Team:NTNU Trondheim/Human Practices

• Team
  Team Members Collaborations
• Project
  Description Design Model Results Demonstrate
• Lab
  Lab Journal Protocols Measurement Safety
• Parts
  Parts Overview New Part
• InterLab
• Human Practices
  Integrated Human Practices Education and Engangement
• Acknowledgements
  Sponsors Attributions
• Awards

Human practices allowed us to reach out to our community and increase awareness of the various problems caused by biofilm.

We chose our project with the idea of combating antibiotic resistance. It has been well documented that bacteria found in biofilms are more resistant to antibiotic treatment than other bacteria, making them very difficult to get rid of. Having little idea about the impact biofilm has on our society, we decided to investigate further. Initially we wanted to find one area of application, but at the beginning we were not aware of the broadness of issues caused by biofilm. After doing several interviews we decided that the purpose of our project is to prove a principal and to address the broad variety of issues that needs individual solutions. This is important when assessing the delivery method for our CRISPR-system. This way we got an idea about the applicability of our system and which considerations that have to be made related to the individual problems.

We also spoke with experts for advice regarding our lab procedures, as we were uncertain of which aspects to focus on regarding biofilm formation. We wanted the biofilm assay design to have similar conditions as in nature. The discussions we had are described in the timeline below.

Interactive Timeline

Click on the header of each bullet point to read more about how this interaction contributed to our project

Each person has a gold or silver medal based on how which criteria they contributed to. A gold medal indicates that they contributed to the Gold criteria of Integrated Human Practices, while a silver medal indicated they contributed to the Silver criteria.

For each interview, you can read a short list of the key aspects of how this person contributed to Integrated Human Practices, a short list of key insights from the interview, and at last, a summary of the interview as a whole.

Marthe Lind Kroknes

Raffael Himmelbach

Kåre Bergh

Sven Even Borgos

Eivind Witsø

Hanne Tobiassen

Odd Atle Tveit

Hans P. Kleppen

Hilde Mellegård

Conclusion

The human practices part of our project provided us with a lot of new knowledge and ideas that we applied to our project. More importantly, it allowed us to come into contact with our community and increase both our and public awareness on the various problems caused by biofilm-producing bacteria.

The main idea at the start of our project was to reduce biofilm formation in bacteria and thus make them more susceptible to antibiotics. During the course of the Human Practices part of our project we learned a great deal about other areas in need of an effective biofilm reducing method, such as dental care, diabetes, prostheses surgery, diagnostics and aquaculture. A variety of treatments for biofilm are available as of today, however they all seem to have their limitations. Our approach to biofilm removal is unique in that bacteriophages have an ability to evolve, meaning that bacteria most likely wouldn’t be able to develop resistance for a long time. If successful, our method could be a potential breakthrough in the field of biofilm-reducing agents.

We want to thank all the experts for taking the time to talk with us. Their perspectives opened up our eyes for a whole new world of biofilm-related problems that we had no idea about prior to our project. We also got quite a few useful tips that we implemented in our lab procedures, such as the use of Crystal Violet Assay for biofilm quantification.

Marthe Lind Kroknes

Biomedical laboratory scientist at St.Olavs hospital

Conclusion for Integrated HP

• Initially we wanted to find one area of application, but at the beginning we were not aware of the broadness of issues caused by biofilm.
• Marthe gave valuable information that made it clear that infections related to prosthetics is a problem caused by biofilm.
• To learn more about different aspects of this problem, she told us to contact Eivind Witsø, an orthopaedic surgeon and Kåre Bergh, a microbiologist. This way we could learn about how bacteria interacts in a biofilm and an orthopaedics view as well.
• These interviews provided valuable insights in assessing the applicability of our project. Biofilm is hard to discover and diagnose, and therefore hard to treat. It requires extensive research to discover a delivery method for our CRISPR-system that is safe and usable. In addition we need to know that biofilm is present in the first place and the nature of the bacteria.

