Our Goal
The aim of Healios is to benefit the growing need for alternatives to antibiotic treatments for bacterial infections. With antimicrobial resistance causing 1.27 million deaths in 2019 alone[3], it is clear something needs to change. We hope that Healios can continuously be altered to attack a multitude of bacterial pathogens, with our plasmid design making this possible. The current target for us is Staphylococcus aureus. S. aureus are gram-positive bacteria that produce exfoliative toxins[1] that can cause skin infections if not properly combated. Thus, increasing the likelihood of these infections in immunocompromised patients. The infection can be deadly when it enters the bloodstream or other parts of the body and releases its other toxins, enterotoxins and/or toxic shock syndrome toxin-1[1]. The emergence of antibiotic-resistance in S. aureus strains is concerning, the most notable being methicillin-resistant Staphylococcus aureus (MRSA). MRSA is especially prominent in hospital settings,[3] where some of our most vulnerable populations are located, resulting in further medical hurdles. The need for a change is clear, but what can we do? Healios seeks to bridge the gap in treatments for antibiotic-resistant S. aureus and prevent the emergence of new antibiotic-resistant strains. By using a genetically modified dye-bound M13 bacteriophage and helper bacteriophage, paired with photodynamic therapy (PDT), the answer is closer than ever. Through outreach, speaking with industry leaders, and surveying the population, our understanding and influence of how to combat the world of antibiotic-resistant bacteria has increased substantially.
Project Development: Speaking Viability into Reality
At the beginning of our project exploration, an in-depth analysis led us to discover two interesting treatment options: bacteriophage therapy and photodynamic therapy (PDT).
Phages and Focus
Our team is privileged to have a phage biology expert, Dr. Paul Kirchberger, to consult. Dr. Kirchberger received his Ph.D. in Microbiology and Biotechnology from the University of Alberta in Dr. Yann Boucher’s lab. He furthered his research through a postdoctoral fellowship at the University of Texas at Austin in Dr. Howard Ochman’s lab where he published multiple works on phages from the family Microviridae. In recent years, he has been an assistant professor at Oklahoma State University and continues his work on small bacteriophages, with a particular focus on using synthetic biology techniques to engineer these viruses. With such a skilled professor to consult, we felt confident he would be the best person to aid us in harnessing the power of phage therapy.
Dr. Kirchberger provided us immense amounts of advice throughout the project, but to start he helped us to decide on S. aureus as our model bacteria. He explained that phage therapy can be incredibly beneficial, but it is important to note that phage delivery can be problematic. This is where S. aureus was decided on. A superficial Staph infection would allow us to add a phage with significantly more ease. The next hurdle faced was with the best phage to be utilized for the applicability to future bacterial infections we wanted to target. This is where we decided on the M13 bacteriophage. The M13 bacteriophage normally attaches to E. coli, but by altering its genome we could cause it to attach to S. aureus and, theoretically, other bacterial pathogens. The primary issue faced would then be the M13 bacteriophage not retaining its original infection.
Bright Solutions
Our next stop to understand the best solution for our project was Dr. Ihsiu Huang. Dr. Huang received his Ph.D. in Microbiology from Oregon State University. This was followed by post-doctoral fellowships at the University of Oklahoma Health Sciences Center and the University of Texas Health Sciences Center at Houston. He continued his work as an assistant professor at National Cheng Kung University and is presently an assistant professor at Oklahoma State University Health Sciences Center where his primary research focus is bacterial pathogenesis. Out of the works he had published, the most interesting to us was on PDT that utilizes ROS generating photoreactive dyes.
Speaking with Dr. Huang on his experience with photodynamic therapy. The primary issue Dr. Huang saw was the lack of specification when targeting the bacterial infection. “Keeping healthy cells unharmed is especially important when working with immunocompromised patients.” In turn, the plan to combine the benefits of photodynamic therapy and phage therapy was decided. By using a dye-bound bacteriophage to specifically target the bacteria of interest, it becomes possible to target the infection while mitigating harm to healthy surrounding host tissues and the beneficial skin microbiome. Additionally, Dr. Huang aided us in deciding the correct wavelength for the light to properly react with the photosensitizing dye while lessening negative effects to surrounding healthy cells.
A Rosy Outlook
With our solution decided, the next thing we needed was an expert who could help with the chemical conjugation of Rose-Bengal to the M13 bacteriophage. This is where Dr. David Miller was an immense amount of help. Dr. Miller is an organic chemist who received his Ph.D. in Chemistry from Princeton University while working under Dr. Robert Knowles. This was followed by a postdoctoral fellowship under Nobel Prize winner Dr. Frances Arnold. Dr. Miller is currently an assistant professor at Oklahoma State University where his research focuses on biocatalysis. Overall, Dr. Miller was an incredible resource for our project.
The organic chemistry expertise of Dr. Miller provided us with proper insights into the best conjugation process. He explained that for our project, selective labeling of the Rose-Bengal dye would not be necessary. The localization of the singlet oxygen from the dye containing a carboxylate group would allow for the efficient killing of the infection when paired with the photodynamic therapy. He also provided us with proper safety information regarding necessary reagents for the procedures. For example, “repeated exposure to peptide-coupling reagents like EDC will cause people to develop an allergic reaction to the [1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide].”
Final Blueprint
After our final conversations with the experts in our fields of interest, we had a final idea for our project. The plan was genetic modification of a Rose-Bengal dye-bound M13 bacteriophage, paired with a helper phage to create the structural proteins. The photodynamic light would then be applied using blue light between 450-500 nm causing the reaction with the Rose-Bengal dye releasing the singlet oxygen responsible for lysing the pathogenic bacterial cell.
