Team:IISc-Bangalore/Intergrated HP
Integrated Human Practices
Overview
Antibiotic resistance is a global challenge, and proposing a solution to it via improvement in phage therapy which is still not completely understood presented to us a tricky situation which required considerable thought and discussion. The motivation which led PhageShift to take its current form came from our active outreach and dialogue with the community. Human Practices was thus an integral part of PhageShift, from the very beginning, till the very end!
Searching for problems
India is the second most populous country, and as a developing country, it faces several challenges on a wide variety of issues. We held discussions within the team and with our PIs, and decided that our project idea would be on utilizing synthetic biology to solve such a real-world challenge. We implemented this decision in a unique way; our brainstorming sessions involved discussions on news articles and social issues, and prospective synbio based solutions to them! This helped us in multiple ways. Apart from providing a starting point to think of solutions and experiments, it also helped us visualize how Human Practices, software, hardware and various other aspects of the iGEM project could be built around a particular social issue.
On January 30, a report by The Bureau of Investigative Journalism bought to light how usage of antibiotics, particularly the last-resort drug colistin, in the poultry industry as weight boosters was playing a major role in contribution of worldwide drug resistance and warned of emergence of superbugs [1]. This was picked up by several newspapers which brought it to our attention.
This provided the seed for us to read more about colistin resistance, and to see what we could do to tackle it. We then learnt about the mobilised colistin resistance (mcr-1) gene, which confers plasmid mediated resistance to colistin[2]. Since it can be horizontally transferred between different strains of bacteria, it poses a serious threat in rendering the last-hope antibiotic colistin obsolete. The mechanism of resistance is through addition of phosphoethanolamine to lipid A in the cell membrane, which decreases the affinity of colistin for lipid A and thus lowers the antimicrobial activity of the drug. We decided to look into alternatives to antibiotics, and on reading about phage therapy, we planned out how bacteriophages could be improved to exploit antibiotic resistance. And thus, on this note our project idea of PhageShift began, with a special emphasis on colistin resistance via PACMAN.
Survey to gauge perception
As we read further about the causes of antibiotic resistance in India, it was clear that the problem was the rampant misuse in the medical and poultry sectors [2][3]. To know more about how aware the Indian population was on antibiotics and phage therapy, we decided to conduct a survey that would also be helpful in identifying key concerns which people would have with bacteriophages. The reason this was important was that bacteriophages are viruses, and viruses have a very negative connotation for a large part of the populace who associate them diseases and danger. The questions were designed keeping iGEM's advise on scientific surveys in mind, and we put out the survey for the general public to fill, and especially invited many doctors and healthcare professionals to participate in it.
The survey saw 193 responses, 93 of whom were in the medical profession. Some had over 15+ years of experience, and some more were just starting out. 34 respondees were involved in scientific research and another 64 said they were in professions besides medicine and research (including students). The diversity and number of reposnses helped us greatly in assessing the viewpoints of a broad community whom we identified as stakeholders in our project. Our survey report summarises our findings, and how we intended to take it forward. Two things emerged from the survey that had a large impact on our project. Firstly, that lot of people were unaware about phage therapy, and among those who had heard of it, there was significant concern on the effects of using phages would have on our bodies. Secondly, that cases of antibiotic resistant bacterial infections were clearly on a rise, which we could correlate to the higher than average antibiotic consumption in India. This prompted us to develop a series of videos which explained briefly what phages were and how they affect bacteria, as well as increase awareness on the consequences of misuse of antibiotics.
How PAIR came to be
To further explore the role of PhageShift in therapeutics, we sought to understand what are the challenges that antibiotics faced in the last few decades as the primary form of treatment of bacterial infections. We studied articles and clinical cases that brought some of these points forth. Among them, the one that stood out to us was the problem of endotoxin release due to antibiotic induced lysis of gram negative bacterial cell membrane, which has severe consequences in patients with multi-organ failure (MOFs) and low immunity[4]. Inflammation is also a problem in young patients especially.
At this stage, it was important for us to understand the real-life consequences of antibiotic usage which was happening around us. We visited and interacted with doctors in our institute's Health Center and then contacted medical professionals in nearby hospitals, and spoke to them on what and how severe the problem of antibiotic induced endotoxin release was. We also sought their views on phage therapy and antibiotic resistance. Our discussions with them highlighted the problem of endotoxin release, and they encouraged us to see if we could mitigate this problem in our project. It was clear to us that this problem would not go away even in phage therapy, as the process of bacterial lysis remained.
