Team:Waterloo/Translate

From 2014.igem.org

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     <li><a href="#view0">Project Inspiration</a></li>
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     <li><a href="#view0">Commercialization</a></li>
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     <li><a href="#view1">Market Analysis</a></li>
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     <li><a href="#view1">Safety</a></li>
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     <li><a href="#view2">Ethics</a></li>
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     <li><a href="#view2">Adaptations</a></li>
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     <li><a href="#view3">Scalability</a></li>
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     <li><a href="#view5">References</a></li>
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    <li><a href="#view4">Project Interests</a></li>
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       <h2>Project Inspirations</h2>
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       <h2>Commercialization</h2>
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<p>Based on the design of Staphylocide, we developed an assembly of practices that attempts to implement a therapeutic microbe into a topical medication. We considered the marketability, production, and scalability of such a medication. Dr. Marianna Foldvari, a professor whose research focuses on the development of intelligent drug delivery systems, guided our understanding of current practices in medicine and pharmaceuticals. We also analyzed the safety, quality control, and ethical issues that are presented. Preventing contamination and maintenance of the viability of S. epidermidis in a large bioreactor are examples of issues that need to be considered when scaling up. A list of excipients for the drug, a method for the scaled production of freeze-dried cells, and a protocol for administration of the drug are suggested. These practices are essential to the healthcare infrastructure in terms of Staphylocide’s real-world application.</p>
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<p>At this time, the increase in cases of MRSA and CA-MRSA infections places a high demand on a novel product for treatment without relying solely on developing new antibiotics. Interest in research for novel antibiotics has only recently increased, but the treatment of MRSA and CA-MRSA infections still relies on the use of antibiotics (Liu et al 2011). Our hope is that if our product were to be commercialized and approved for sale, it would be sufficient to treat MRSA and CA-MRSA infections when used in conjunction with conventional antibiotics. So far our system have demonstrated promising results in lab settings; but in order for this system to be developed into a treatment option, there are many questions that need to be answered. Over the course of our project, our HP team focused on addressing the following two questions.</p><br><br>
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<p><b>Our first question is how do we turn our bacterial system into the scaled up commercial product, Staphylocide? What special considerations need to be addressed wth regards to the development of this product?</b></p>
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      <h2>Market Analysis</h2>
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<p>With these questions in mind, we spoke to Dr. Marianna Foldvari from School of Pharmacy, University of Waterloo regarding the product design of Staphylocide. Dr. Marianna Foldvari provided key input that guided us in our research approach, and in our considerations for topical medication ingredients. Our consultation with her allowed us to develop our product design to a greater depth.  Furthermore, we used the exposure to address components of drug development. For example by adopting Dr. Foldvari’s considerations regarding the shelf-life of a drug, we are better informed in our process of determining the ingredients of our ideal topical medication. Although the expertise needed to integrate our lab design’s machinery with compatible drug components was limited, Dr. Foldvari’s contributions have been significant and valuable to our product.</p>
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<p> If a CRISPR-RNAi-based drug designed to treat MRSA infections were to enter the marketplace, there would be few competing technologies that could offer alternative solutions to the same problem. While new antibiotics such as NAI-107 (NBI, 2013) are being developed for MRSA infection treatment, the number of antimicrobial agents approved for human use by the FDA has dwindled down to only seven from the years 2003-2012 (IDSA, 2011). The European Centre for Disease is challenging world governments to develop ten novel antibiotics by 2020 (Edward-Jones, 2013).</p>
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<p>Although discovering new antibiotics is not an impossible task, much of the ‘low-hanging fruit’ has already been commercialized (Davies, 2006). Pharmaceutical companies must spend more time and resources for discovery. Currently, only the five largest pharmaceutical firms have antibiotic discovery divisions (Davies, 2006). Constraints on revenue, expenses, and time have slowed the discovery of novel antibiotics.</p>
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<h3>Pharmaceutical design</h3>
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<ul><li style="list-style:circle;"> Use freeze-dried Staphylocide (therapeutic microbe). (Lian, 2012)</li>
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<li style="list-style:circle;"> Include minimum nutrients for S. epidermidis. (M. Foldvari, personal communication, August 1, 2014)</li>
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<li style="list-style:circle;"> Prepare an ointment-type for the base, in accordance to the practice of matching the lesions with preparation characteristics. (M. Foldvari, personal communication, August 1, 2014) </li>
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<li style="list-style:circle;"> Prohibit the use of preservatives and packaging.(Lian, 2012) </li>
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<li style="list-style:circle;"> Compare the toxicity profile of other excipients to S. epidermidis. (M. Foldvari, personal communication, August 1, 2014) </li></ul>
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<p>Antimicrobial agents are usually only prescribed for a short period of time, and are no longer required once the infection has been addressed.  This limited duration allows only a few doses from which pharmaceutical firms can recuperate costs.  Alternatively, these companies are entering research in developing “quality of life” drugs that treat chronic illnesses, and require prolonged administration (Davies, 2006).</p>
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<h3>Product transport</h3>
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<p>Since Staphylocide contains live Staphylococcus epidermidis cells, the transport of our product to health care facilities and preparation must be considered. </p>
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<p>Given that many of the easily-discovered antibiotics are already commercialized, the time and research required to discover novel antibiotics that can be proven to be safe for human use drives up pharmaceutical research costs. This venture is hence less worthwhile now than when it was first established (Davies, 2006). On the contrary, bacteriophage therapies of MRSA infections are growing in popularity, have the advantage of being able to treat internal infections, and are estimated to be less expensive to administer than antibiotic treatments (Abedon et al., 2011).</p>
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From our research, the ideal form for our product is an ointment. Using a topical medium for administration would be the most effective for Staphylocide because most MRSA infections originate as skin infections and Staphylocide can easily be applied to patient skin.  First, during production, regular assessment of our engineered S. epidermidis cells must be performed to ensure that the plasmid is maintained. This can be accomplished by using an auxic strain.   By requiring the presence of a gene for organismal survival, adding a gene essential to growth on the plasmid with the non-functional gene found on the bacteria's genome, the bioreactor’s environmental conditions could be set such that if the plasmid were not essential for survival, the cell line would lose the plasmid in attempt to reduce its metabolic load.
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<p>
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      <h2>Ethics</h2>
