Background

Treatment of burn wounds is an expensive and major process which causes a lot of pain to those affected. Skin transplantations, surgeries, permanent scars and infections are just some stages that burn victims have to go through. The high infection risk of the wounds is not only painful but it also gives rise to another major problem; antibiotic resistant bacteria. ESKAPE is a group of bacteria that is resistant to antibiotics and often occur in these types of wounds. In view of these problems we made the decision to aid in the battle against infections in wounds, such as burn wounds, by creating an antimicrobial bandage. Our goal is to eradicate the bacteria in ways that diminish the need for antibiotics. We also brought inspiration for our CBD-peptide fusion protein, connected with a linker containing a thrombin cleavage site, from the previous iGEM teams Imperial 2014, Edinburgh 2015 and Ecuador 2018. Inspiration for our model came from NCTU Formosa 2017.

During our journey we have reached out to a number of experts in the field. We have had close contact with the National Burn Care Center (BRIVA) in Linköping and Dr. Johan Junker, PhD at the Center for Disaster Medicine and Traumatology. In addition, we have reached out to EMT staff from emergency medical centers as well as talked with both former and currently treated burn patients. All the input we have received from integrated human practice has been the key in developing and steering our project towards a product that can be utilized in the treatment of wounds. The desired result is a product developed together with the community and hopefully Novosite will prove to be a valuable asset in the battle against infections in wounds.

Integrated Human Practice - Integrating the Communities

Interview with Lasse Gustavsson, a connection to the affected
- ”There is a person behind each injury”

In the middle of the night, in May 1981, the local fire department is dispatched to tend to a gas leak in Gothenburg’s oil harbor. Lasse, who was 24 years old at the time and his comrade, who was driving, steer towards the site. Unaware of how big the leak is, they end up driving into the cloud of gas. Before they were able to back out of the cloud the truck dies and an enormous explosion occurs. The two firefighters get out of the truck and begin running through the flames back towards safety.

After two months Lasse wakes up from a controlled, medically induced coma. He has endured burn wounds over 40% of his body. Half of these burn wounds were of the third degree. By the time he came out of the coma transplantations had already been done on his head and arms. From that point he underwent transplantation to the back of his head and legs. After a while, more functional surgery was executed in order to provide Lasse with a thumb grip as well as eyelids.

Lasse spent an entire year in the hospital as his wounds healed, undergoing physical therapy, surgeries and transplantations - constantly working towards regaining movement, coordination and strength. According to Lasse, the most difficult parts of his recuperation was the physiotherapy and that he felt as though he was back to square one again after every new surgery.

Replacement of bandages and wound dressings were also painful matters since the bandages would dry up and stick to the wounds, thereby tearing the wounds upon removal. In view of this, we have aimed to utilize our wound dressing in a way that will not cause extra distress to the wounds. Our thought is that the bandage itself will not be kept on the wound long enough to dry up and stick. Instead it will only be in contact with the wound long enough for the antimicrobial agents to be released. Infection in his wounds were also a recurring problem during the healing stages, especially along the edges of the wounds. They were treated with different ointments, intending to halt the infections. Lasse also mentioned the use of “Jelonet” a Vaseline covered mesh wound dressing which is placed in direct contact with the open wounds and reduces the risk of other bandages sticking to the wounds as it has a lubricating effect.

Today Lasse is 63 years old. As he has aged; his skin has become less elastic and has therefore had to undergo some new surgeries, but has otherwise not suffered from many recurring struggles. When asked about what he would like for the future of burn wound healing and treatment Lasse says that it is important for the medical field, doctors, nurses and even researchers, to see the entire person. He explains how nice it was to be included in the treatment process by his doctors and nurses. Lasse believes that it is important for the general public to have a decent, respectable and friendly disposition towards burn victims. “No one is normal” Lasse says and although there is some prejudice, burn victims still need to be able to strive to be themselves. Lasse mentioned the existens of a Swedish Facebook page “Burning Spirits”. It is a group for burn victims but remains open to the general public as well and is a good forum offering support and community, where the members can share their experiences. Lasse has his own website with more information about himself as well as podcast episodes.

