<h2 class="SubSectionTitle">Antibiotic resistance is one of the greatest threats to global public health</h2>
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<p>Antibiotic resistance poses a serious threat to global public health, with over <span class="grouped">23 000</span> people dying annually in the United States alone as a result of antibiotic resistant bacterial infections<sup>[1]</sup>.
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Additionally, antibiotic
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resistant bacterial infections cause extensive strain on global healthcare systems. For instance, <span class="blobs"><span class="wobblybobbly"></span><a href="https://2018.igem.org/Team:Leiden/Human_Practices" class="popoutBlob" target="_blank"> Read more about these
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patients on our Integrated Human Practices page</a><span> Dutch patients</span></span> infected with multidrug resistant tuberculosis need to receive near daily treatments lasting up to two years, of which they spend up to six months in
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quarantaine. In 2014, <span class="grouped">480 000</span> cases of multidrug resistant tuberculosis occured globally<sup>[2]</sup>. Worst of all, the frequency of multidrug resistant bacterial infections is expected to rise greatly in the
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coming decades. Estimates
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suggest over 10 million people will die annually due to antibiotic resistance by the year 2050<sup>[3]</sup>. This has lead organisations such as the World Health Organization and United Nations to proclaim antibiotic resistance one of the
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greatest threats to global public health<sup>[4][5]</sup>. </p>
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<h2 class="SubSectionTitle">Opportunities are missed in current antibiotic research</h2>
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<p> To avert this crisis, new antibiotics are acutely needed to allow for the treatment of multidrug resistant bacterial infections. However, despite extensive research, discovery of new antibiotics is sparse<sup>[6]</sup>. These poor results
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are putting pharmaceutical companies off from investing in the search for new antibiotics<sup>[7]</sup>. Therefore, we believe new strategies to revitalize research are needed in the search for antibiotics. To that end, we delved into current
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antibiotic research by speaking to researchers and companies attempting to discover new antibiotics. During these investigations we identified two key missed opportunities in antibiotic research. First, all compounds incapable of killing
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bacteria are discarded. However, such compounds may still hold unknown abilities to damage bacteria - without killing them - which could be utilized in combination therapies.
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<br><br>
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Secondly, research into the mode of action of novel
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antimicrobials is only performed at late stages of development and takes a considerable time investment. This way, opportunities for the enhancement of bactericidal capabilities are missed. To address these two missed opportunities, we have
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developed a novel screening platform for detecting bacterial stress.</p>
<p>Our screening system is able to visualize specific bacterial stresses by coupling stress activated promoters to visual markers. During our project we have successfully isolated and screened 26 promoters - originating from stress related
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pathways in <i>E. coli</i> - for their stress responsive capabilities (see figure below).</p>
<p> Based on discussions with future users of our system, we have created two versions of our stress responsive system. Firstly, we have created a system producing <span class="blobs"><span class="wobblybobbly"></span><span class="popoutBlob" style="cursor:default"> A conjugated
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protein with a pigmented prosthetic group
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</span><span> chromoprotein</span></span> markers - visible to the naked eye - in response to cellular stresses. This system can be used in combination with bacterial overlays, a technique used to identify bacteria that produce
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antimicrobials,
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responsible for the identification of many currently used antibiotics. Traditional overlay screening only identifies bacterial colonies producing lethal concentrations of antibiotics. Our chromoprotein system upgrades traditional overlays to
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allow for detection of possible antibiotics at a much lower concentration and to enable screening for stressful substances. Additionally, information is given on the mechanism of action of the candidate compound.</p>
<p>Secondly, we have created cell lines producing fluorescent markers in response to cellular stresses. This allows for screening towards multiple distinct cellular stresses in one test, by using a different fluorescent marker for each stress
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activated promoter. Moreover, results can be acquired using standard fluorescence detection machinery present in most labs. We have further characterized the responses of these promoters using a repertoire of known antibiotics. As these
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reporter cell lines are more readily quantifiable, we have designed a screening system using these fluorophore cell lines to be used in laboratories worldwide.</p>
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<h2 class="SubSectionTitle"> 50S.O.S. enables easy large scale bacterial stress screening</h2>
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<p>Based on the aforementioned screening system, we have developed an easy-to-use kit to enable rapid testing of candidate compounds by anyone with access to an ML-1 lab. This kit was developed to cater to the needs of our various future users,
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who may either use our system for small scale
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testing of a specific compound, or for large scale screening of compound libraries. Additionally, extensive safety discussions aided in the creation of the final design of our kit.
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The kit will contain sealed strips and holds four different freeze dried <i>E. coli</i> cell lines, each with three stress-related promoters fused to fluorophores. Therefore, a total of 12 stress related promoters, representing 5 stress
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pathways in
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bacterial cells, will be included in our kit.
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Strips are inserted into a provided testing plate - designed with the same proportions as 96 well plates - , which can hold enough strips to test up to 23 different compounds and a negative control.
