Antibiotic resistance is a natural phenomenon that emerged chaotically due to our overconsumption of antibiotics. Nowadays, it represents one of the biggest threats to our world for alimentary safety, public health, and development.
Antibiotic resistance is the phenomenon by which bacteria become resistant to antibiotics.
Antibiotics are mostly synthetic molecules but can also be natural or semi-synthetic, introduced in our society with the discovery of Penicillin by Alexander Flemming in 1928. Those molecules, ineffective against viruses and fungi (antifungals are used against them), are specific to one or a group of bacteria and can slow down their growth or even kill them. Different types of antibiotics exist, determined by their way of action (some will act on DNA replication, others on essential proteins synthesis...).
Figure 1 : Explanation of antibiotics and antibiotic resistance mechanisms.
Over the last century, they helped to fight and drastically reduce the fatality of once-deadly diseases, including ear infections.
But bacteria exposed to antibiotics evolve and develop defense mechanisms, allowing them to escape their actions. This phenomenon affects both the bacteria that cause infections (pathogenic bacteria) and the generally harmless bacteria that are naturally present in our bodies (commensal bacteria). But it also concerns bacteria in animals (pets and, to a greater extent, food-producing animals) and in the environment. Once resistance has developed in one or more of these bacterial species, it can be transmitted to other species, contributing to the spread of the phenomenon. Antibiotics thus become ineffective, and we can no longer treat infections caused by resistant bacteria.[7]
This is not a new phenomenon. Since the discovery of penicillin, each new generation of antibiotics has seen the emergence of corresponding resistance mechanisms. The first resistance to penicillin appeared in 1940. The first multi-resistant bacteria (MRB), which resists multiple antibiotics, appeared in the 1970s, while highly resistant bacteria (HRB), which resists almost all antibiotics, emerged in the 2000s. [4]
Antibiotic resistance is spreading extremely rapidly, affecting everything and everyone, making it one of the greatest threats to global public health, animal health, food safety, biodiversity, and the global economic balance.
Global public health insecurity is exacerbated by the lack of new antibiotic discoveries over the last 20 years, a situation which, according to the AMR, does not seem to be improving, as this sector is unattractive to pharmaceutical companies. [6]
Figure 2 : Pharmaceutical R&D investments in 2003 and 2013
124 806
Number of patients with resistant bacterial infections in France in 2019
1,3 millions
Number of deaths worldwide due to antibiotic resistance in 2019
10 millions
Estimated number of deaths worldwide due to antibiotic resistance in 2050
Figure 3 : All-age rate of deaths attributable to and associated with bacterial antimicrobial resistance by GBD region, 2019, [2]
Antibiotic resistance is not only a health threat but also a threat to global economic equilibrium. One of the UN's main objectives for 2030 is to end extreme poverty in the world. Given the costs associated with the increasingly complex and lengthy treatment of patients suffering from bacterial infections and the impact of high mortality, this objective could be called into question. According to the World Bank, the diversity of this phenomenon makes it one of the greatest threats to the economy. It could cause economic damage on a scale at least comparable to that caused by the 2008 financial crisis.
The impact on poverty would be particularly significant. By 2050, antimicrobial resistance could force another 28.3 million people into extreme poverty, including 26.2 million in low-income countries.
"The scale and nature of this economic threat could wipe out hard-won economic development gains and move us further away from our goals of ending extreme poverty and promoting shared prosperity."
World Bank Group President Jim Yong Kim
"if nothing is done to curb antibiotic resistance, it is the poor who will suffer most. This is why we need to treat this problem as a crucial development issue," says Margaret Chan, Director General of the World Health Organization (WHO).
Figure 4 : Forecasts of the economic impact of antibiotic resistance in 2050, graph inspired by the 2016 World Bank Group study [5].
Figure 5 : Mapping the spread of KPC,NDM and OXA-48 producing bacteria, 2017 [3]
Carbapenems are antibiotics known to be broad-spectrum. They are effective against many different types of bacteria, especially ones already resistant to other antibiotics. This is why they are seen as the "last chance" antibiotics. However, with the widespread use of these antibiotics, the prevalence of carbapenem-resistant bacteria has increased rapidly.
Infections caused by bacteria producing carbapenemases, an enzyme that can hydrolyze carbapenems, have experienced unprecedented intercontinental spread and proliferation and continue to present a therapeutic challenge.
The most effective carbapenemases for carbapenem hydrolysis and geographic spread are the KPC, VIM, IMP, NDM, and OXA-48 types.