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It is also important to preserve the viability of our engineered S.epidermidis cells.  Production, transportation, and storage could decrease the number of viable cells decreasing its effectiveness.  Considering the shelf-life of Staphylocide after production, the use of refrigeration during transport and storage would prevent excessive growth of the engineered S. epidermidis cells, but still keep them viable.  As this product would only be available by prescription, transport will only be from the production site to a health care facility.</p>
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      <p>Three main parties hold the onus to consider the ethical implications of synthetic biology projects into society: the researching scientist, the government/regulatory bodies, and the general public.</p>
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<h3>Scalability</h3>
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We assessed management of the project’s transition from the laboratory work into society’s functional use.
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<i><p>While we argue that characterization of a CRISPRi/RNAi conjugation mechanism has intrinsic value in the knowledge of genetic approaches to address antibiotic resistances, this project undoubtedly has instrumental value in offering much needed alternatives to one of the greatest potential public health concerns.</i></p>
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Our proposal for scaling up the project is as follows:
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<p>We are responsible to ensure that any research conducted is done so in an ethical manner, primarily to avoid causing harm while seeking scientific knowledge (Preston, 2013).   Endless arguments have been held about whether or not the research of synthetically engineered organisms holds intrinsic value, primarily because these values are subjective in nature, and therefore nearly impossible to arrive at a socially-accepted unanimity (Preston, 2013).</p>  
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Process highlights for the scaled production of donor cells
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(Lian, 2012)
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<i><p>We acknowledge the lengthy time requirements for our project to be developed, gain regulatory approval, and be commercialized, in addition to the risk that the product may fail to pass any of these required steps (Mandle, 2013).  By extension, there exists a requirement that medical practitioners make MRSA-infected patients aware that the product uses live, recombinant organisms, and address any potential risks or stigma that is associated with the treatment. </i></p>
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<p>Any regulatory bodies, and the entire government by extension, sets policies to ensure public safety, avoid public harm, and avoid discriminatory distribution of benefits and risks  associated with ‘new-to-world’ technologies (Jennings, 2013). When considering this level of governance, policy makers must carefully consider the use of rhetoric, and avoid both describing synthetic biology advances as such radical changes as to inspire fear, or as unexceptionally commonplace as to culture complacency (Jennings, 2013).  Either stage of hyperbole will alienate the public or researchers, respectively, and cause losses to societal welfare and well being (Jennings, 2013).</p>
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<i><p>We have made efforts to educate the next generation of synthetic biologists, through our efforts with the high school students via on-site visits and the Shad Valley Program, as well as a YouTube series exploring basic techniques used in the lab.  In addition to the publicly available information on the Wiki, we are attempting to add a level of transparency and social responsibility in efforts to reduce the stigma surrounding synthetic biology.</i></p>
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<p>The public is by far the largest subset of stakeholders when considering the ethics of synthetic biology.  Researchers explore avenues as to benefit societal well being (Preston, 2013), and policy makers look to protect the public interest (Jennings, 2013).  It is thus the responsibility of the public to be aware of how these changes could possibly affect them (Mandle, 2013).  When this fails, it is often communicated in the form of public outcry from a small, but highly vocal  subset of the population (Mandle, 2013).  While it is important for researchers and government bodies to educate the public and dispel rumors of perceived risks (Jennings, 2013), the onus is on the public to be receptive, internalize and find value in the new knowledge (Mandle, 2013).  The public also must be critical, and understand the contexts in which public information is presented, and the underlying biases which may be present (Mandle, 2013).</p>
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      <h2>Scalability</h2>
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<img src="https://static.igem.org/mediawiki/2014/0/0a/Waterloo_Overview.png" alt="overview of project scaling"/>
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      <p>
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We assessed management of the project’s transition from the laboratory work into society’s functional use.</p>
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<p>Our proposal for scaling up the project is as follows:</p>
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<h4><u>Process highlights for the scaled production of donor cells</u></h4>
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<ol><li>Establish dedicated manufacturing equipment and facilities.</li>
<ol><li>Establish dedicated manufacturing equipment and facilities.</li>
<li>Choose a strain for the “master seed lot” to preserve desired characteristics.</li>
<li>Choose a strain for the “master seed lot” to preserve desired characteristics.</li>
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<li>Test the batch quality under the same criteria above. This may also include animal testing.</li></ol>
<li>Test the batch quality under the same criteria above. This may also include animal testing.</li></ol>
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<h4><u>Scheme for Point of Care</u></h4>
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<ul><li>Package the base (vehicle) and donor cells separately (as a multi/single dose). (Lian, 2012)</li>
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      <h2>Safety</h2>
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<li>Screen for adverse complications (i.e. immunodeficiency) and confirm MRSA infection. (Sanofi Pasteur Limited, 2002)(Public Health Agency of Canada, 2012)</li>
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<p><b>Safety: The second question we want to address is what are the safety concerns pertaining to the use of this drug? What are measures we can take to make Staphylocide an effective, easy to use and safe product?</b></p>
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<li>Train administrators to handle and discard materials according to Biosafety Level 1 practices. (ATCC, 2014)</li>
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<li>Instructions: Reconstitute bacteria and incubate.  Inoculate base (vehicle).  Apply Staphylocide in appropriate dose(s) for an optimized period of time as determined by plasmid conjugation, and apply the β-lactam antibiotics.</li> 
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<li>Store at 2°C to 8°C until expiration. (Sanofi Pasteur Limited, 2002)(Public Health Agency of Canada, 2012)(ATCC, 2014)</li>
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<li>Short life-span for drug after reconstitution. (Sanofi Pasteur Limited, 2002)</li></ul>
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<h4><u>Pharmaceutical design</u></h4>
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<h3> Administrative controls recommended at health facilities</h3>
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<ul><li>Use freeze-dried Staphylocide (therapeutic microbe). (Lian, 2012)</li>
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<li>Include minimum nutrients for S. epidermidis. (M. Foldvari, personal communication, August 1, 2014)</li>
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<li>Prepare an ointment-type for the base, in accordance to the practice of matching the lesions with preparation characteristics. (M. Foldvari, personal communication, August 1, 2014)</li>
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<li>Prohibit the use of preservatives and packaging.(Lian, 2012)</li>
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<li>Compare the toxicity profile of other excipients to S. epidermidis. (M. Foldvari, personal communication, August 1, 2014)</li></ul>
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<h3>Product Analysis</h3>
