SuperBugBuster aims to eliminate antibiotic resistance in bacteria that has become overabundant in the environment and the human body due to our antibiotic use and activities. We will now explore how the tool we have developed can be effectively utilized to contribute to the fight against antibiotic resistance.
We have currently focused on beta-lactam resistance. This type of resistance is carried by a wide range of bacterial strains. Typically, these resistances are found on plasmids (multi-copies) and rarely on chromosomes. These bacteria (CRE) reside in the host's gastrointestinal tract before becoming pathogenic as they breach human barriers. But resistance can also be transmitted through tools used during hospital procedures. (reference)
Our tool could therefore be used preventively in the human or animal intestinal flora to eliminate resistance in bacteria that may become potentially pathogenic later on, or even as a preventive measure in the environment. By preventing the spread of resistance and preserving the intestinal flora from resistances, a person suffering from a bacterial infection could receive antibiotic treatment when necessary.
We have opted for a non-destructive tool for bacteria to preserve the balance of ecosystems, whether in the stomach or in the environment (Goal #15 of the Sustainable Development Goals).
High-copy resistance plasmids:
The pOXA48 plasmid we utilized is a low-copy conjugative plasmid. However, if the resistance gene is present on a high-copy plasmid, the quantities of Cas9 and guide RNA produced must be sufficient to target all copies of the plasmid. This is why we placed Cas9 and the guide RNAs under the control of a highly inducible promoter (the induction rate of the Tet system is approximately 300 X).
Form of the preventive tool:
We have considered various forms for the administration of our tool. The first option is in the form of a probiotic. To achieve this, we have contemplated the lyophilization of bacteria to preserve them for later reconstitution in the patient's digestive tract. Once in the target organ of resistance, we can facilitate conjugation to minimize the targeted resistance.
The issue of off-target effects when using a mutagenic module arose. Therefore, we made efforts to minimize this concern. To achieve this, we considered using our software to design target motifs for the resistance we aim to eliminate. Once these motifs are identified, the software will then be employed to assess whether these motifs could induce a mutation in the human genome and mitochondria. Only designs causing no damage will be retained.
Inherent Physical Limitations Ensure Isolation of Engineered Proteins:
It is important to note that due to the size of the CRISPR protein, as well as that of the PROTAC, it is physically impossible for these molecules to cross the cell membrane of human cells or bacteria. Therefore, even in the event of protein release during the death of a bacterium, including those we have designed, they will remain confined inside the cell or the specific microenvironment of the deceased bacterium, without the risk of entering other cells or causing undesirable effects.
Strategic Control and Short Lifespan Minimize Off-Target Risks:
The short lifespan of our biological device significantly reduces the risk of off-target effects. Furthermore, we have increased control over plasmid transfer, which can only occur through the donor bacteria we introduce into the organism. Importantly, the mobilizable plasmid we use does not contain the cellular components necessary for autonomous transfer. Moreover, the donor bacteria we introduce are auxotrophs for diaminopimelate (DAP) and are unable to survive in absence of DAPincapable, limiting the persistence of the plasmid in the environment.
Precision Control of the CRISPR Module:
Regarding the CRISPR module, it is strictly regulated by an inducible promoter sensitive to anhydrotetracycline, allowing us to exert precise control over its activation. Thus, we avoid continuous activation that could potentially lead to unwanted non-specific effects. In addition our CRISPR-base editor contains a C-terminal degradation tag that addresses our tool to the proteasome and limits its half-life.
Balancing Dysbiosis Concerns with Antibiotic Resistance Fight:
The donor bacteria introduced into the organism are auxotrophs for diaminopimelate (DAP) and are unable to survive in absence of DAP, which means they will not induce dysbiosis (an imbalance in the biodiversity of our intestinal flora). This is because their presence can only minimally disturb the intestinal flora for a short period of time. Furthermore, as we do not induce the death of the bacteria but only a mutation in the gene responsible for resistance, this reduces the risks of dysbiosis. This was one of our major concerns, given the essential role of our intestinal flora in our well-being.
Dysbiosis can occur when carbapenems are used to treat an infection. However, in this specific case, a careful risk-benefit assessment is necessary. The use of carbapenems predates the emergence of resistance, and historically, it had not been associated with any harmful consequences. Moreover, dysbiosis is a common occurrence when antibiotics are used, and currently, there is no solution available other than the use of probiotics to reinforce the intestinal flora.
In essence, the potential for a short-lived disruption of the intestinal flora due to our intervention is a minor concern when weighed against the urgent need to combat antibiotic resistance and the relative, transient nature of any disruption.
Enhancing Plasmid Selection Precision with Multi-Guide RNA Approach:
Using a resistance gene, as we did, to enable the selection of clones that have acquired our plasmid during transformation may seem counterproductive. That's why we devised a plasmid containing four guide RNAs: two for targeting carbapenemase genes and two for targeting the chloramphenicol resistance gene, which is the antibiotic that facilitates selection. This approach ensures that we do not inadvertently transmit chloramphenicol resistance, as it becomes active concurrently with the carbapenemase genes.
Optimizing Long-Term Usage of Our Preventive Tool:
For our tool to be effective in humans, it needs to be used over an extended period. The short lifespan of the plasmid, implemented for the sake of controlling its spread, reduces conjugation efficiency. Therefore, the tool should be used continuously to maximize the duration of contact among various bacteria, thus increasing the likelihood of successful conjugation. Furthermore, as the tool is intended for preventive use, it should be employed regularly, as individuals are not immune to encountering resistant bacteria that can readily propagate resistance within the host's digestive tract.
In summary, we envision a preventive tool that must always be accompanied by a comprehensive awareness campaign on antibiotic resistance to ensure its long-term viability. This tool aims to eliminate resistance in the intestinal flora and prevent its spread, while remaining non-destructive to preserve the balance of the flora. Our top priority is to thoroughly understand all safety implications associated with this tool. Additionally, further considerations are required to enhance the tool's effectiveness, given that the intestinal flora environment is much more complex than what can be replicated in vitro.
Although additional work and research are needed to develop a strain suitable for widespread human therapeutic use, we believe that this goal is achievable.