Key insight

• Biofilm increases the risk of protheses-related infections.
• Surgery might be a new application for the project, mainly regarding implants and prostheses.
• An infection related to an implant has big consequences for the patients, and the surgery has to be redone. There is no way to remove the biofilm caused by the infection, and a new implant is needed.
• A biofilm consisting of non-pathogenic bacteria provides a hospitable environment for pathogenic bacteria.

Summary of interview

We met Marthe Lind Kroknes, a biomedical laboratory scientist with a master in molecular medicine, at St. Olavs Hospital in Trondheim. She gave valuable information about infections in hip prosthesis and where she believes biofilms increase the risk of infections.

In 2017, about 750 prostheses surgeries was performed at St. Olavs hospital in Trondheim and 19 of the patients had an infection after the surgery. Those infections did not necessarily occur because of biofilm formation, but it might very well have been a contributing factor.

Kroknes told us that in some cases where infections around prostheses are untreatable with antibiotics, they usually remove the implant and redo the surgery. It is unfortunate for the patients to redo the surgery as it is a huge burden to go through another procedure.

Biofilms are not only a problem because of the pathogenic bacteria, but it also makes it difficult to run diagnostics. Even when the biofilms consist mainly of non-pathogenic bacteria, it makes a very hospitable environment for pathogenic bacteria. Thus, it could be difficult to kill even a small part of pathogenic bacteria in the biofilm. Those bacteria are even more dangerous considering that some of the patients have a somewhat weakened immune system.

An efficient method for prevention of biofilm formation would be of great benefit for both doctors and patients. From the interview we learned about how biofilms can increase risk of infections and how big the consequences can be for patients. This gave us new insight about the possible applications of our project.

A photo from our meeting with Marthe LInd Kroknes. From left to right: Hege Hetland Pedersen, Anja Rosvold From, Marthe Lind Kroknes, Paulius Cigriejus.

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Raffael Himmelbach

Coordinator, Research and Innovation, Centre for Digital Life Norway

Conclusion for Integrated HP

• Raffael changed the way we wanted to investigate the different issues related to biofilm by suggesting the interview methodology instead of a survey.
• By doing interviews we gained more insight and understood the broadness of biofilm-related issues.

Key insight

• Get an overview of potential users, and learn more about our stakeholders.
• Map out potential users and talk to them. The people we think would have use of our project results would not necessarily say the same thing.
• Consider the side effects versus the positive effects and based on that choose the application area.
• An interview methodology is more flexible than doing a survey when exploring views of a small number of respondents.

Summary of Interview

We met with Rafael Himmelbach, a Coordinator responsible for Research and Innovation at Digital Life Norway with the goal of learning about gathering public opinion and other possible applications of our project. He advised us to get an overview of potential users for our project solution. He told us that usually, what we think some people may think and what they actually think is not necessarily the same. Therefore, it’s important to distinguish between what you know and what you just think you know. It’s also important to understand users preferences and adapt a method to meet the needs of its potential users. Every application has its benefits and shortcomings, and we have to make sure that our method meets unmet needs while limiting its potential side-effects. He also advised us to make sure that we think about every possible effect our method can have on the environment, such as the effect a non-biofilm producing bacteria would have in a bacterial population consisting of other biofilm-producing bacteria. When assessing where the project can be applied, if it has more significant side effects than positive effects it would be wise to change the application area.

Regarding public opinion, he advised us to consider the interview methodology instead of a survey. Gathering insights with a survey is methodologically challenging and requires extensive testing of the questionnaire and the target groups. This is to avoid over-representing one group of people and to gather information that is useful. The interview methodology is more flexible when exploring views of a small number of respondents. Following his advice, we decided to gather public opinion from interviews with people representing different groups of potential stakeholders.

A photo from our meeting with Raffael Himmelbach. From left to right: Adrian Marinovic, Raffael Himmelbach, Amanda Sande, Hege Hetland Pedersen.

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Kåre Bergh

Professor at «Institute of Clinical and Molecular Medicine» at St. Olavs Hospital in Trondheim, Norway. Working with diagnostics.