Motivated to tackle this, we studied the ability of our body's natural immune cells in getting rid of bacteria, and found out that the endotoxin release here was considerably lesser. We then spoke to Prof. Dipshika Chakravortty in the Microbiology and Cell Biology department of IISc, who has worked on molecular pathology and immunology to know more about this. Equipped with this understanding, and inspired to ensure phage therapy would not contribute to the problem of endotoxin release, we designed PAIR, the second component of our project. Later on, we interviewed Dr. Nirmala (IISc Health Center) and Dr. Abhishek Bhalerao (Bowring and Lady Curzon Hospital) on why they believe endotoxin release is a problem that needs to be tackled, and the interview is available on our Youtube channel.
Speaking with GangaGen and researchers
The idea of PAIR was to use lysis deficient phages to recruit the body's immune cells to clear bacteria which would help treat the infection while minimizing endotoxin release. To know more about lysis deficient phages, we visited GangaGen, a biotechnology startup in Bangalore which has two patents on this and is India's leading company on phage research. It also presented to us an opportunity to know more about the rules which govern the development of new therapeutic agents.
This meeting proved to be highly informative, as we learnt about the work GangaGen did on phages, and they were very kind in listening to our idea and providing valuable inputs. Among other things, they talked to us about how the overcoming of phage resistance in APES was unlikely (which we later proved through our modelling), discussed the feasibility of our PAIR and PACMAN ideas, and gave some helpful tips on working with phages. They spoke about the lengthy process of getting a medicine approved which helped us realise the impact of our project on the biotechnology and pharmaceutical industry, and how one of the problems with phage therapy is the uncertain dosage for safe, therapeutic action. We took the idea of dosage forward by carrying out experiments on the MIC for bacterial lysis.
We also sought to incorporate feedback from scientific researchers, another community we identified as a stakeholder in our work. To achieve this, we discussed our ideas with Prof. Ry Young (Texas A&M University), and Prof. Venigalla B. Rao (Catholic University of America), both of whom are well known names in bacteriophage research. We discussed our PAIR idea with Prof. Ry Young, a pioneer in lysis deficient phages. We had planned a fourth component of the project, called CARE (CRISPR-cas9 assisted Resistance Elimination), where we aimed to deliver Crispr-cas9 system into the pathogen using a phage. However, Prof. Rao pointed out that ~500 bp is the maximum sequence length that can be added to the T4 genome safely without any encountering problems in packaging. Considering this, we decided not to pursue CARE further.
Motivation to review laws
Our meeting with GangaGen made us curious about the regulations which govern topics such as antibiotic resistance and phage therapy, and whether we were complying with the framework in our proposal of PAIR, PACMAN and APES. We comprehensively read the "Scoping Report on Antimicrobial Resistance in India" prepared for the Department of Biotechnology (DBT), Government of India, and Research Councils United Kingdom (RCUK) by the Center for Disease Dynamics, Economics & Policy, India (CDDEP).
To our surprise, we found no legal guidelines on working with phages, and what the legal status of phage therapy in India is. We contacted CRI Kasauli, India's only government funded lab which is allowed to do bacteriophage testing. We then reviewed the Drug and Cosmetics Act, 1940 which governs the manufacturing and sale of drugs in India, to see how they apply to phages. This was in stark comparison to Eastern European countries where there are clear laws on phage therapy, and USA, where the Food and Drug Administration has cleared clinical trials. This provided the spark for us to prepare a comprehensive review on phage therapy in the Indian context. This review has been described in our public engagement page.
User testing
Persistence in work, in spite of the number of failures we faced, paved a way for the team to carry out their work without any major roadblocks.
References
[1] Davies, M. "A game of chicken: how Indian poultry farming is creating global superbugs", The Bureau of Investigative Journalism, 30 January 2018.
[2] Patel, Isha, et al. "Antimicrobial resistance in India." Journal of pharmaceutical policy and practice 10.1 (2017): 27.
[3] Bhutia, L. "How India Became The Antibiotics Capital Of The World And Wasted The Wonder Cure", Huffington Post, 31 October 2015.
[4] Holzheimer, R. G. "Antibiotic induced endotoxin release and clinical sepsis: a review." Journal of Chemotherapy 13.sup4 (2001): 159-172.