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<p>The engineered S. epidermidis cells require a unique treatment protocol that healthcare practitioners must be familiar with. It is important to note that the use of Staphylocide alone will not be able to kill MRSA cells. It must be used on alternating cycles with conventional β-lactam antibiotics to effectively treat the infection. It is therefore crucial for patients to follow their course of antibiotic treatment. Staphylocide will only be available by prescription for patients who have been infected with MRSA. Treatment will administrated at a health care facility where healthcare practitioners can oversee the entire course of treatment. </p>
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<p>
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<p>At this time, the increase in cases of MRSA and CA-MRSA infections requires a novel product for treatment without relying solely on developing new antibiotics. Interest in research for novel antibiotics has only recently increased, but the treatment of MRSA and CA-MRSA infections still relies on the use of antibiotics (Liu et al 2011). Our hope is that if our product were to be commercialized and approved for sale, it would be sufficient to treat MRSA and CA-MRSA infections when used in conjunction with conventional antibiotics. Our product, Staphylocide, can be analyzed from the production, transport, patient and application aspects.</p>
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At this time, we do not know how effective Staphylocide will be as this information would be obtained through lab testing and clinical trials. Relevant factors from the application of our product that would influence the efficacy include the conjugation efficiency of the CRISPRi system, the quantity of donor cells provided during application, and the subsequent use of antibiotics.</p>
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<h4>Production</h4>
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<img class="icon floatRight" src="https://static.igem.org/mediawiki/2014/8/8f/Waterloo_Production.png" />
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<p>Since Staphylocide contains live Staphylococcus epidermidis cells, the transport of our product to health care facilities and preparation must be considered. From our research, the ideal form for our product is an ointment that contains our engineered S. epidermidis with the CRISPRi system. Using a topical medium for administration would be the most effective for Staphylocide as a solid surface is required for conjugation and can easily be applied to patient skin where MRSA infections occur.</p>
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  <p>
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It is important to consider the viability of our engineered S.epidermidis cells as production, transportation, and storage could decrease the number of viable cells, and therefore decrease its effectiveness. A greater viability of our engineered S. epidermidis cells during production and transportation would be highly beneficial. Greater viability would require fewer additions to the product during production, and would also ensure that the concentration of S.epidermidis cells would be sufficient for effective treatment. As a result, the components of Staphylocide must be able to maintain viable cells for as long as possible.
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As a result, health care practitioners must apply Staphylocide liberally over the infected area and allow treatment time. If Staphylocide must be removed, the necessary actions such as cleaning the infection, assessing the infection, and/or replacing the dressings should be performed as soon as possible, before re-applying the ointment on the infection.</p>
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</p>
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<p>
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<p>Another consideration is the continued propagation of the plasmid after rounds of growth in the bioreactor. During production, regular assessment of our engineered S. epidermidis cells must be performed to ensure that the plasmid is maintained. This can be accomplished by adding a gene essential to growth on the plasmid with the non-functional gene found on the bacteria's genome, and setting the bioreactor environmental conditions to require the presence of the gene for organismal survival. Otherwise, if the plasmid were not essential for survival, the cell line would lose the plasmid in attempt to reduce its metabolic load.</p>
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Healthcare practitioners should also perform regular microbiological testing of the infection site to monitor the progress of conjugation. When microbial testing indicates that all cells in the area are susceptible to β-lactam antibiotics, a transition from Staphylocide to using antibiotics can be made.</p>
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<h4>Product Transport</h4>
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Here is a product use guideline we have created outlining the major steps health care professionals must follow when using our product.</p>
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<img class="icon floatLeft" src="https://static.igem.org/mediawiki/2014/0/03/Waterloo_Production_part_2.png" />
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<ul>
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<li style="list-style:circle;">Screen for adverse complications (i.e. immunodeficiency) and confirm MRSA infection. (Sanofi Pasteur Limited, 2002, Public Health Agency of Canada, 2012)</li>
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<li style="list-style:circle;"> Train administrators to handle and discard materials according to Biosafety Level 1 practices. (ATCC, 2014)</li>
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<li style="list-style:circle;"> Instructions: Reconstitute bacteria and incubate. Inoculate base (vehicle). Apply Staphylocide in appropriate dose(s) for an optimized period of time as determined by plasmid conjugation, and apply the β-lactam antibiotics.  </li>
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<li style="list-style:circle;"> Store at (2°C to 8°C) until expiration. (Sanofi Pasteur Limited, 2002, Public Health Agency of Canada, 2012, ATCC, 2014)</li></ul>
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      <h2>Adaptations for This Project Beyond iGEM</h2>
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<p>The results of Waterloo iGEM’s project would be of interest to the following groups and industries:</p>
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<p>We must also consider the shelf-life of Staphylocide after production. It is imperative to use refrigeration during transport and storage. This would prevent excessive growth of the engineered S. epidermidis cells, but still keep them viable. As this product would only be available by prescription, transport will only be from the production site to a health care facility.</p>
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<h3>Academia/Research</h3>
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<p>Our project incorporates systems and procedures that those in academia can apply to their own research. These include the CRISPRi system that inhibits mecA (the gene responsible for β-lactam antibiotic resistance), RNAi, and conjugation in gram positive bacteria such as <i>Staphylococci</i>.</p>
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<h4>Patient Perception Considerations</h4>
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<h4>RNA Interference (RNAi) systems</h4>
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<p>Within the application perspective, the product can be further analyzed from the patient and healthcare practitioner perspective as well as for effectiveness. The reception by patients and healthcare practitioners to treatment using our engineered S. epidermidis cells could be an obstacle for widespread adoption and patient compliance. Both parties must be assured that Staphylocide is a safe and effective treatment method.</p>
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<p>RNA interference is the silencing of genes using small RNA molecules (Daka and Peer 2012). It has many potential uses where the synthesis of specific proteins must be prevented. Therapies using RNAi to treat diseases include those for glaucoma, hepatitis C virus, HIV, various forms of cancer, and diseases of the central nervous system have been investigated (Deng et al, 2013), (Ramachandran Keiser and Davidson, 2013). The versatility of RNAi allows for applications in fields beyond healthcare, such as agriculture. RNAi can be used to improve the nutritional value of crops and bioremediation potential. Engineering pathways in plants can be improved by increasing anabolic pathways while decreasing catabolic pathways for the desired end products (Tang and Galili, 2004). For example, the flavenoid and carotenoid contents of tomato plants can be increased by silencing the DET1 gene (Davulrui GR et al 2005). RNAi can also be used to improve bioremediation of contaminated areas. The accumulation of arsenic in the shoots of Arabidopsis plants was increased by up to 16 times by silencing the arsenate reductase gene (Dhankher et al 2005). Silencing arsenate reductase in the shoots allows greater mobility for arsenic to move from the roots into the shoot tissues.</p>