Conclusion for Integrated HP

Bacteria and biofilm formation can be unpredictable in nature in comparison to in the laboratory. We have to assess different strains, glucose levels, pH, and medium nutrients when doing biofilm assays, and try to imitate the conditions in nature.

His insight made us decide to do a test on LB and M63B1 medium to discover which glucose and pH values that were optimal for biofilm formation in E. coli Dh5α and TG1 strains.

His advice benefited us a lot by allowing us to produce significant amounts of biofilm and therefore enabling us to see whether CRISPRi had an inhibitory effect or not.

Kåre made us aware of the flaws in our original protocols for biofilm quantification, and consequently we went with the alternative protocol that he had recommended.

Key insight

• It is hard to diagnose biofilm. Routine-diagnostics is based on planktonic growth. In biofilm the bacteria multiply slower, there are less metabolic activity, gene expression changes and the bacteria is less sensitive to antibiotics. To diagnose it, they have to try to remove and crush the biofilm matrix, before growing the bacteria.
• It is the low-virulent bacteria that causes problems, because there is few symptoms. The consequence is a buildup of bacteria in the biofilm so the implant has to be removed.
• Biofilm formation depends heavily on the growth media, especially the glucose levels.
• Provided a better protocol on Crystal Violet Assay for biofilm measurements.

Summary of Interview

We interviewed Kåre Bergh, professor at «Institute of Clinical and Molecular Medicine» at St. Olavs Hospital in Trondheim, Norway. Kåre has previously worked with infections, microbiology and immunology, and currently works with diagnostics. In our interview, Kåre gave us interesting and detailed insights regarding prosthetics operations and the risk of bacterial contamination during the operation. Infections can actually occur years after a prosthetics surgery, and can be discovered due to indications that the prosthetics has detached.This is caused by low-virulent bacteria in biofilm that gives rise to non or modest symptoms of inflammation. This makes it difficult to diagnose and the biofilm would have to be discovered, removed and grown in the lab to be able to identify the bacteria. Only 1-2 % of the patients with hip prostheses is infected with biofilm-producing bacteria, but it is very unfortunate for those who do. After a few years, often between 1-2 years, the prosthetic has accumulated dangerous levels of bacteria in a biofilm matrix. Consequently, the prosthetic must be removed through another surgery and replaced with a new one. This is very stressful for the patient, as well as expensive. There is no revolutionary way to treat biofilm on prostheses.

We were curious to know Kåres opinion on the E. coli strain (TG1) that we chose as our biofilm producing bacteria to be used in our future Crystal Violet Assay experiments (Biofilm Quantification). Kåre told us that there is no guarantee that a bacterium known for a low, medium or high biofilm production actually will produce the expected amount of biofilm. It largely depends on the growth medium that we use. According to Kåre, a proper glucose concentration is the most important criteria to induce the right amount of biofilm production in our chosen E. coli strain. Biofilm formation in nature and in the laboratory is not the same. In addition, Kåre made us rethink our experimental procedure in quantifying biofilm produced by E. coli. We initially planned to use a protocol for a Crystal Violet Assay and described the approach to him. He questioned the protocols reliability and found a new protocol which he found more reliable (See Protocols -> Crystal Violet).

We gained more knowledge about biofilm and the problems it can cause on prosthetics. In addition, Kåre made us aware of the flaws in our original protocols for biofilm quantification, and consequently we went with the alternative protocol that he had recommended. As another result of the interview, we performed a test on LB and M63B1 medium to see what glucose and pH values were optimal for biofilm formation in E. coli Dh5α and TG1 strains. His advice benefited us a lot by allowing us to produce significant amounts of biofilm and therefore enabling us to see whether CRISPRi had an inhibitory effect or not.

A photo from our meeting with Kåre Bergh. From left to right: Tom Strømsen Haugland, Kåre Bergh, Hege Hetland Pedersen, Carmen Chen.

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Sven Even Borgos

Senior Research Scientist at the Department of Biotechnology and Nanomedicine at SINTEF Industry

Conclusion for Integrated HP

• His insight was significant when choosing incubation times and growth media (M63B1 in addition to LB).
• By doing a cell viability assay we can learn more about the biofilm formed in our plates, and not just that there is less biofilm.