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<br><br>
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Our 50S.O.S. screening procedure
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consists of 5 easy steps. Firstly, the desired number of strips are inserted into the testing plate (Step 1 and 2). Next, MilliQ is added to the freeze dried bacteria to revive them (Step 3). Subsequently,
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candidate compounds can be added for testing (Step 4). After an incubation period, the testing plate can be inserted into any 96 well plate compatible device designed to measure fluorescence (Step 5). The read-out will contain information on
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the stress inducing capabilities of the tested compounds on five categories of bacterial cell stress.</p>
<p>Previous research has determined a synergistic effect for certain combinations of antimicrobial agents<sup>[8]</sup>. We believe that newly discovered stressful substances can be combined to search for synergistic effects, in order to
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establish lethal
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combination therapies.
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Our system could be used to assess the effectiveness of compound combinations in enhancing bacterial stress levels. Besides providing novel treatment options, antibiotic combination therapies have also been indicated to reduce the chance of
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resistance forming<sup>[9]</sup>, something previously shown in HIV and cancer combination therapies<sup>[10][11]</sup>. Thereby, such therapies would tackle the antibiotic resistance crisis twofold.</p>
<p>Pharmaceutical companies are abandoning the search for novel antibiotics due to increasing difficulties in antibiotic discovery. 50S.O.S. aims to revitalize this pursuit. By targeting two major missed opportunities, iGEM Leiden will make a
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contribution in the fight against antibiotic resistance by stimulating antibiotic discovery. You can read about our proof of concept screening experiment on <a href="https://2018.igem.org/Team:Leiden/Demonstrate" class="colouredlink">our
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demonstration page</a>.
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<br><br>
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During discussions with experts of antibiotic resistance, we have also realised this issue cannot be solved by just discovering new antibiotics. Our conversations with experts in the field and representatives of
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government agencies showed us that public awareness of antibiotic resistance is just as vital. Therefore, we have developed an extensive <a href="https://2018.igem.org/Team:Leiden/Public_Engagement" class="colouredlink" target="_blank">outreach
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program</a> to promote public consciousness on the topic of the antimicrobial resistance crisis and improve handling of antibiotics.</p>
<p id="Ref3">[1]: Center for Disease Control and Prevention (CDC). (2018, September 10). Antibiotic / Antimicrobial Resistance (AR / AMR). Retrieved October 9, 2018, from https://www.cdc.gov/drugresistance/index.html
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</p>
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<p id="Ref4">[2]: World Health Organisation (WHO). (2018, February 15). Antimicrobial Resistance. Retrieved October 9, 2018, from
<p id="Ref4">[3]: Ventola, C.L. (2015). The Antibiotic Resistance Crisis: Part 1: Causes and Threats. Pharmacy and Therapeutics, 40(4), 277–283.
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</p>
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<p id="Ref4">[4]: World Health Organisation (WHO). (2018, February 5). Antibiotic Resistance. Retrieved October 9, 2018, from http://www.who.int/en/news-room/fact-sheets/detail/antibiotic-resistance
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</p>
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<p id="Ref3">[5]: World Health Organisation (WHO). (2016, September 19). Antimicrobial Resistance: Global action plan on antimicrobial resistance. Retrieved October 9, 2018, from
<p id="Ref4">[6]: Silver, L. L. (2011b). Challenges of Antibacterial Discovery. Clinical Microbiology Reviews, 24(1), 71–109. https://doi.org/10.1128/cmr.00030-10
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</p>
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<p id="Ref4">[7]: Hu, C. (2018, 21 juli). Pharmaceutical companies are backing away from a growing threat that could kill 10 million people a year by 2050. Business Insider. Retrieved from
<p id="Ref4">[8]: Acar, J. F. (2000). Antibiotic synergy and antagonism. Medical Clinics of North America, 84(6), 1391-1406. https://doi.org/10.1016/S0025-7125(05)70294-7
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</p>
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<p id="Ref4">[9]: Xu, X., Xu, L., Yuan, G., Wang, Y., Qu, Y., & Zhou, M. (2018). Synergistic combination of two antimicrobial agents closing each other’s mutant selection windows to prevent antimicrobial resistance. Scientific Reports, 8(1).
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https://doi.org/10.1038/s41598-018-25714-z
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</p>
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<p id="Ref4">[10]: Yardley, D. A. (2013). Drug Resistance and the Role of Combination Chemotherapy in Improving Patient Outcomes. International Journal of Breast Cancer, 2013, 1–15. https://doi.org/10.1155/2013/137414
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</p>
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<p id="Ref4">[11]: Katlama, C. (1996). Safety and Efficacy of Lamivudine-Zidovudine Combination Therapy in Antiretroviral-Naive Patients. JAMA, 276(2), 118. https://doi.org/10.1001/jama.1996.03540020040027