Figure 6 : Sum up of the carbapenems classification [1]
Our project aims to prevent the spread of antibiotic resistance while focusing on carbapenemase, an enzyme produced by carbapenem-resistant bacteria. To achieve this, we developed a plasmid carrying a CRISPR dCas-9 fused with a mutagenesis module as well as a BacPROTAC system
Our ultimate goal is to create a system capable of combating different antibiotic resistances. By targeting a specific bacterial strain, we aim to transfer the system through conjugation to eliminate antibiotic resistance in a bacterial population.
Figure 7: Overview of our Project with 2 tools: CRISPR and BacPROTAC
The aim is to use donor bacteria that will transmit a plasmid containing the CRISPR-dCas9 system as well as BacPROTAC to resensitize the recipient bacteria to an antibiotic instead of just killing it.
SuperBugBuster operates on two fronts: a synthetic biology approach aimed at reversing past errors by eliminating and preventing the spread of resistance. And an education and awareness front aimed at teaching people how to use antibiotics more effectively so that technical efforts work in the long term. Sustainability is indeed the watchword of our project. We need a solution that works quickly but, above all, lasts over time.
You can find all aspects of our study leading up to our awareness campaign on the HP and Education pages.
We chose to focus on carbapenem resistance and, more specifically, on the OXA-48 carbapenemase. We aspire to contribute to the scientific advances that will enable existing carbapenems to retain their clinical efficacy and thus save lives. Using synthetic biology, we are trying to design a genuine tool for combating resistance: we combine different methods and improve them.
Our biotechnological tool was created in several major stages, which you can find on our experiments page:
- Construction of the plasmid containing the guide RNAs
- Deletion of unnecessary sequences in the plasmid containing the origin of replication and the origin of transfer
- Assembly of the TetR_dCas9_CDA1_Ugi system
- Assembly of various intermediate plasmids by Gateway cloning
- Modeling of the nanobody structure.
- Insertion of the nanobody into an overexpression plasmid vector.
- Overproduction of the three best nanobody variants.
- Construction of a plasmid containing OXA48 for CRISPR testing.
- Use of the CRISPR tool on the plasmid containing OXA48.
- Characterization of CRISPR tool efficiency:
- Assessment of the activity of the CRISPR tool on the OXA48 target.
- Measurement of genomic editing or target inhibition efficiency.
Figure 8 :Comic strip taking up the concept of SuperBugBuster: repairing the mistakes of the past with synthetic biology and educating sustainably with human practices
Our team comprises very diverse profiles, a strength on which we have based our project by integrating much more than just synthetic biology. All our achievements have been possible thanks to the constant support of our IP and advisors.
Figure 9: picture of our software
The design of our biological tool was constantly linked to our human practices study. We conducted numerous interviews with experts in antibiotics, doctors, veterinarians, ethicists, and biodiversity specialists. We aimed to confront SuperBugBuster with their opinions to perfect it, make it safer and even more sustainable.
Our team has designed a software program to create the guide RNAs needed for binding to resistance genes. This software is applicable to any gene and any context. It is also compatible with various CRISPR tools: CRISPR-dCas9, CRISPRi, and CRISPR-dCas9 with a cytidine deaminase. This software was essential to support the design of the plasmid used for biological manipulations and to guarantee the safety of our tool, which will not induce mutations in other genes of the bacterial genome.
Figure 10: One of our members in an interview with Claude Mabilat
Figure 11: Simulation of our IBM model
Our team also carried out mathematical modeling to predict the proper functioning of our in silico tool. The model then makes choosing initial conditions for the test phases possible, ensuring optimal operation.
With all of that, SuperBugBuster is much more than just a biological construction. Our tool is defined based on 5 major concepts to answer a problem like antibiotic resistance.
Figure 12: SuperBugBuster key characteristics
Figure 13 : Big steps of SuperBugBuster
Our first step is to modify the bacteria's genome to eliminate the resistance.
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Secondly, we plan to use the Bacprotac system to degrade the resistance proteins.
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As the resistance of the bacteria comes from a gene carried by a plasmid or located in the DNA, the best solution was to inactivate this gene. Indeed, if it is inactivated, the proteins won't be produced, and it will conduce to the loss of the resistance.
To do so, we planned to use the molecular scissors CRISPR. However, cutting a gene would lead to the death of the bacteria. So, it was better to induce a mutation on the gene to inactivate it, resulting in a truncated non-functional protein. Thanks to that, when bacteria die and release their DNA, other bacteria in the ecosystem cannot integrate the functional carbapenem resistance gene in their genome by natural transformation.