Key insight

• It may be difficult to inhibit all pathways in bacterial communication, due to its complexity.
• Optimize the biofilm measurements by providing information regarding incubation times and growth media. Recommended a cell viability assay in combination with the crystal violet assay.
• In modeling we should only include parameters that can be measured, like pH, O₂-levels, concentration of inducer.

Summary of interview

After the interview with Kåre Bergh, we were still unsure about what the optimal experimental setup would be for measuring the biofilm production. To further investigate this, we scheduled a meeting with Sven Even Borgos who is a Senior Research Scientist at the Department of Biotechnology and Nanomedicine at SINTEF Industry here in Trondheim. Sven has among other things knowledge within molecular genetics, biochemistry and system biology.

Sven was very curious and showed great interest in our approach to reduce bacterial biofilm formation using CRISPRi and bacteriophages. He informed us that it may be difficult to fully inhibit all pathways involved in the bacterial communication due to its great complexity. Still, he said that targeting important quorum sensing molecules may be a possible solution to reduce the bacteria’s ability to communicate, and thereby decrease their biofilm production. Sven was very helpful in the making of the framework of our experimental procedure. He told us that an optimal setup would be to combine measurements of the biofilm production with a cell viability assay. Furthermore, Sven put us in contact with his colleague Anne Tøndervik who was eager to help us with some tips and tricks regarding the theoretical and practical aspects of our project. They both helped us find recipes for growth mediums, incubation times and informed us about a potential cell quantification kit (BacTiter-Glo).

Lastly, Sven had some comments regarding the modelling of our project. He reminded us that biofilms are not homogeneous, and that bacteria do not necessarily respond similarly and/or linearly to outer stimuli. Sven said that not all parameters are of interest, and that we only should include those that we can measure (eg. pH, nutrients, O2-levels, concentration of biofilm inhibitor, etc.).

How did this interview shape our project? The interview with Sven Even Borgos influenced how we conducted our experimental procedures. To obtain more valuable results we incorporated his idea to combine cell count with the Crystal Violet assay. We also used the growth media and incubation time suggested by him and Anne Tøndervik, and included some of the suggested parameters into our model.

The interview with Sven Even Borgos gave us further insight into theoretical aspects of bacterial communication and biofilm formation. We also got some excellent advice regarding both the experimental setup and the modelling of our project, which have shaped the demonstration of our concept. His expertise is greatly appreciated!

A photo from our meeting with Sven Even Borgos. From left to right: Paulius Cigriejus, Vanessa Solvang, Amanda Sande, Sven Even Borgos.

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Eivind Witsøe

Orthopaedic Surgeon at St. Olavs Hospital in Trondheim

Conclusion for Integrated HP

• A new area of application is related to diabetic foot ulcers. Biofilm is assumed to be responsible for chronic wound infections and open wounds that won't heal sufficiently on its own.
• See more in Conclusion of Marthe Lund Kroknes

Key insight

• Chronic infections require a surgery redo as there is no safe and effective method to remove biofilm. Repeating the surgery increases the risk of more infections, and can in the worst case scenario lead to amputations.
• Besides foreign implants, there is also a need for an effective biofilm treatment for diabetic foot ulcers.
• In modeling we should only include parameters that can be measured, like pH, O₂-levels, concentration of inducer.
• Cystic Fibrosis is the first known biofilm-associated medical condition.
• Finding a method for prevention of biofilm is an urgent and important issue. There is a lot of research on different materials that limit biofilm growth, especially since bacteria in biofilm quickly develops resistance against antibiotics.
• A treatment should be compatible to pseudomonas and not be to invasive in terms of changing the microflora composition.

Summary of interview

We met with Eivind Witsø, an orthopaedic surgeon at St. Olavs Hospital in Trondheim for information about biofilm-related problems in orthopaedic procedures. Eivind has extensive experience with biofilm in his career and provided us with a lot of interesting and useful information.