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Patients may have a strong aversion to treating their MRSA/CA-MRSA infection with engineered S. epidermidis, with the worst case scenario being possible refusal of treatment. This attitude would impede treatment and must be addressed by having health care practitioners educate patients and address questions concerning their treatment. Patients must understand that MRSA infections cannot be treated effectively by many antibiotics and therefore require treatment with Staphylocide.</p>
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<h4>Application Considerations</h4>
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<h4>Improving bacterial conjugation </h4>
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<img class="icon floatRight" src="https://static.igem.org/mediawiki/2014/f/f9/Waterloo_Drug.png" />
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<p>Greater opportunities for the transfer of specific genes through a bacterial population could result from improved conjugation efficiency. In bioremediation,  conjugation can be used to restore contaminated environmental areas. Many plasmids are able to provide additional metabolic capabilities to bacteria, and can therefore recipient bacteria to have enhanced abilities for the degradation of contaminants (Top Springael and Boon 2002). The ability to transfer genes from donor cells to indigenous microorganisms via conjugation allows increases survival of the indigenous microorganisms, and also increases their ability to degrade contaminants (Ikuma and Gunsch 2012). This was suggested in a Pseudomonas species in Antarctica that has acquired the ability to degrade polyaromatic hydrocarbons such as phenanthrene and naphthalene (Ma, Wang and Shao 2006).</p>
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<p>The engineered S. epidermidis cells require a unique treatment protocol that healthcare practitioners must be familiar with. Staphylocide will only be available by prescription for patients who have been infected with MRSA, and treatment will occur at a health care facility and be administered by healthcare practitioners. Proper application of this product at the site of infection is critical for effective treatments. </p>
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<p>
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At this time, we do not know how effective Staphylocide will be as this information would be obtained through lab testing and clinical trials. Relevant factors from the application of our product that would influence the efficacy include the conjugation efficiency of the CRISPRi system, the quantity of donor cells provided during application, and the subsequent use of antibiotics.
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  </p><p>
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High conjugation efficiency on the skin surface is critical for a successful treatment. While conjugation efficiency may be optimized in the lab, it would likely be lower in situ. Conjugation efficiency and donor concentration also influence the duration of treatment as time is required for the transfer of the CRISPRi system throughout the MRSA population.
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</p><p>
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As a result, health care practitioners must apply Staphylocide liberally over the infected area and allow treatment time. If Staphylocide must be removed, the necessary actions such as cleaning the infection, assessing the infection, and/or replacing the dressings should be performed as soon as possible, before re-applying the ointment on the infection.
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</p><p>
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It is important to note that the use of Staphylocide alone will not be able to kill MRSA cells. It must be used on alternating cycles with conventional β-lactam antibiotics to effectively treat the infection. It is therefore crucial for patients to follow their course of antibiotic treatment. For this reason, Staphylocide should be used only at healthcare facilities where health care practitioners are able to oversee the entire treatment.
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</p><p>
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Healthcare practitioners should also perform regular microbiological testing of the infection site to monitor the progress of conjugation. When microbial testing indicates that all cells in the area are susceptible to β-lactam antibiotics, a transition from Staphylocide to using antibiotics can be made.</p>
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<h3>Pharmaceutical Industry</h3>
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<p>Waterloo iGEM’s project findings lead could to the future development of a drug that can treat MRSA infections. With the production of a new drug, the pharmaceutical industry has a new product on the market with a comparative advantage over existing MRSA treatments, and this product would subsequently be useful to healthcare workers.</p>
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<h3>Threats challenging the completion of the work</h3>
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<h3>Healthcare</h3>
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<h5>Source Review and Methodology</h5>
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<p>MRSA quickly emerged as a deadly pathogen in intensive care units after methicillin was used as an antimicrobial agent in the 1960s (David & Daum, 2010). Since MRSA is prevalent in healthcare facilities such as hospitals and clinics, healthcare workers would be interested in Waterloo iGEM’s solution to eliminate MRSA. If a drug or disinfectant is eventually developed, the technology can be applied to treat patients and clean facilities.</p>
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<p>The recurring challenge encountered in pursuing this work was the novelty of employing donor cells as the active ingredient in a topical medication. While it is theoretically sound, the extent to which we can claim viability of the schemes is limited by the resources available to anticipate problems in practice. Therefore, using heavily modified practices from historiography, source analyses were conducted to evaluate the values and limitations of evidence needed to complete the results according to their origin and purpose.  Current pharmaceutical practices and information gathered from Dr. Marianna Foldvari were used to demonstrate this because they are major contributors to several parts of the project. </p>  
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<p>
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Although most individuals get MRSA infections from healthcare facilities, contact with MRSA can happen anywhere, and anyone can become infected. Since Waterloo iGEM’s project aims to make MRSA cells sensitive to antibiotics, public health and safety can be improved, and therefore garner public interest.</p>
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<h5>Drug delivery system focused researcher</h5>
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<h3>Livestock Operations</h3>
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<p>Dr. Marianna Foldvari provided key input that guided us in our research approach, and in our considerations for topical medication ingredients. Our consultation with her allowed us to develop our product design to a greater depth. Furthermore, we used the exposure to address components of drug development. For example by adopting Dr. Foldvari’s considerations regarding the shelf-life of a drug, we are better informed in our process of determining the ingredients of our ideal topical medication. Although the expertise needed to integrate our lab design’s machinery with compatible drug components was limited, Dr. Foldvari’s contributions have been significant and valuable to our research.</p>
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<p>80% of all antibiotics in the United States are used for livestock operations (Casey et al 2013). The use of antibiotics in livestock operations has resulted in the development of pathogens that have resistance to methicillin and other antibiotics. Livestock animals can act as reservoirs for the independent development of MRSA (Spoor et al 2013). Furthermore, livestock associated MRSA (LA-MRSA) cells have the potential to spread to humans, as shown in livestock operation workers, slaughterhouse workers, veterinarians, and in contaminated food (Rinsky et al 2013), (Van Cleef et al 2010), (Garcia-Graells et al 2011), (Boost et al 2013), (Pu, Wang, and Ge 2011). The use of Staphylocide can reduce the spread of MRSA among livestock populations, and prevent the spread to livestock workers.</p>
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<h5>Proximation through industry standard</h5>
 
