we address this important issue is by developing a fast, inexpensive and mobile system in which we can identify antibiotic resistant bacterial infections in urinary tract infections before antibiotics are prescribed. This allows for the proper administration of antibiotics in order to reduce antibiotic misuse (which would increase the likelihood of the development of antibiotic resistance) and to reduce patient discomfort by accurately prescribing effective clinical treatments. Our goal is to both develop a fast, easy-to-use diagnostic test and integrate it with a mobile phone- app. Thus, making the test available for medical personnel both in the field as well as the clinic.

Extended spectrum beta - lactamase: We have focused our diagnostic tool to be able to detect β - lactamase activity. This enzymes conveys resistant to many types of beta - lactam antibiotics by hydrolysing the beta lactam ring. This ring is our main focus in our detection test. We have chosen to focus on a specific β - lactamase family called extended spectrum β - lactamase. These bacteria are increasing in incidents throughout the world in the last decade.

Diagnostic test: We created a diagnostic test that detects ESBL producing bacteria in urinary tract infections. The biochemistry of this test was first established as a penta Well test by Mura et. al. (2015), which developed a colorimetric assay showing changes from yellow to red when a resistant bacterium is present. The color change is due to the cleavage of the β-lactam ring in Nitrocefin. A main advantage of this test is that with additional reagents, it can discriminate between several classes of β-lactamases.

Smartphone app: We have also incorporated the molecular-biology portions of the test with a 3D printed device and a smartphone app – PhoneLab. The app will read the color change generated from the hydrolysis of Nitrocefin with the smartphone camera, and will tell if resistant bacteria are present or not. Detection of the color change by this method is much more accurate than the human eye and the detection threshold is much lower, allowing for a more rapid result. Color changes are then compared with internal controls and cross-referenced with a database. This is information is then delivered to the doctor as a list of any antibiotic resistances found and which antibiotics not to give.

Future development: In the future, we envision to expand the capabilities of this system to a wider range of applications. Our plan for this is to use a simplified CRISPR/Cas9 system to link base-pairing to a detectable signal. The system will be comprised of a tracr/crRNA towards the dangerous resistance genes and a modified Cas9 protein. This will enable us to screen for resistance on the genetic level. Conceptually; the tracr/crRNA would be linked to a null-nuclease Cas9 that doesn´t cleave the target genes. Bound to the Cas9 is half of a detectable enzyme. By designing two, separate such constructs that recognize adjacent parts of the same gene, the two halves of a split-enzyme assay can be brought together and form a detectable construct. We imagine galactosidase as the effector in this split assay, which could detect for example an added X-gal derivative. PhoneLab would then become a functioning diagnostic tool on both a phenotypic and a genotypic level with a much broader functionality.

Urinary Tract Infection

This text will contain facts about drugs and treatments that have a base in Norway. Different countries often use different drugs and treatments.

Cystitis, urinary tract infection and painful bladder disease are all names for the same disease. About 80-90% of the time, the infection is caused by the bacteria Escherichia Coli (E. Coli) - a gram-negative bacteria that lives in our intestines. When E. Coli (or another bacteria) makes its way through the ureteral and to the urinary bladder, we have an urinary tract infection. Urinary tract infection can be divided into two categories; upper urinary tract infection (which include infection of the kidneys, also called pyelitis), and lower urinary tract infection (which only infect the ureteral and the bladder). You may also call it uncomplicated urinary tract infection or complicated urinary tract infection.

The infection affects more women than men. It is a 100 times more common for women to get cystitis, than it is for men. The reason for that is biology and anatomy. Women have a shorter ureteral and therefore it is easier for the bacteria to infect the bladder. Also, after menopause, women often experiences that the mucosa gets dry. This makes it easier for the bacteria to infect organs.

So, how do we treat cystitis?

In Norway we have national guidelines that doctors use in the treatment of different kinds of infection. The guidelines are made to keep the total amount of antibiotics down, and to give the best and most effective treatment to the patient. It is based on the principle that we must use narrow-spectrum antibiotics before broad-spectrum antibiotics. This is to keep the antibiotic resistance at a distance.

Uncomplicated urinary tract infections and complicated urinary tract infections are treated differently. The body can often take care of an uncomplicated cystitis by itself, but many use antibiotics to shorten the duration of the disease.

As many as 40% of the E. Coli found in urinary tract infections in Norway have been seen to be resistant against amino penicillin, such as amoxicillin. The other amino penicillin have shown to be intermediate sensitive, and therefore amino penicillin are no longer first hand treatment against cystitis.

