Carbapenems have emerged as “last-line agents” or “antibiotics of last resort” in the treatment of life-threatening infections involving multi-resistant strains of bacteria([1,2]). Therefore, carbapenemase-producing organismspresent a significant challenge to the continued clinical efficacy of existing carbapenem antibiotics. In this context, the goal of the SuperBugBuster project is to re-sensitize bacteria that have become resistant to carbapenems, thus paving the way for new strategies to prevent or treat infections caused by carbapenem-resistant bacteria (Description).
The use of antibiotic-resistant strains of bacteria, antibiotics, and other chemicals in the laboratory was required to conduct the experiments, leading to the construction of our tool. To be able to handle such materials while ensuring the safety of people workingat the laboratory, but also to preventany leakage of hazardous materialin the environment, strict regulations had to be respected ([3]).
The experiments were conducted in a biosafety level 2 laboratory, using various safety gear and equipment to ensure containment of biological material.
Additionally, all the members of our team who worked at the laboratory went through safety training beforestarting experiments. We have submitted check-in forms and consulted experts about managing risks (especially regarding the spread of antibiotic resistance). According to safety regulations, we modified our experimental design and methodology and evaluated countermeasures against hazardous organismsand parts. Our Principal Investigator and laboratory researchers supervised experiments, with an institutional biological safety officer ensuring safety.
Use a lab coat made out of non-flammable materials.
Do not smoke, drink, or eat during experiments.
Wash hands as often as needed, especially before and after experiments to avoid contamination.
Wash surfaces: benches, tables, and other devices that come into contact with biological material (bacteria, ...) and that could be contaminated.
Several chemicals were used during experiments. Some were more hazardous than others, so we had to take into account the different preventive measures associated with the use of these chemicals ([3], [4]).
* CMR : Carcinogenic, Mutagenic, Reprotoxic (negative effects on fertility)
A diverse ensemble of E. coli strains were used in our experiments, each with its unique role within our experimental strategies.
None of those strains could be environmentally hazardous except for the TOP10/pOXA48 E. coli strain, which carries a gene coding for a carbapenemase-inducing carbapenem resistance. When this bacterial strain is used to test the efficiency of the tool we designed, strict safety measures are respected, especially regarding waste disposal.
The following flow chart summarizes chemical waste management at the laboratory where all experiments were conducted, except for antibiotic-contaminated medium disposed of as biological waste.
After sorting chemical wastes, a specialized company retrieves them for treatment.
All the experiments were conducted in an L2 biosafety laboratory under a microbiological safety station with protective clothing, and all the tools used were autoclaved. Moreover, the biological wastes were evacuated through the DASRI chain. All the Lyon 1 University DASRI are sent for incineration so they can not contaminate the environment or laboratory workers. After each use, everything was also cleaned with a powerful agent, the Virkon, which is very efficient against bacteria.
Our projectaims to fight against severe infections caused by Gram-negative antibiotic-resistant organisms such as the bacteria Klebsiella pneumoniae ([1]),which is responsible for urinary infections, pneumonia, and other severe nosocomial infections.
As of right now, our tool can not fulfill this purpose. However,if it were to be developed furtherfor real-world use after the iGEM project stage, it should be able to improve human health. It could also be used in other strategies involving antibiotic-resistant microorganisms, such as:
Our tool and modified bacterial stains should not be able to spread autonomously in the environment. We used biocontainment strategies to prevent it.
First, regarding the system we designed, we have chosen to put the dCas9 fused to the cytidine deaminase module under the control of an anhydrotetracycline-inducible promoter, which will allow us to have control over our tool. The inducible promoter also allows for a shorter induction period for our tool. Since the longer the tool is active, the more probable off-targets are, using an inducible promoter reduces the chances of targeting a sequence it should not.
Our tool will be put on a miniF plasmid deleted from its partition machinery, which implies that this plasmid is unstable and rapidly loss during bacterial division. In addition, the miniF plasmid carries the RP4 origin transfer but does not carry the RP4 conjugative machinery and thus is not self-transmissible. So after the initial conjugation between the donor strain MFDpir/ miniF and the recipient strain Top10/pOXA48, our miniF tool could not be transferred by conjugation to another bacteria, making it a plasmid with a short lifespan.
Finally, a negative selection gene, sacB, was also integrated into our device. In the presence of saccharose, the bacteria carrying our tool expresses sacB, a lethal gene to E. coli. By staying on the plasmid that carries our tool, the sacB gene reduces the probability of spreading our tool in the environment. Initially, sacB was used as a selection method, but we plan to replace it either with a reporter gene (mCherry, GFP, enabling conjugation to be visualised), or with the second tool we have tried to design: the BacProtac system targeting carbapenemases (Description).