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<p>Current pharmacy practices for large-batch manufacture of therapeutic bacteria were referenced from Taiwan Epidemiology Bulletin’s 2012 Seed History and In-process Control for Freeze-dried BCG Vaccine Produced in Taiwan.  Similarly, account for special storage instructions, high expiration sensitivity, as well as other post-production concerns was derived from Package Insert: BCG VACCINE (FREEZE-DRIED) from Sanofi Pasteur Limited.  The BCG (Bacillus Calmette–Guérin) vaccine was chosen as a model for designing the scaled production of Staphylocide donor cells.  Also, using live bacteria as the active therapeutic ingredient, our topical medication should be subject to similar, if not identical standards for controlling medicine quality.  These sources were valuable in our understanding of important details and practices needed, for example, to set parameters for usage and administration. As a limitation, it should be noted that the strictness for these criteria may not be necessary to use in <i>S. epidermidis </i> production, since Staphylocide will be applied topically and not intradermally as in the BCG vaccine.  Parties may be interested in pursuing this limitation to reduce costs accordingly.</p>
 
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       <h2>Project Interests</h2>
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       <h2>References</h2>
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      <p>
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Abedon, S.T., Kuhl, S.J., Blasdel, B.G., & Kutter, E.M. (2011).  Phage Treatment of Human Infections.  Bacteriophage, 1(2): 66-85.<br><br>
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        The results of Waterloo iGEM’s project would be of interest to the following groups and industries:</p>
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<h5>Academia/Research</h5><p>
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ATCC (2014).  Staphylococcus epidermidis (Winslow and Winslow) Evans (ATCC®12228™). Retrieved from https://www.atcc.org/products/all/12228.aspx?slp=1.<br><br>
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Waterloo iGEM’s project incorporates systems and procedures that those in academia can apply to their own research. These include the CRISPRi system that inhibits mecA (the gene responsible for β-lactam antibiotic resistance), RNAi, and conjugation in gram positive bacteria such as Staphylococci.</p>
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<h5>Potential Technological Applications</h5><p>
+
Bassetti, M., Baguneid, M., Bouza, E., Dryden, M., Nathwani, D., and Wilcox, M. (2014). European perspective and update on the management of complicated skin and soft tissue infections due to methicillin-resistant Staphylococcus aureus after more than 10 years of experience with linezolid. Clinical Microbiology and Infection. 20  Suppl 4:3-18<br><br>
-
Our project has utilized several different techniques to remove methicillin resistance in MRSA cells. As a result, we believe our project can provide benefits to health care research in the future. In addition, these techniques are versatile and can be used extensively in the field of synthetic biology. Using RNA interference and conjugation can be applied beyond the field of health care.</p>
+
-
<h6>RNA Interference (RNAi)</h6><p>
+
Boost, M.V., Wong, A., Ho, J., O'Donoghue, M. 2013. Isolation of methicillin-resistant Staphylococcus aureus (MRSA) from retail meats in Hong Kong. Foodborne Pathogens and Disease. 10(8):705-710<br><br>
-
RNA Interference is the silencing of genes using small RNA molecules (Daka and Peer 2012). As a result, this could have many potential uses where the synthesis of specific proteins must be prevented. Therapies using RNAi for diseases include glaucoma, hepatitis C virus, HIV, different types of cancer and for diseases of the central nervous system have been investigated (Deng et al 2013), (Ramachandran Keiser and Davidson 2013). The versatility of RNA interference allows for applications beyond healthcare. A promising field for the use of RNAi would be in agriculture for improving the nutritional value of crops and bioremediation potential. Engineering pathways in plants can be improved by increasing anabolic pathways while decreasing catabolic pathways for the desired end products (Tang and Galili 2004). For example, the flavenoid and carotenoid content of tomato plants can be increased silencing the DET1 gene (Davulrui GR et al 2005). RNA interference can also be used to improve bioremediation of contaminated areas. The accumulation of arsenic in the shoots of Arabidopsis plants was increased by up to 16 times by silencing the arsenate reductase gene (Dhankher et al 2005). Silencing arsenate reductase in the shoots allowed for greater mobility of arsenic from the roots to move into the shoot tissues.</p>
+
-
+
-
+
-
<h6>Conjugation</h6><p>
+
-
Greater opportunities for the transfer of specific genes through a bacterial population could result from improved conjugation efficiency. An area of potential use is for bioremediation to restore contaminated areas. Many plasmids are able to provide additional metabolic capabilities to bacteria and therefore could allow for enhanced abilities for recipient bacteria to degrade contaminants (Top Springael and Boon 2002). The ability to transfer genes from donor cells to indigenous microorganisms through conjugation would allow for greater survival of the indigenous microorganisms and also increase their ability to degrade contaminants (Ikuma and Gunsch 2012). This has been suggested in Pseudomonas species in Antarctica who have acquired the ability to degrade polyaromatic hydrocarbons such as phenanthrene and naphthalene (Ma, Wang and Shao 2006).</p>
+
-
<h5>Pharmaceutical Industry</h5><p>
+
Casey, J.A., Curriero, F.C., Cosgrove, S.E., Nachman, K.E., and Schwartz, B.S. 2013. High density livestock operations, crop field application of manure, and risk of community-associated methicillin-resistant Staphylococcus aureus infection in Pennsylvania. JAMA Internal Medicine. 173 (21): 1980-1990.<br><br>
-
Waterloo iGEM’s project findings lead could to the future development of a drug that can treat MRSA infections. With the production of a new drug, the pharmaceutical industry has a new product on the market with a comparative advantage over existing MRSA treatments, and this product would subsequently be useful to healthcare workers.</p>
+
-
<h5>Healthcare</h5><p>
+
Centers for Disease Control and Prevention (2013, September 10). Treating MRSA Skin and Soft Tissue in Outpatient Settings.  Retrieved October 6, 2014 from http://www.cdc.gov/mrsa/community/clinicians/index.html.
-
MRSA quickly emerged as a deadly pathogen in intensive care units after methicillin was used as an antimicrobial agent in the 1960s (David & Daum, 2010). Since MRSA is prevalent in healthcare facilities such as hospitals and clinics, healthcare workers would be interested in Waterloo iGEM’s solution to eliminate MRSA. If a drug or disinfectant is eventually developed, the technology can be applied to treat patients and clean facilities.</p>
+
Garcia-Graells, C., Antoine, J., Larsen, J., Catry, B., Skov, R., and Denis, O., (2012). Livestock veterinarians at high risk of acquiring methicillin-resistant Staphylococcus aureus ST398. Epidemiology and Infection. 140(3):393-389.<br><br>
-
<h5>Public</h5><p>
+
David, M.Z. and Daum, R.S. (2010). Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clinical Microbiology Reviews, 23(3), 616–87. doi:10.1128/CMR.00081-09<br><br>
-
Although most individuals get MRSA infections from healthcare facilities, contact with MRSA can happen anywhere, and anyone can become infected. Since Waterloo iGEM’s project aims to make MRSA cells sensitive to antibiotics, public health and safety can be improved, and therefore garner public interest.</p>
+
 