Follow the link to Lasse's website Lasse Gustavsson and visit the facebook group Burning Spirits

Meeting Emergency Medical Personel

Emergency medical teams offer the first form of medical assistance when disaster strikes. They encounter numerous types of wounds and injuries, not to mention burns, during their careers. We felt that it was important to gain their input on our project and listen to their perspectives regarding burn care and wound treatment.

They explained to us the routines that they employ when dealing with burn victims. Their focus is mainly on providing the care needed to ensure the survival of the patient until they are received by the hospital, for example the burn care center in Linköping. Respiration is a priority for EMTs, but after that the burn wounds are cared for by the application of “burn shield”. Burn shield is a patch that offers moisture and a cooling effect. Cooling and adding moisture to a fresh burn wound is essential in order to prevent the spread of detrimental heat to underlying layers of tissue. Typical disinfectant is also used to rid the wounds of the worst debris, but their main role was to assist in pain management. The staff also explained the series of events that usually follow: from the site of the accident the patients are transported to the emergency department and from there moved to suitable departments, for example the burn care center in Linköping.

Center for Disaster Medecine and Traumatology (KMC)

Dr. Johan Junker is a senior researcher at the Centre for Teaching and Research in Disaster Medicine and Traumatology (KMC). He has developed a wound chamber which enables the administration of medicine such as peptides and antibiotics directly to a wound. This increases concentrations in the area of interest and boosts the effectivity compared to systemically administered medications. Junker’s research focuses on effective healing of wounds, making sure infections are not an issue, therefore making potential transplants and other treatments of the wound easier. He’s also currently developing a wound model which could replace animal testing in which human skin is co- cultured with bacteria to simulate an infected wound.

We were fortunate enough to discuss our project with Johan Junker in the early stages of our planning process and his expert advice helped us establish more detailed aspects of our project and guided us in our research. During our first meeting Junker answered our questions regarding antimicrobial peptides, different wound properties and characteristics, as well as which types of bacteria are the most important and relevant to target.

We asked what thoughts he had on antimicrobial peptides and Junker explained that if they are used in combination with antibiotics they could make antibiotics more effective and thereby reduce the amounts of antibiotics needed to kill bacteria. Junker mentioned that there are many different types of bacteria that can exist in a wound and could be relevant targets. In particular, a group of bacteria called ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.). These constitute a group of pathogenic bacteria that are able to develop resistance towards antibiotics. He also thought that we should focus on eradicating all bacteria in the wound to create a completely sterile wound. In view of this information we have tried to choose antimicrobial agents that can specifically targets and combat this group of bacteria. At the meeting we also contemplated if continuously cellulose bound antimicrobial agents is the best and it was from this discussion that our thrombin cleavage mechanism was uncovered. Before this meeting we had also entertained the idea of incorporating biofilm degrading enzymes but after our conversation with Junker we realized that biofilm did not need to be a point of focus. Mainly due to the fact that the treatment and eradication of bacteria would be set in motion before biofilm can be produced.

At the time of our second meeting with Johan Junker we had established more aspects of our project. We presented the concept of our project and the mechanism of action that we planned to utilize. Overall he was pleased with the idea and gave us some tips of different methods we could use to test the antimicrobial activity of our antimicrobial agents.

BRIVA, a connection to society and care

Discussions with BRIVA

The National Burn Care Center in Linköping (BRIVA) has several years of experience in advanced burn care as well as cutting edge research in the field. BRIVA is one of two national burn care centers in Sweden with the other one being in Uppsala (Uppsala University Hospital). Each of these two hospitals serve half of the Swedish population’s burn injuries. Our team was lucky enough to engage in meetings with Dr. Moustafa Elmasry (Head of Surgery at BRIVA), Ingrid Steinvall, PhD and Ahmed El-Serafi, PhD, who are involved in the research of regenerative medicine and wound care.

The meetings proved to be helpful and generated great amounts of input and advice, which we took into consideration as we continued to plan our project. We headed into the first meeting with many questions about burn care and burn wounds in general, for example what the main difficulties were when it came to burn wounds, if infections in the wounds were a problem, how infections in the wounds are handled today and what they would like to see in the future.