The cytosine deaminase, a base editor tool, was ideal for introducing a stop codon in the gene coding for carbapenemases. So we fused it with the CRIPSR-dCAS9 system, which would lead the editor tool to the right sequence thanks to its gRNA designed specifically for that. To achieve this goal, we designed a modular system that consists of four independent plasmids:
The four independent modules are flanked by the Gateway recombination motifs (attR and attL) and should be assembled in the end in a unique LR reaction to create the final plasmid carrying the four modules.
Figure 14 : Our construction for Gateway Assembly
PROTACs are recent chemically synthesized molecules that promote the degradation of a specific protein. They have been synthesized mainly for eukaryotic cells. The latter are cells that contain a nucleus and other membrane-bound organelles, whereas prokaryotic cells, like bacteria, don't have them. In eukaryotic cells, PROTACs address the target protein to the cell proteasome (a complex that will degrade unneeded or damaged proteins by proteolysis) by recruiting a molecule that will add a recognition signal: a polyubiquitin chain.
It's only in 2022 that PROTACs have been produced for bacteria and mycobacteria (prokaryotic) [9]. For this type of organisms they are called BacPROTACs. It was a significant advance since the bacterial degradation system is completely different from the eukaryotic one with the ubiquitin system. Indeed, not all bacteria have a proteasome system: they can only have proteases (smaller molecules with catalytic domains that allow them to break down proteins). However, 80% of them possess the Clp protease system, so we decided to use it.
But how does it work? We wanted to target a specific protein: the OXA48 carbapenemase protein. For that, our BacPROTAC will be linked to the targeted protein but also to the bacterial degradation system, which is, for the bacteria that we are interested in, ClpX:ClpP, a multi-enzymatic complex. This complex will unfold and then degrade our targeted protein.
Through bioinformatics and especially structural biology and molecular docking, we had to find a moiety that binds to the OXA48 carbapenemase and a moiety that binds to the ClpX (once the binding activates ClpX, it'll recruit ClpP and degrade the carbapenemase). To link those two moieties, we'll use a linker.
However, instead of injecting it into the bacteria, we want the bacteria to produce it. Inserting the BacPROTACs DNA sequence along the CRISPR system DNA sequence in a plasmid, it will be transferred by conjugation in the targeted bacteria. When activated, it will be translated into protein, and then it will degrade the carbapenemases that are already present in the cytoplasm.
To adapt the BacPROTACs to other proteins, we only need to change the nanobody we're using. Technically, the ClpX-ClpP system is not present in all the bacteria, but it is the most present of all degradation systems. [10]
Figure 15 : Function of our BacPROTAC system
For the BacPROTAC system, we designed a plasmid with a gene coding for a bifunctional molecule that looks like a PROTAC. This molecule is made of 2 parts: the Nanobody (Oxa48 ligand), which binds to OXA48, the protein of interest, and the second part with the ClpX protein, which is part of the ClpX:ClpP protein complex serving as a bacterial proteasome. A linker links these two parts.
It works as follows: Nanobody recruits OXA48, and by proximity to ClpX, OXA48 is brought to ClpP, which is activated when bound to ClpX so that OXA48 is degraded. One advantage is that the Nanobody-linker-ClpX molecule is recyclable and will, therefore, degrade all the OXA48 present in the bacteria.
One possible application of our project is to enable gene repression in a bacterium easily. Indeed, it can be useful to repress gene expression to understand relationships between certain genes and their functions. By designing a specific guide RNA, our tool allows mutations in the gene we want to repress by creating a nonsense codon. Therefore, depending on the mutation position in the gene, the expressed protein will be non-functional.
Furthermore, the resistance gene on the plasmid carrying the construct will allow for the selection of mutants. Additionally, the plasmid has been designed to be quickly lost, which has the advantage of no longer expressing the plasmid genes after a certain period.
In conclusion, our tool enables the repression of a desired gene by specifying a guide RNA, and it allows for the selection of mutants without altering the genetic expression of the bacterium, except for the desired gene.
Figure 16: Illustrations of SuperBugBuster application fields
We have developed a proof of concept, but it is possible to envision future therapeutic uses for our project. Indeed, our project could, for example, help combat bacterial resistance to antibiotics. Let's take carbapenems, which are antibiotics from the beta-lactam family, as an example. Most bacteria resistant to these antibiotics are found in the digestive tract, which is also the site of their propagation. These resistances are primarily present on a conjugative plasmid. Our tool can target these resistance genes and thus disable the bacteria's ability to resist antibiotics.
This example can be applied to carbapenems but can be extrapolated to many other antibiotic resistances that complicate patient treatment. Another application could be in dermatology to combat the appearance of skin blemishes.