Other penicillin such as mecillinam penicillin can still be used, as about 91% of E. Coli are still sensitive. Less than 2% are resistant.

Trimethoprim, along with pivmecillinam and nitrofurantoin are considered as first hand treatment against lower urinary tract infections. Usually there is no need to treat the patient for more than three days. According to the national guidelines for antibiotics in Norway the first hand treatment looks like this:

- Trimethoprim: 160 mg 2 times a day or 300 mg in the evening for 1-3 days
- Nitrofurantoin: 50 mg 3 times a day for 3 days
- Pivmecillinam: 200 mg 3 times a day for 3 days

All these treatments are considered as equally adequate treatment. Note that this guide is considered for adults who are not pregnant.

A new study also suggests that NSAIDs can be used in the treatment of lower urinary tract infection. It is considered to be equally as good as quinolones.

Treatment against pyelitis is the same as for uncomplicated cystitis, but the treatment is of longer duration.

- Trimethoprim: 160 mg 2 times a day or 300 mg in the evening for 5-7 days
- Nitrofurantoin: 50 mg 3 times a day for 5-7 days
- Pivmecillinam: 200 mg 3 times a day for 5-7 days

Note that treatment depends on severity and response of treatment. There are other guidelines for pregnant women and children.

The doctors also have to consider many factors when deciding which treatment is the most adequate for the patient; such as kidney function, allergies, pregnancy (which trimester?), breastfeeding, age and so on.

PhoneLab

Software

APP DESCRIPTION

The app developed for use with the PhoneLab was devised with the thought of measuring the color change of the urine samples by using the diode in our camera as the photoreceiver. Since the color change might be minute and only small amount of bacteria are antibiotic resistant we want to be able to register these small amounts of color change in the samples.

At first we considered a full spectrophotometer but decided that just taking a picture under the right lighting circumstances would be adequate to measure color changes quite accurately.

So the IT department designed the app based on these criteria and chose to develop the app with Android SDK using Java's API to handle the pixel readout and baseline comparisons that would have to be made to account for the different nuances of urine.

More precisely explained the app has to account for the base sample of urine and store the unique RGB value of the urine sample without any reagents, so that it can be compared to the samples with reagent. After this comparison is made the color change from the pixel’s RGB values are evaluated as either significant enough color change to determine antibiotic resistance or not significant enough color change to determine antibiotic resistance.

The reason for using a camera diode instead of just looking with the naked eye is because of the sensitivity of the camera combined with controlled light environment should equate to more reliable measurements.

APP DESIGN

The app was created in Android SDK and AIDE (Android IDE) it features very simplistic design with some information about antibiotic resistance to help doctors choose suitable antibiotic alternatives in the case of a sample containing antibiotic resistant bacteria towards conventional antibiotics.

The application takes a picture into the phoneLab device and runs a quick test on the resulting bitmap from the picture taken.

The points of reference are the 7 dots on the example picture above, where the bottom sample has no reagent added to the urine so that the bottom sample is the color change reference point. The color change is then measured from this baseline RGB value and compared with the 6 other samples to evaluate if there has been a significant color change in any of them.

APP FEATURES:
The app also features a information button that gives guidelines to health personnel. It includes information from the norwegian “legemiddelhandboka” an official document issued by the norwegian government.

This information can help health personnel to make decisions concerning various infections including urinary tract infections.

App improvements and ideas for the future: From the beginning of developing we were considering a spectrophotometer to detect the color change instead of using the pixel value. We determined that the pixel value would be sufficient and proceeded with this approach since a spectrophotometer would be slightly more advanced and difficult to design. However after our cooperation with Waag society at the cutting edge festival (http://cuttingedgefestival.no/) we were presented a great design for a spectrophotometer that could be incorporated instead of pixel readout for a theoretically more precise diagnostic on color change.

The spectrophotometer that inspired what could possibly be the next generation of our diagnostics test: https://biohackacademy.github.io/biofactory/class/7/pdf/2%20Spectrometer%20design.pdf

The PhoneLab module could also be geo-tagged and stored in a cloud open access. If this information accumulates, algorithms can be developed to predict the bacterial infection.