They expressed concerns that bandages tend stick to the wounds if left applied for too long. We continued to discuss this problem and eventually came to the conclusion that the cellulose bandage (Epiprotect, S2Medical) that they are currently testing could be a candidate to use. BRIVA kindly provided us with this cellulose bandage for our experimental use. They also expressed that the bandage might need to be changed regularly depending on the burn. Therefore, they inspired us to consider Novosite as a carrier of antimicrobial agents. According to this, the bandage will be applied to the wound long enough to ensure thrombin cleavage and release of the antimicrobial agents, something expected to take around 24 hours on third grade burn wounds. Thereafter, a new cellulose bandage can be reapplied or substituted with a different kind of wound dressing depending on the situation.

The vision for the future focuses on helping the body heal itself rather than only killing bacteria. Cellulose assists in this due to its breathable and moist features. BRIVA explained that there are many bacteria included in and needed for a healthy microbiome. These certain bacteria should be considered to be spared. These discoveries indicate that the microbiome might help the healing process. In view of this, we decided to be more specific in the bacteria that we wanted to target with our antimicrobial agents and focus on those that are dangerous, cause infections and are hard to eradicate because of developing antibiotic resistance. Therefore, we have chosen to not only work with antimicrobial peptides, which tend to be more specific, but also bacteriophage enzymes that instead target specific bacteria. The bacteriophage enzymes we chose are highly specific towards A. baumannii and S. aureus, two members of the ESKAPE group of bacteria. The meetings and conversations with these experts gave us the possibility to modify plans in our project and steered us early in a good direction.

Visiting BRIVA

In July we had the wonderful opportunity to shadow the doctors and nurses of the Burn Care Center in Linköping. It was rewarding and humbling to be able to observe firsthand, the treatments of mild to severe burn wounds.

In the wound clinic, patients with both burns and other wounds were treated, whereas in the intensive care department patients with severe burns were cared for. During these visits we were able to talk to nurses, doctors and patients. We learned that the silver wound dressings used were expensive and that their efficiency was widely disputed. As we observed the change of wound dressings in burns, we were able to ask the doctors and nurses questions and absorb their experienced opinions and thoughts. Here we came in contact with the product Jelonet, a Vaseline and paraffin coated cotton mesh dressing which is applied closest to the wound before other bandages are placed on top. This is to try to prevent the dressings sticking to the wounds. This product was also used in the treatment of Lasse Gustavsson, the former firefighter from Gothenburg. In conclusion, Jelonet could be an option and solution to this problem and if made antimicrobial would provide two important functions. One would be its antimicrobial capacity and the other its lubricating qualities. Since the Jelonet material consist of cotton (which contains 90-95% of cellulose) we tried binding our fusion proteins to the material via the cellulose binding domain (CBD). Therefore, we tested binding our CBD to Jelonet. We learned that burn wounds are quite damp and moist the first couple days and dry up in later stages of healing. When we asked if it is important to have a moist/lubricating gauze we received some different opinions and perspectives. During the initial stages it might not be crucial to have a lubricating wound dressing because the wound itself is naturally moist. However, it became clear to us that this lubricating quality is important in order to not cause more distress or create more problems should the bandage dry up and stick to the wound

During our visit we also had the opportunity to observe skin transplant surgeries and the preparation of skin grafts. Skin grafts were prepared and placed in a quilted fashion over the intended area. Jelonet was once again applied over the grafts after the surgery, with other bandages layered on top. However, during one operation there was a lack of available grafting options and the cellulose bandage we tested (Epiprotect, S2Medical) was used as a substitute and Jelonet was not used. This was especially interesting for us since our project was founded on the bases of a cellulose bandage.

Academic Hospital Burn Care Center Uppsala

The burn care center at the Academic hospital in Uppsala is one of two burn care centers in Sweden. They have a long experience in treatment of burn wounds and conduct research within the field. Our team had the opportunity to have a meeting with Marie Lindblad (research nurse) to ask some questions about the treatment of burn wounds and problems they may encounter when dealing with these types of wounds.

From this meeting we learned a lot about the different bacteria that is commonly present in these types of wounds and which stages they present themselves in. Lindblad told us that initially the bacteria Staphylococcus Aureus, a gram positive bacteria, is most common in the wounds. However, this bacteria is easily treated with antibiotics as long as it does not enter the blood system. More infamous and harder to treat are the bacteria Acinetobacter Baumannii and Pseudomonas Aeruginosa. These are gram negative bacteria that also are classified as ESKAPE-bacteria, meaning they easily develop resistant to antibiotics. She expressed concern about the gram negative bacteria and explained that these are hard to eradicate and should be a priority for future research.