In his experience, many infections arise during implantation of foreign parts into the body, such as hip surgery. These chronic infections require a surgery redo as there isn’t an effective and safe way of removing biofilm. A secondary surgery involves an even greater risk of infection, and patients often find themselves having to go through multiple surgeries, or in worst case live without hip joints or undergo amputation. Antibiotic treatments are ineffective and there is a great need for an alternative biofilm removing solution.

Biofilm-related problems are well known in medicine, with Cystic Fibrosis being the first known biofilm-associated medical condition. Besides foreign implants, there is also a need for an effective biofilm treatment for diabetic foot ulcers. Biofilm is assumed to be responsible for chronic wound infections, leading to open wounds that can’t manage to heal sufficiently by themselves.

He also told us that there is a lot of ongoing research revolving around biofilm removing substances, but none of those substances have been implemented at St. Olavs Hospital. Large biotech companies are being reluctant in developing new antibiotics due to bacteria in biofilms developing resistance in a commercially short period of time. This makes finding an alternative method for biofilm removal an even more important and urgent task.

Eivind was also kind enough to provide us with some insight regarding our project. He told us that the bacterium known to be responsible for most infectional problems is pseudomonas and that a biofilm removing method has to be compatible with this bacterium. In addition a potential bacteriophage treatment has to be minimally invasive in microflora composition. There has been done research suggesting that many medical conditions are a result of faulty microflora composition, and if this is indeed the case then a disturbance in its composition might do more harm than good. We believe that if our method is adapted to pseudomonas this shouldn’t be a problem as pseudomonas is a pathogenic bacteria that is, with the exception of the gastrointestinal tract essential, unessential to our microflora.

A photo from our meeting with Eivind Witsø. From left to right: Adrian Marinovic, Eivind Witsø, Hege Hetland Pedersen, Vanessa Solvang.

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Hanne Tobiassen

Quality Manager at SalMar, World leading Salmon Production Company

Conclusion for Integrated HP

• A new area of application is avoiding biofilm formation in the production chain in the production of seafood and therefore also in other areas of food production.

Key insight

• It is a challenge to avoid biofilm in every part of the production chain. Biofilm usually do not grow on smooth surfaces, and this makes it harder to prevent and remove.
• At the production site there is always high humidity levels which can lead to an increased risk of bacterial growth.
• In the seafood industry the main enemy is the Listeria bacteria. There is some future concerns about the bacteria developing resistance against disinfectants that are used in cleaning processes.
• For future methods there are strict requirements in terms of risk assessments. The use of GMO is also highly restricted by international regulations, especially when it comes to food and nutrition.

Summary of interview

After some interviews regarding biofilm as a health issue we were curious about how biofilm is a problem in other industries, especially the aquaculture industry. By doing an interview we could discover other pros and cons about biofilm, and which aspects that should be considered for a solution at such production sites.

We had a telephone interview with Hanne Tobiassen, the quality manager in SalMar in Norway. SalMar is one of the world´s largest and most efficient producers of farmed salmon. She works on making sure the product is safe for consumers by monitoring every part of the production chain. She is very familiar with biofilm, and told us that it is a challenge to avoid biofilm formation on equipment in the factory. Cleanliness is a big priority at the facility, and it is something they do on a daily basis. The humidity levels at the facility are relatively high which increases the risk of bacterial growth. SalMar’s measures against biofilms per now are good cleaning and maintenance procedures, quality equipment and communication with distributors. There are many stages in the production, and it is a problem to make sure that they avoid biofilm formation in any of the steps. For a future solution there would probably need to be several biofilm reducing products that can be used for each step, because the production in total is very complex.

Hanne told us that the main enemy is the Listeria bacteria. Listeria bacteria is a bacteria that is almost everywhere in nature. Listeria rarely cause infections, but when the amount of bacteria is significant it can cause illness especially for people with weak immune systems There is some research on this bacteria in particular, to understand if it arrives to the factory with the fish or if it appears under production. Apart from the problems with pathogenic bacteria, Hanne told us that there is some future concerns about the development of resistance against disinfectants used for cleaning. It would be a major issue in the future if the cleaning steps fail to work. Even when the biofilm only consists of non-pathogenic bacteria it can still affect the welfare of the fish and may cause negative changes to the end product. Hanne is sure in her answer when she said that there is a need for more measures and research to avoid the formation of biofilm.