Hardware

It all started with our iGEM team wanting to build a diagnostic device to deal with the problem of antibiotic misuse. After deciding we were going to work with beta lactamase enzymes in urinary tract infections, we started with our first idea; detection of changes in pH caused by hydrolysis of the beta lactam ring found in all beta-lactam antibiotics. To do this, we initially imagined to design and use an ISFET device (Ion Sensitive Field Effect Transistor), but due to poor readouts with our lab equipment we decided after a while to move away from the pH-measurements and go for an optical solution.

Figure 1. The first sketches of Prothenius - an ISFET device run by an arduino computer
along with a simple display, to detect the hydrolysis of beta lactam ring.

The optimal candidate for optical detection of beta lactamase proved to be nitrocefin, a molecule with a beta-lactam ring that does not function as an antibiotic. When beta lactamase hydrolyse its beta-lactam ring, nitrocefin turns red. This enabled us to do an optical measurement of the color change to determine the presence of bacteria resistant to beta-lactam antibiotics.

The first idea to tackle this problem of measuring colorchange was to build a spectrometer using Arduino computer and 3D printing, but due to casts, this would have been less viable to use in for example third world countries.

We therefore decided to utilize the power that recides in the mobile devices many carry around every day; the smartphone. The modern smartphones generally have very good cameras, powerful processor calculations, and it is easy to develop apps for them. To do so, we needed to design and make hardware that allowed us to interface our samples with a mobile camera, and further design software (an app) to work with this hardware.

Figure 2. The first sketches of Phonelab. The left sketch is the very first version which was
changed to the middle sketch due to some optics problems. The sketch to the right is a single cuvette test.

The purpose of this design is to create a stable way to interface samples with the app. The app collects all the information needed to read and analyse the color distribution between cuvettes, based on the contents of each tube. It then compares the results to a pre-made list containing information on what we are looking for. It would be convinient to have a login-option with an interface where you can add projects and patients, with the possiblilty to add notes. It could also be possible to allow doctors to send prescriptions for the suitable medicine. This could, for example, be done with a QR code on sms. For more information about the app, see the “Software”-section.

Figure 3. The first drafts of the app that would analyze the colors that phonelab detects.

As in any project, there were several prototypes before arriving at our final design. The assembly of the first prototype of PhoneLab was used to find design flaws and proving that the project was possible. The problems we encountered were the focusing on the cuvettes in such a way that we get the most accurate color readout, and the color difference of each diode. We decided that the next model would feature a different geometry to fix these issues.

Figure 4. The assembly of the electronics for PhoneLab prototype 1.

The dimensions and shape of the PhoneLab Optics design with 5 tubes for the nitrocefin penta well test are shown below.This design is made with an exchangeable phone adapter to fit with many different phones. A small LED is included in each slot, to ensure a homogenous and stable illumination of the samples. The LEDs run of an internally installed battery pack, and the light intensity is adjustable. As you see there is equal distance to every slot, and light from the LEDs are emitted radially through the slots where the sample is placed, towards the origin where the lence is placed.

Figure 5. The geometry of PhoneLab Optics, made for perfect 3D prints and cuvette exposure to camera.

To further improve on our device, we imagine to coat it with a hydrophobic layer that repels polar molecules, thereby ensuring it stays clean when being used. We also want this device to autoclavable without being destroyed. In this way it can be easily sterilized, when using it on bacterial samples, and it can be reused in a safe manner. The current material is PLA plastic, meaning that we would have to change the material to make it autoclavable. The cool part about this product, in addition to the box itself, is that it interfaces of biological samples to a phone and gives the userbase the ability to store, geotag, and share information in such an easy way through our software.

To summarize; through a series of prototypes, we have designed and 3D-printed a device that interfaces urine samples, or any sample one has software to and interest in analyzing, with our PhoneLab.

Design

The 3D-model is made in the online 3D-modelling software at https://www.tinkercad.com. The 3D-model represent a E. coli bacterium and shows its DNA, plasmid, ribosomes, pili and flagellum.

The 3D-model was first meant to be a helpful tool during the lectures at “Ungforsk”. Unfortunately, the 3D-printer we had available had difficulties printing different parts of the model. Especially the parts sticking out of the core of the model, e.g. the pili and flagellum, where not compatible with our printer. This meaning that we could not print and use it during our project, but the 3D-model is available online, and we have given the file to the “Biologylab” at Department of Biosciences. Hopefully, they can use another printer and use it for educational purposes. The 3D-model represent a E. coli bacterium and shows different parts which can be helpful during lectures about e.g. protein synthesis and gene transfer.