It is possible to imagine solutions to combat antibiotic resistance in the environment, agriculture, and other fields. Another application is in the industrial sector. Antibiotic resistance cassettes are often used in the bioproduction of molecules of interest through biotechnology. Our tool would allow for the removal of these resistance cassettes, thereby improving the safety of individuals who come into contact with bacteria producing these molecules of interest.
As biology students, we are particularly concerned about the issue of antibiotic resistance. But this issue goes further than that. We are all consumers of antibiotics and can be affected by the dangerous phenomenon of antibiotic resistance. Antibiotic resistance is the modern world's new pandemic, and we are all aware that anyone near or far from us could one day find themselves in a critical situation due to a lack of functional antibiotics.
What's more, this problem deeply affects poor populations and underdeveloped countries with low levels of medicalization. It is essential to explore the human implications of this phenomenon to understand how it might be battled on a global scale.
This problem affects human health, animals, and the environment with water pollution, for example. Therefore, every member of our team is genuinely involved in this project and impacted by the problem we are trying to tackle with our tool.
According to Courrier International, antibiotic resistance kills around 3,500 people every day. We are committed to studying the causes and consequences of this problem to combat it more sustainably and contribute to the fight against it.
A major issue...
... for different reasons...
... that touchs everyone...
We quickly agreed on the subject of antibiotic resistance, and the question was how we would combat this scourge. Many inspirations guided our project. We found them by consulting experts around us and by looking at previous iGEM projects. Here is a non-exhaustive list of our inspirations :
These two promising papers encouraged us in our research, and we built upon them to create the future of the field. To further develop our project, our team also relied on the work done by previous iGEM teams for inspiration :
We began our iGEM adventure with one goal, shared by all our highly motivated student members: to combat antibiotic resistance. Today, we proudly present SuperBugBuster, a safe, sustainable, standardized, open-source, and modular biotechnology tool. With the help of our IP and the laboratories close to our team, we have contributed to achieving the global goal of "Local people solving local problems, using synthetic biology, everywhere around the world". The applications of our tool are very promising, contributing to the progress of science and society against this global scourge.
[1] Entérobactéries productrices de carbapénémase (CRE ou EPC). URL: https://www.hpci.ch/prevention/bases-theoriques/microorganismes-et-pathologies/entérobactéries-avec-carbapénémase-cre-ou
[2] Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis 399, 629–655. doi: https://doi.org/10.1016/ S0140-6736(21)02724-0
[3] Bonomo, R.A., Burd, E.M., Conly, J., Limbago, B.M., Poirel, L., Segre, J.A., Westblade, L.F., . Carbapenemase-producing organisms: A global scourge doi: 10.1093/cid/cix893.
[4] Ministère de la santé et de la prévention, France. L’antibiorésistance : pourquoi est-ce si grave ? URL: https://sante.gouv.fr/prevention-en-sante/ les-antibiotiques-des-medicaments-essentiels-a-preserver/ des-antibiotiques-a-l-antibioresistance/article/ l-antibioresistance-pourquoi-est-ce-si-grave .
[5] Jonas, O.B., Irwin, A., Berthe, F.C.J., Le Gall, F.G., Marquez, P.V., . Drug-resistant infec- tions : a threat to our economic future (vol. 2): final report.
[6] Renwick, M.J., Simpkin, V., Mossialos, E., . Targeting innovation in antibiotic drug discovery and development: The need for a one health – one europe – one world framework doi:28806044.
[7] Werth, B.J., . Carbapénèmes. URL: https://www.msdmanuals.com/fr/accueil/infections/antibiotiques/carbap%C3%A9n%C3%A8mes.
[8] 2021. Reuter A. et al. Targeted-antibacterial-plasmids (TAPs) combining conjugation and CRISPR/Cas systems achieve strain-specific antibacterial activity. Nucleic Acids Res. 2021 Apr 6;49(6):3584-3598
[9] Morreale, Francesca E et al. “BacPROTACs mediate targeted protein degradation in bacteria.” Cell vol. 185,13 (2022): 2338-2353.e18. doi:10.1016/j.cell.2022.05.009
[10] Hoi, David M et al. “Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival.” Cell vol. 186,10 (2023): 2176-2192.e22. doi:10.1016/j.cell.2023.04.009
[11] Armstrong T, Fenn SJ, Hardie KR. JMM Profile: Carbapenems: a broad-spectrum antibiotic. J Med Microbiol. 2021 Dec;70(12):001462. doi: 10.1099/jmm.0.001462. PMID: 34889726; PMCID: PMC8744278