From Bench to Bedside
To reach our ultimate goal of helping patients suffering from S. aureus infections, it is necessary to better understand the viability of our project as a business. Fortunately, we had the opportunity to consult with Elizabeth Pollard and Daniel Will. Elizabeth Pollard is the current CEO and President of the Oklahoma State University Innovation Foundation and Cowboy Enterprises. Additionally, she serves as a Strategic Advisor for xCures, Board Director and Finance Chairperson of YPO, and Chairperson of the Board for Oklahoma Center for Advancement of Science and Technology (OCAST). Daniel Will is the Executive Director for Oklahoma State University’s Cowboy Technologies and Innovation Accelerator. These skilled professionals gave us insight into the process of commercializing our project.
Pollard has served on many committees for products in the medical field and helped with regulatory management, therefore one of her main priorities was explaining the process of receiving EPA and FDA approval. She explained that it isn’t a quick process and “it takes time and effort.” Furthermore, there are more regulations beyond these when you are treating patients directly. Will explained the Intellectual Property process to us. “The complications arise when it comes to who directly owns the rights.” Since we have used outside papers to aid in the design of our project and university funding, it becomes difficult to determine the patentable parts of the project and who will receive compensation. We were provided a direct contact, Russ Hopper who works at the OSU Office of Technology Commercialization at Cowboy Enterprises to better understand our project’s intellectual property and the possibility of a startup company through the Innovation Accelerator.
Educating Ourselves
In order to help others, it is important for us to understand our own biases and work to combat them. There is a prevalence in bacterial infections being related to intravenous drug use, one of the most common of which being caused by Staphylococcus aureus[5]. Since this demographic would be benefitted by our project, we believe any biases that we may have need to be combatted to best serve the population.
To understand this, we conducted a survey that would be sent to other iGEM teams and our own. It is important to note that 4/20 respondents were not from the OKstate iGEM team. The survey consisted of questions surrounding one’s personal opinions surrounding drug use and the perceived stigma in one’s society. In the survey “drugs are defined as an illegal or misused legal substance that alters mood, perception, or behavior.”
85% of respondents believe that there is a stigma towards those that use drugs in their community.
45% of respondents describe their attitude towards those that use drugs as negative.
45% of respondents describe the stigma surrounding intravenous drug use as greater than other methods of drug use and 20% of respondents describing the stigma as much greater.
From this survey, it is clear something needs to change. The stigma surrounding drug use makes it significantly more difficult to seek help[2], thus to combat biases and increase accessibility for our project, all team members are fighting to combat their own biases.
From Lab to Lesson Plans
One important goal for our team is to widen the understanding and world of synthetic biology and the importance of combating antibiotic-resistance. We believe that one of the best ways to accomplish this is by teaching people of all ages about our project and how they can engage in the world of synthetic biology.
Middle School Outreach
Our latest initiative was at our local middle school. We partnered with the One Health Innovation Lab to teach around 120 7th-graders how to extract DNA from their own cheek cells. We explained to them our process of plasmid extraction and the similarities and differences between their procedures and our own.
High School Outreach
The next age group we worked with was at a high school in a surrounding city. We worked with a group of around 50 9th-graders and taught them how to form their own experiment using the fundamentals of the scientific process. Their experiments were incredibly innovative and creative, some of which were related to synthetic biology. Then the class learned how we formed our iGEM project and the relationship between scientific experiments and the engineering design process. One student even came up with a synthetic biology experiment using artificial organs as a diagnostics tool.
University
For one of our team’s fundraisers, we hosted a “pie-a-friend” event. This event allowed students and community members to pay money to pie a friend or iGEM team member with a tin of whipped cream or shaving cream in the face. We also had informational flyers about our club and talked to the community about our project and the benefits of synthetic biology in a fun and engaging way.
References
- Bukowski, M., Wladyka, B., & Dubin, G. (2010). Exfoliative toxins of Staphylococcus aureus. Toxins, 2(5), 1148–1165. https://doi.org/10.3390/toxins2051148
- Crapanzano, K., Hammarlund, R., Ahmad, B., Hunsinger, N., & Kullar, R. (2018). The association between perceived stigma and substance use disorder treatment outcomes: A Review. Substance Abuse and Rehabilitation, Volume 10, 1–12. https://doi.org/10.2147/sar.s183252
- Hassoun, A., Linden, P. K., & Friedman, B. (2017). Incidence, prevalence, and management of MRSA bacteremia across patient populations—a review of recent developments in MRSA management and treatment. Critical Care, 21(1). https://doi.org/10.1186/s13054-017-1801-3
- Murray, C. J., Ikuta, K. S., Sharara, F., Swetschinski, L., Robles Aguilar, G., Gray, A., Han, C., Bisignano, C., Rao, P., Wool, E., Johnson, S. C., Browne, A. J., Chipeta, M. G., Fell, F., Hackett, S., Haines-Woodhouse, G., Kashef Hamadani, B. H., Kumaran, E. A., McManigal, B., … Naghavi, M. (2022). Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis. The Lancet, 399(10325), 629–655. https://doi.org/10.1016/s0140-6736(21)02724-0
- Rehman, S., Arif, S., Ushakumari, L. G., Amreen, J., Nagelli, A., Moonnumackel, S. J., & Nair, A. (2023). Assessment of bacterial infections and antibiotic regimens in intravenous drug users. Cureus. https://doi.org/10.7759/cureus.45716