After telling her about our project we asked her about which aspects we have to consider for such a solution to be implemented. She told us that costs are very significant, especially if there is a series of products that need to be used. More importantly, risk assessments has to be made and considered. There are many restrictions when it comes to providing safe methods and products in the food industry. The use of GMO is highly restricted by international regulations, especially when it comes to food and nutrition.

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Odd Atle Tveit

Head of Department for Water and Wastewater in Trondheim municipality

Key insight

• The department does cell counts to research bacterial growth and assess the results based on drinking water regulations. They don´t research biofilm in particular.
• If the bacteria level is higher than permitted they use methods like flushing water or rinse the tubes with soft sponges. At the treatment facility they use methods like UV and chlorine.
• Biofilm can be used in a positive way by removing pollution from wastewater using biorotors.

Summary of interview

We also wanted to talk to someone working on providing the town with clean water and the treatment of wastewater, as this could be another relevant application area for the project. We had a telephone interview with Odd Atle Tveit, the head of the department for water and wastewater in Trondheim municipality. There is an extensive network for the transportation of clean water to the citizens in addition to treatment facilities. They also treat the waste water before it is led to the ocean. Because of humidity and water levels, bacterial growth is a risk. They don´t research biofilm in particular, but water quality. Based on different levels they assess if the network has to be treated to avoid further growth of bacteria.

The department does cell counts to research bacterial growth and assess the values based on drinking water regulations. We asked him if biofilm is an issue, and he could inform us that it does not cause big problems. If the bacteria levels reaches a certain level, they use flushing of water through the tubes to get a better flow through. They also use soft sponges with the same diameter as the tubes to rinse the walls. In the treatment facility they use chlorine and UV-light for disinfection. He could inform us that many water treatment facilities removes organic substances so there is less nutrition available in the water. This is one way to decrease biofilm formation in water distribution systems. He agreed with the fact that biofilm is complex and important to understand, but that it also can be used in a positive way. They actually uses biofilm on biorotors to remove pollution from the waste water when it is treated.

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Hilde Mellegård

Senior Adviser at the Norwegian Biotechnology Advisory Board

Key insight

• The norwegian law concerning gene technology is quite strict compared to other countries, as the precautionary principle is important.
• The regulation of GMOs is based on the technology used, not the final product.
• Our use of GMOs in the lab is regulated by our university’s rules, therefore we do not need to take further precautions than we already do.

Summary of interview

Hilde Mellegård works as a Senior Adviser at The Norwegian Biotechnology Advisory Board. She has a doctorate in microbiology and a background as a veterinary. We contacted Mellegård to learn more about GMOs in Norway.

The Norwegian Biotechnology Advisory Board is a consultative body that informs the public and initiates debate on biotechnology. From their website: «The main tasks of the Norwegian Biotechnology Advisory Board are to evaluate the social and ethical consequences of modern biotechnology and to discuss usage which promotes sustainable development». The precautionary principle is essential in the gene technology law. [HM1] For a GMO to be approved by the Norwegian government, it needs to be comply criteria linked to health and environment. Norway has its own legislation on GMOs, which is similar to the EU legislation in general, but there are some differences, e.g in definitions of what a GMO is. In Norway and the European union, the regulation of GMOs is based on the technology used, not the final product regardless of what technology is applied, as in some other countries.

