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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.


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: 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:


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.


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.


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