For more information regarding the use of the parts listed below, check out the following link : Parts
| Name | Hazard |
|---|---|
| BBa_K4818000 | No hazardous effect |
| BBa_K4818001 | No hazardous effect |
| BBa_K4818002 | No hazardous effect |
| BBa_K4818003 | No hazardous effect |
| BBa_K4818005 | No hazardous effect |
| BBa_K4818006 | No hazardous effect |
| BBa_K4818007 | No hazardous effect |
| BBa_K4818008 | No hazardous effect |
| BBa_K4818009 | No hazardous effect |
| BBa_K4818011 | No hazardous effect |
| BBa_K4818012 | No hazardous effect |
| BBa_K4818013 | No hazardous effect |
| BBa_K4818014 | No hazardous effect |
| BBa_K4818020 | No hazardous effect |
| BBa_K4818021 | No hazardous effect |
| BBa_K4818022 | No hazardous effect |
| BBa_K4818023 | No hazardous effect |
| BBa_K4818024 | No hazardous effect |
| BBa_K4818025 | No hazardous effect |
| BBa_K4818026 | Hazardous |
| BBa_K4818027 | No hazardous effect |
| BBa_K4818029 | Hazardous |
| BBa_K4818030 | No hazardous effect |
| BBa_K4818040 | No hazardous effect |
| BBa_K4818041 | No hazardous effect |
| BBa_K4818044 | No hazardous effect |
| BBa_K4818045 | No hazardous effect |
| BBa_K4818046 | No hazardous effect |
| BBa_K4818050 | No hazardous effect |
| BBa_K4818053 | No hazardous effect |
| BBa_K4818054 | No hazardous effect |
| BBa_K4818055 | No hazardous effect |
| BBa_K4818056 | No hazardous effect |
| BBa_K4818057 | No hazardous effect |
| BBa_K4818060 | No hazardous effect |
| BBa_K4818070 | Hazardous |
| BBa_K4818071 | Hazardous |
| BBa_K4818081 | No hazardous effect |
| BBa_K4818085 | No hazardous effect |
| BBa_K4818090 | No hazardous effect |
| BBa_K4818091 | No hazardous effect |
| BBa_K4818101 | No hazardous effect |
| BBa_K4818102 | No hazardous effect |
| BBa_K4818103 | No hazardous effect |
| BBa_K4818104 | No hazardous effect |
| BBa_K4818105 | No hazardous effect |
| BBa_K4818106 | No hazardous effect |
| BBa_K4818120 | Hazardous |
The Super BugBuster system aims at fighting antibiotic-resistance.
We focused on designing two complementary strategies based on the CRISPR and BacPROTAC systems. The goal was to combine the two strategies in one plasmid to increase the overall system's efficiency in re-sensitizing carbapenem-resistant bacteria . (Description).
The E. coli K12 Top10/pOxa48 strain used to test our system's efficiency has a particular genotype that allows us to control its spread in the environment. This bacteria is auxotrophic for leucine, which means it can not synthesize leucine, an amino acid essential for its growth, so it should not survive or multiply on a medium not supplemented in leucine. Thanks to this auxotrophy, the bacteria should not be able to spread antibiotic resistance in the environment.
A) BacPROTAC-based system
The efficiency of the BacPROTAC system had to be tested on the OXA-48 carbapenemase. This protein was overproduced thanks to a specific bacterial strain (BL21) carryinga plasmid in which the gene codingfor OXA- 48 was inserted but without the signal sequence addressing the protein to the periplasm.
This particular design resulted in an inactive form of the OXA-48 protein, which conferred no resistance to ß- lactam antibiotics and could safely be used for our experiments. This also allowed us to handle a bacterial strain (BL21) with diminished risks of spreading carbapenemase resistance.
B) CRISPR-based system
To prove the efficiency of the CRISPR-based strategy, we planned to transfer, by conjugation, the plasmid carrying our tool to an antibiotic-resistant bacteria. However, this plasmid still carries a gene coding for chloramphenicol resistance, a remnant of its construction process.
Since we didn't want to spread any more antibiotic resistance by transferring a functional gene coding for chloramphenicol resistance in the carbapenem-resistant bacterial strain Top10/pOxa48 (recipient strain), a solution had to be found. On top of the gRNAs already targeting the gene coding for a carbapenemase responsible for resistance to ß-lactam antibiotics such as carbapenems and penicillins, another gRNA, this time targeting the chloramphenicol resistance gene was added to our tool. This way, when the SuperBugBuster system is induced, the carbapenem-resistant bacteria that received our tool loses its carbapenem resistance as well as the chloramphenicol resistance that was transferred along with the tool. By using this strategy, when our system is induced, the recipient bacteria should neither be able to spread carbapenem resistance nor chloramphenicol resistance in the environment.
[1] Pitout, Johann D D et al. “The Global Ascendency of OXA-48-Type Carbapenemases.” Clinical microbiology reviews vol. 33,1 e00102-19. 13 Nov. 2019, doi:10.1128/CMR.00102-19
[2] Jean, Shio-Shin et al. “Global Threat of Carbapenem-Resistant Gram-Negative Bacteria.” Frontiers in cellular and infection microbiology vol. 12 823684. 15 Mar. 2022, doi:10.3389/fcimb.2022.823684
[3] https://www.legifrance.gouv.fr/loda/id/JORFTEXT000000465273/
[4] https://echa.europa.eu/fr/regulations/clp/clp-pictograms