 +
Davies, J. (2006). Where have all the antibiotics gone?  Canadian Journal of Infectious Disease and Medical Microbiology, 17(5). 287-290.<br><br>
 +
 
 +
Davuluri, G.R., van Tuinen, A., Fraser, P.D., Manfredonia, A., Newman, R., Burgess, D., Brummell, D.A., King, S.R., Palys, J., Uhlig, J., Bramley, P.M., Pennings, H.M.J., and Bowler, C. (2005). Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavenoid content in tomatoes. Nature Biotechnology. 23(7):890-895<br><br>
 +
 
 +
Deng, Y., Wang, C.C., Choy, K.W., Du, Q., Chen, J., Wang, Q., Lu., L., Chung, T.K.H., and Tang, T. (2014).  Therapeutic potentials of gene silencing by RNA interference: Principles, challenges, and new strategies Gene. 538(2):217-227.<br><br>
 +
 
 +
Dhanker, O.P., Rosen, B.P., McKinney, E.C., Meagher, R.B. (2006). Hyperaccumulation of arsenic in the shoots of Arabidopsis silenced for arsenate reductase (ACR2). Proccedings of the National Academy of Sciences of the United States of America. 103(14):5413-5418.<br><br>
 +
 
 +
Edward-Jones, V. (2013, January 27).  New Antibiotics are a Matter of Life or Death.  The Telegraph, Retrieved October 6, 2014 from http://www.telegraph.co.uk/finance/newsbysector/pharmaceuticalsandchemicals/9827232/New-antibiotics-are-a-matter-of-life-or-death.html.<br><br>
 +
 
 +
Ikuma, K. and Gunsch, C.K. (2012). Genetic bioaugmentation as an effective method for in situ bioremediation: functionality of catabolic plasmids following conjugal transfers. Bioengineered. 3(4): 236-241.<br><br>
 +
 
 +
Infectious Diseases Society of America. (2011). Combating Antimicrobial Resistance: Policy Recommendations to Save Lives.  Clinical Infectious Disease, 52(5):S397-S428.<br><br>
 +
 
 +
Jennings, B.  (2013). Biotechnology as Cultural Meaning: Reflections on the Moral Reception of Synthetic Biology.  In Kaebnick, G.E. & Murray, T.H. (Eds.), Synthetic Biology and Morality: Artificial Life and the Bounds of Nature (149-176). Cambridge, MA, USA: MIT Press.<br><br>
 +
 
 +
Lian, W.C., Hsieh, W.C., Yang H.C., and Lo, Y.W. (2012, January 10). Seed History and In-process Control for Freeze-dried BCG Vaccine Produced in Taiwan. Taiwan Epidemiology Bulletin, Vol.28(No.1).  Retrieved from
 +
http://www.cdc.gov.tw/english/downloadfile.aspx?fid=7C54BEBDB7680E83.<br><br>
-
<h5>Livestock Operations</h5><p>
+
Lim, D. and Strynadka, N.C.J. (2002). Structural basis for the β-lactam resistance of PBP2A from methicillin-resistant Staphylococcus aureus. Nature Structural & Molecular Biology, 9:870-876.<br><br>
-
Livestock operations present a great opportunity to prevent the spread of MRSA. 80% of all antibiotics in the United States are used for livestock operations (Casey et al 2013). As a result, the use of antibiotics in livestock operations has resulted in the development of pathogens that have resistance to methicillin and other antibiotics. It has been shown that livestock can act as reservoirs for the independent development of S. aureus that are methicillin resistant (Spoor et al 2013). Furthermore, the livestock associated methicillin-resistant <i>Staphylococcus aureus</i> (LA-MRSA) has the potential to spread to humans as already evidenced in livestock operation workers, slaughterhouse workers and veterinarians (Rinsky et al 2013) (Van Cleef et al 2010) (Garcia-Graells et al 2011). The use of Staphylocide could be used to reduce the spread of MRSA among livestock populations and prevent the spread to livestock workers.
+
-
      </p>
+
     </div>
     </div>
   </div>
   </div>

Latest revision as of 22:42, 21 November 2014

Translate

Commercialization

At this time, the increase in cases of MRSA and CA-MRSA infections places a high demand on a novel product for treatment without relying solely on developing new antibiotics. Interest in research for novel antibiotics has only recently increased, but the treatment of MRSA and CA-MRSA infections still relies on the use of antibiotics (Liu et al 2011). Our hope is that if our product were to be commercialized and approved for sale, it would be sufficient to treat MRSA and CA-MRSA infections when used in conjunction with conventional antibiotics. So far our system have demonstrated promising results in lab settings; but in order for this system to be developed into a treatment option, there are many questions that need to be answered. Over the course of our project, our HP team focused on addressing the following two questions.



Our first question is how do we turn our bacterial system into the scaled up commercial product, Staphylocide? What special considerations need to be addressed wth regards to the development of this product?

With these questions in mind, we spoke to Dr. Marianna Foldvari from School of Pharmacy, University of Waterloo regarding the product design of Staphylocide. Dr. Marianna Foldvari provided key input that guided us in our research approach, and in our considerations for topical medication ingredients. Our consultation with her allowed us to develop our product design to a greater depth. Furthermore, we used the exposure to address components of drug development. For example by adopting Dr. Foldvari’s considerations regarding the shelf-life of a drug, we are better informed in our process of determining the ingredients of our ideal topical medication. Although the expertise needed to integrate our lab design’s machinery with compatible drug components was limited, Dr. Foldvari’s contributions have been significant and valuable to our product.

Pharmaceutical design

  • Use freeze-dried Staphylocide (therapeutic microbe). (Lian, 2012)
  • Include minimum nutrients for S. epidermidis. (M. Foldvari, personal communication, August 1, 2014)
  • Prepare an ointment-type for the base, in accordance to the practice of matching the lesions with preparation characteristics. (M. Foldvari, personal communication, August 1, 2014)
  • Prohibit the use of preservatives and packaging.(Lian, 2012)
  • Compare the toxicity profile of other excipients to S. epidermidis. (M. Foldvari, personal communication, August 1, 2014)

Product transport

Since Staphylocide contains live Staphylococcus epidermidis cells, the transport of our product to health care facilities and preparation must be considered.