In our project we have heavily focused on the infections of the wounds however, from this meeting our eyes were also opened to the problem of sepsis. Sepsis occurs when an infection has spread throughout the whole body which happens when bacteria enters the bloodstream. Lindblad described to us that sepsis is a major problem and is especially common when it comes to larger wounds. She also explained that patients with major burn wounds can experience sepsis multiple times during their hospitalization because of nosocomial infection. At the hospital, screening for antibiotic resistant bacteria is a routine. It is most common to find antibiotic resistant bacteria in elderly patients since they have acquired Methicillin-resistant Staphylococcus aureus (MRSA) bacteria during their long lifetime. After talking to Marie Lindblad we gained a better understanding of which types of bacteria are important for our antimicrobial agents to be able to target.

An Industrial Perspective - S2Medical

A young medical company is developing in Linköping - focusing on the treatment and healing of burns and chronic wounds. We had the privilege to converse with the COO and co-founder of S2 Medical, Mårten Skog. He relayed to us the industrial perspective of medical product development, explaining that when creating a medical product it will fall under different categories depending on its intended purpose and how it achieves that purpose. A product could either fall under medical devices or administered therapeutics for example. Skog urged us to think through all the aspects of product design when creating a product that is aimed for the medical market. Similar to information from BRIVA, Skog suggested that we should focus on our construct rather than the bandage material itself.

Prevent, Protect and Prosper

After talking to the different organizations and individuals above, we realized that Novosite should aspire to meet three different criteria based on the feedback from all the meetings. The needs expressed could be summarized by three words: prevent (prevention of scarring and antibiotic resistance), protect (protection from patient infections and microbiome) and prosper (increase patient prosperity by having the bandage easily applied/have on the skin and reduced pain). We named these points the three P criteria. The three criteria are explained below and also displayed with the corresponding meetings that influenced these criteria and aspirations.

Prevent
KMC and BRIVA helped us with this criterion by discussing and sharing their experiences with wound healing and antibiotic resistance.
• By choosing cellulose (therefore our CBD-construct) as a carrier we hope that in further testing Novosite will prove to assist in reducing scarring. By eliminating bacteria and preventing infections, as well as influencing the duration the wound is open, tissue remodeling will be reduced which helps to decrease scarring.
• Antibiotic resistance will be prevented by Novosite’s antibacterial properties, leading to either the replacement or reduction of antibiotics used in treatment. By keeping it applied and switching it regularly with a new one, Novosite will maintain a steril environment, preventing the recurrence of infection and aiding in the healing of the wound.

Protect
KMC, BRIVA and Uppsala university hospital assisted in this criterion by explaining their difficulties with infections, particularly pertaining to the ESKAPE-group of bacteria. BRIVA especially pointed out that they also thought that protecting the host microbiome was important. We tried to follow this by using specific lysins. These lysins would be specific towards certain bacteria of the ESKAPE-group, eradicating them but leaving the rest of the host microbiome untouched. KMC was more interested in eradicating all bacteria and we took this into consideration by incorporating broad spectrum antimicrobial peptides.
• By choosing antimicrobial peptides and enzymes we can protect wounds from infections.
• The lysins will be more specific, therefore hopefully sparing the microbiome.

Prosper
When talking to the EMT-staff, BRIVA, Uppsala University Hospital and Lasse Gustavsson we understood that today’s bandages can be painful to change and can be hard to apply to joints and other flexible parts of the body.
• In view of this aspect, we opted to move forward with a cellulose bandage (variants of which are being tested clinically, such as S2Medical's Epiprotect) and a carbohydrate binding domain (CBD). The goal was to create a practical, painless, flexible and functional antimicrobial bandage.
• The CBD can also be used for different materials if needed depending on the injury - proving Novosite's modularity. Many different types of bandage materials exist and are being developed and we felt that a modular construct would be a great asset, making it possible to give different types of materials antimicrobial features.

Team:Linkoping Sweden/Human Practices