These days, the Advisory Board is working on a statement on future regulation of GMOs in Norway through the Gene Technology Act. The Act is now 25 years old, and there has been a rapid technological development since then. 25 years ago, the divide between gene technology and conventional breeding techniques was clear. Today, with new technologies giving a much larger range of possibilities, these lines are becoming increasingly blurred. According to Mellegård, the technological development presents new opportunities and challenges. Therefore, the law that regulates the GMOs might be due for an upgrade. The aim of the work of the Board is to develop appropriate and robust regulatory frameworks that facilitate the harnessing of the potential of gene technology, while also avoiding harm to health and the environment, as well as promoting sustainability, societal benefit and ethics. There is a lot going on in the field of gene editing technologies. CRISPR has become a very popular tool as it is rather easy and cheap compared to other techniques. It is used in several science projects and experiments these days, with plants, animals, microorganisms and even humans. There are also many projects on the way concerning gene therapy. Especially cancer is an illness of interest where scientists are working on ways to cure cancer using gene therapy applying CRISPR technology.

Surveys have shown that a majority of the public is sceptical to GMOs, although the younger generation might be more open-minded. The younger citizens of Norway generally care about the environment and sustainability, and GMOs that might contribute positively in this area have been developed or are in the pipeline, such as corn that sustains drought better than natural corn. Some investigations also suggest that when GMOs contribute to animal welfare, the public is more positive.

We wanted to know if there was anything we should be specifically aware of during our project about GMOs, but since the use of genetically modified bacteria is restricted to our lab (contained use), the requirements are different than if we were to use GMO in for example hospitals (deliberate release).

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Hans P. Kleppen

Biologist and Phage Expert

Key insight

The interview with Hans Petter Kleppen was very informative and interesting. He informed us about different aspects of bacteriophages by sharing his experiences with the use of them in his work. Among others he told us about their negative effects in diary facilities, but also about their positive sides as alternatives to antibiotics and as microbiome controlling agents. Hans also notified us about the relatively low level of scepticism that he had experienced during his research, as well as the various restrictions related to the use of bacteriophages. In addition, he suggested a potential futuristic improvement to our experimental setup.

Summary of interview

Our initial thought was to use bacteriophages to deliver the CRISPRi system targeting the luxS gene, which is involved in the bacterial quorum sensing. To learn more about the use of phages and their potentials we reached out to Hans Petter Kleppen, one of relatively few with phage experience here in Norway.

Hans has a solid background in Molecular Biology, which he gained through his master’s degree at The University of Bergen. In his doctoral degree he studied the occurrence of bacteriophages in diary facilities, where bacteriophage infections can reduce both yield and quality of the desired fermented products. Despite this encounter with the negative effects of bacteriophages, Hans also recognized their potentials as something positive. Today Hans works as the research director in Nordli Holding, which is a firm aiming to increase the health and welfare of fish. As a part the solution, they use bacteriophages as an alternative to antibiotics. He also informed us about the nanobiome (viriome), which is the viral counterpart to the microbiome. The nanobiome is essential to maintain the balance of the microbiome in an organism and prevents the development of dominance by one single bacteria. With the knowledge of the nanobiome, it may be possible to specifically control the microbiome to increase the health and wealth of the host organism.

Even though Hans has mainly worked with natural bacteriophages, he still thinks our approach to constructing and using recombinant phages may theoretically be possible. He also gave us some input to improvements of our experimental setup. In addition to the biofilm reducing selective marker, he suggested us to design a second gRNA that targets a specific antibiotic resistance gene carried by the host bacteria. The degree of antibiotic sensitivity after phage infection can then be used as a measure of the phage infectivity and the effectiveness of the CRISPRi system. This may be an easier and more accurate method compared with the Crystal Violet assay. We probably do not have time to conduct this suggestion, but this could be implemented in future work.

Lastly, Hans gave us some insight into the public scepticism and legal restrictions regarding the use of bacteriophages. He informed us that he had met an impressively low amount of scepticism – The key is to spread knowledge about the natural occurrence of phages and the positive effects they can have. Regarding legal restrictions he told us that it strongly depends on the usage and claims of the product. Since our potential product falls within the therapeutic section, it is among others essential to extensively evaluate it’s positive and negative effects on both the environment and health as well as document the therapeutical effects it claims to have. Due to our product being GMO, he told us that it may be even more regulations involved in legalizing our phage delivered CRISPRi system for medical usage. This led us on to dig deeper into the GMO restrictions here in Norway, and we decided to contact Bioteknologirådet (The Biotechnology Council).

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