From our research, the ideal form for our product is an ointment. Using a topical medium for administration would be the most effective for Staphylocide because most MRSA infections originate as skin infections and Staphylocide can easily be applied to patient skin. First, during production, regular assessment of our engineered S. epidermidis cells must be performed to ensure that the plasmid is maintained. This can be accomplished by using an auxic strain. By requiring the presence of a gene for organismal survival, adding a gene essential to growth on the plasmid with the non-functional gene found on the bacteria's genome, the bioreactor’s environmental conditions could be set such that if the plasmid were not essential for survival, the cell line would lose the plasmid in attempt to reduce its metabolic load.

It is also important to preserve the viability of our engineered S.epidermidis cells. Production, transportation, and storage could decrease the number of viable cells decreasing its effectiveness. Considering the shelf-life of Staphylocide after production, the use of refrigeration during transport and storage would prevent excessive growth of the engineered S. epidermidis cells, but still keep them viable. As this product would only be available by prescription, transport will only be from the production site to a health care facility.

Scalability

We assessed management of the project’s transition from the laboratory work into society’s functional use.

Our proposal for scaling up the project is as follows: Process highlights for the scaled production of donor cells (Lian, 2012)

  1. Establish dedicated manufacturing equipment and facilities.
  2. Choose a strain for the “master seed lot” to preserve desired characteristics.
  3. Maintain a succession of cultures so that live bacteria will be available for each new batch, transferring cells to a new culture at predetermined intervals.
  4. Inoculate vessels containing a nutrient medium. At regular intervals, evaluate the condition of cultures for preservation of desired characteristics, growth, and sterility. Growth is monitored through comparisons to the ideal optical density and, if applicable, the pH of the growth medium. Meanwhile, sterility requires the collection and subculture of a sample. Then, examined with an approved identity test using microbiological techniques and/or molecular biology techniques, S. epidermidis should be the only microbe present in the cultures.
  5. Follow the freeze-drying procedure appropriate for S. epidermidis. Evaluate the condition of cultures for growth and sterility in the same manner outlined in step 5, with the addition of weighing the freeze-dried pellet.
  6. Package the dormant therapeutic donor cells according to the standards to ensure viability when it reaches the point of care.
  7. Test the batch quality under the same criteria above. This may also include animal testing.

Safety

Safety: The second question we want to address is what are the safety concerns pertaining to the use of this drug? What are measures we can take to make Staphylocide an effective, easy to use and safe product?

Administrative controls recommended at health facilities

The engineered S. epidermidis cells require a unique treatment protocol that healthcare practitioners must be familiar with. It is important to note that the use of Staphylocide alone will not be able to kill MRSA cells. It must be used on alternating cycles with conventional β-lactam antibiotics to effectively treat the infection. It is therefore crucial for patients to follow their course of antibiotic treatment. Staphylocide will only be available by prescription for patients who have been infected with MRSA. Treatment will administrated at a health care facility where healthcare practitioners can oversee the entire course of treatment.

At this time, we do not know how effective Staphylocide will be as this information would be obtained through lab testing and clinical trials. Relevant factors from the application of our product that would influence the efficacy include the conjugation efficiency of the CRISPRi system, the quantity of donor cells provided during application, and the subsequent use of antibiotics.

As a result, health care practitioners must apply Staphylocide liberally over the infected area and allow treatment time. If Staphylocide must be removed, the necessary actions such as cleaning the infection, assessing the infection, and/or replacing the dressings should be performed as soon as possible, before re-applying the ointment on the infection.

Healthcare practitioners should also perform regular microbiological testing of the infection site to monitor the progress of conjugation. When microbial testing indicates that all cells in the area are susceptible to β-lactam antibiotics, a transition from Staphylocide to using antibiotics can be made.

Here is a product use guideline we have created outlining the major steps health care professionals must follow when using our product.

  • Screen for adverse complications (i.e. immunodeficiency) and confirm MRSA infection. (Sanofi Pasteur Limited, 2002, Public Health Agency of Canada, 2012)
  • Train administrators to handle and discard materials according to Biosafety Level 1 practices. (ATCC, 2014)
  • Instructions: Reconstitute bacteria and incubate. Inoculate base (vehicle). Apply Staphylocide in appropriate dose(s) for an optimized period of time as determined by plasmid conjugation, and apply the β-lactam antibiotics.
  • Store at (2°C to 8°C) until expiration. (Sanofi Pasteur Limited, 2002, Public Health Agency of Canada, 2012, ATCC, 2014)

Adaptations for This Project Beyond iGEM

The results of Waterloo iGEM’s project would be of interest to the following groups and industries:

Academia/Research

Our project incorporates systems and procedures that those in academia can apply to their own research. These include the CRISPRi system that inhibits mecA (the gene responsible for β-lactam antibiotic resistance), RNAi, and conjugation in gram positive bacteria such as Staphylococci.

RNA Interference (RNAi) systems

RNA interference is the silencing of genes using small RNA molecules (Daka and Peer 2012). It has many potential uses where the synthesis of specific proteins must be prevented. Therapies using RNAi to treat diseases include those for glaucoma, hepatitis C virus, HIV, various forms of cancer, and diseases of the central nervous system have been investigated (Deng et al, 2013), (Ramachandran Keiser and Davidson, 2013). The versatility of RNAi allows for applications in fields beyond healthcare, such as agriculture. RNAi can be used to improve the nutritional value of crops and bioremediation potential. Engineering pathways in plants can be improved by increasing anabolic pathways while decreasing catabolic pathways for the desired end products (Tang and Galili, 2004). For example, the flavenoid and carotenoid contents of tomato plants can be increased by silencing the DET1 gene (Davulrui GR et al 2005). RNAi can also be used to improve bioremediation of contaminated areas. The accumulation of arsenic in the shoots of Arabidopsis plants was increased by up to 16 times by silencing the arsenate reductase gene (Dhankher et al 2005). Silencing arsenate reductase in the shoots allows greater mobility for arsenic to move from the roots into the shoot tissues.

Improving bacterial conjugation

Greater opportunities for the transfer of specific genes through a bacterial population could result from improved conjugation efficiency. In bioremediation, conjugation can be used to restore contaminated environmental areas. Many plasmids are able to provide additional metabolic capabilities to bacteria, and can therefore recipient bacteria to have enhanced abilities for the degradation of contaminants (Top Springael and Boon 2002). The ability to transfer genes from donor cells to indigenous microorganisms via conjugation allows increases survival of the indigenous microorganisms, and also increases their ability to degrade contaminants (Ikuma and Gunsch 2012). This was suggested in a Pseudomonas species in Antarctica that has acquired the ability to degrade polyaromatic hydrocarbons such as phenanthrene and naphthalene (Ma, Wang and Shao 2006).

Pharmaceutical Industry

Waterloo iGEM’s project findings lead could to the future development of a drug that can treat MRSA infections. With the production of a new drug, the pharmaceutical industry has a new product on the market with a comparative advantage over existing MRSA treatments, and this product would subsequently be useful to healthcare workers.

Healthcare

MRSA quickly emerged as a deadly pathogen in intensive care units after methicillin was used as an antimicrobial agent in the 1960s (David & Daum, 2010). Since MRSA is prevalent in healthcare facilities such as hospitals and clinics, healthcare workers would be interested in Waterloo iGEM’s solution to eliminate MRSA. If a drug or disinfectant is eventually developed, the technology can be applied to treat patients and clean facilities.

Although most individuals get MRSA infections from healthcare facilities, contact with MRSA can happen anywhere, and anyone can become infected. Since Waterloo iGEM’s project aims to make MRSA cells sensitive to antibiotics, public health and safety can be improved, and therefore garner public interest.

Livestock Operations

80% of all antibiotics in the United States are used for livestock operations (Casey et al 2013). The use of antibiotics in livestock operations has resulted in the development of pathogens that have resistance to methicillin and other antibiotics. Livestock animals can act as reservoirs for the independent development of MRSA (Spoor et al 2013). Furthermore, livestock associated MRSA (LA-MRSA) cells have the potential to spread to humans, as shown in livestock operation workers, slaughterhouse workers, veterinarians, and in contaminated food (Rinsky et al 2013), (Van Cleef et al 2010), (Garcia-Graells et al 2011), (Boost et al 2013), (Pu, Wang, and Ge 2011). The use of Staphylocide can reduce the spread of MRSA among livestock populations, and prevent the spread to livestock workers.

References

Abedon, S.T., Kuhl, S.J., Blasdel, B.G., & Kutter, E.M. (2011). Phage Treatment of Human Infections. Bacteriophage, 1(2): 66-85.

ATCC (2014). Staphylococcus epidermidis (Winslow and Winslow) Evans (ATCC®12228™). Retrieved from https://www.atcc.org/products/all/12228.aspx?slp=1.

Bassetti, M., Baguneid, M., Bouza, E., Dryden, M., Nathwani, D., and Wilcox, M. (2014). European perspective and update on the management of complicated skin and soft tissue infections due to methicillin-resistant Staphylococcus aureus after more than 10 years of experience with linezolid. Clinical Microbiology and Infection. 20 Suppl 4:3-18

Boost, M.V., Wong, A., Ho, J., O'Donoghue, M. 2013. Isolation of methicillin-resistant Staphylococcus aureus (MRSA) from retail meats in Hong Kong. Foodborne Pathogens and Disease. 10(8):705-710

Casey, J.A., Curriero, F.C., Cosgrove, S.E., Nachman, K.E., and Schwartz, B.S. 2013. High density livestock operations, crop field application of manure, and risk of community-associated methicillin-resistant Staphylococcus aureus infection in Pennsylvania. JAMA Internal Medicine. 173 (21): 1980-1990.

Centers for Disease Control and Prevention (2013, September 10). Treating MRSA Skin and Soft Tissue in Outpatient Settings. Retrieved October 6, 2014 from http://www.cdc.gov/mrsa/community/clinicians/index.html. Garcia-Graells, C., Antoine, J., Larsen, J., Catry, B., Skov, R., and Denis, O., (2012). Livestock veterinarians at high risk of acquiring methicillin-resistant Staphylococcus aureus ST398. Epidemiology and Infection. 140(3):393-389.

David, M.Z. and Daum, R.S. (2010). Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clinical Microbiology Reviews, 23(3), 616–87. doi:10.1128/CMR.00081-09

Davies, J. (2006). Where have all the antibiotics gone? Canadian Journal of Infectious Disease and Medical Microbiology, 17(5). 287-290.

Davuluri, G.R., van Tuinen, A., Fraser, P.D., Manfredonia, A., Newman, R., Burgess, D., Brummell, D.A., King, S.R., Palys, J., Uhlig, J., Bramley, P.M., Pennings, H.M.J., and Bowler, C. (2005). Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavenoid content in tomatoes. Nature Biotechnology. 23(7):890-895

Deng, Y., Wang, C.C., Choy, K.W., Du, Q., Chen, J., Wang, Q., Lu., L., Chung, T.K.H., and Tang, T. (2014). Therapeutic potentials of gene silencing by RNA interference: Principles, challenges, and new strategies Gene. 538(2):217-227.

Dhanker, O.P., Rosen, B.P., McKinney, E.C., Meagher, R.B. (2006). Hyperaccumulation of arsenic in the shoots of Arabidopsis silenced for arsenate reductase (ACR2). Proccedings of the National Academy of Sciences of the United States of America. 103(14):5413-5418.

Edward-Jones, V. (2013, January 27). New Antibiotics are a Matter of Life or Death. The Telegraph, Retrieved October 6, 2014 from http://www.telegraph.co.uk/finance/newsbysector/pharmaceuticalsandchemicals/9827232/New-antibiotics-are-a-matter-of-life-or-death.html.

Ikuma, K. and Gunsch, C.K. (2012). Genetic bioaugmentation as an effective method for in situ bioremediation: functionality of catabolic plasmids following conjugal transfers. Bioengineered. 3(4): 236-241.

Infectious Diseases Society of America. (2011). Combating Antimicrobial Resistance: Policy Recommendations to Save Lives. Clinical Infectious Disease, 52(5):S397-S428.

Jennings, B. (2013). Biotechnology as Cultural Meaning: Reflections on the Moral Reception of Synthetic Biology. In Kaebnick, G.E. & Murray, T.H. (Eds.), Synthetic Biology and Morality: Artificial Life and the Bounds of Nature (149-176). Cambridge, MA, USA: MIT Press.

Lian, W.C., Hsieh, W.C., Yang H.C., and Lo, Y.W. (2012, January 10). Seed History and In-process Control for Freeze-dried BCG Vaccine Produced in Taiwan. Taiwan Epidemiology Bulletin, Vol.28(No.1). Retrieved from http://www.cdc.gov.tw/english/downloadfile.aspx?fid=7C54BEBDB7680E83.

Lim, D. and Strynadka, N.C.J. (2002). Structural basis for the β-lactam resistance of PBP2A from methicillin-resistant Staphylococcus aureus. Nature Structural & Molecular Biology, 9:870-876.

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