Exploring the CRISPR-Cas9 potential to revert beta-lactam resistance in clinically relevant gram-negative bacteria

The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of Enterobacteriaceae family. One innovative approach to tackle AMR is the use of CRISPR-Cas9 system which has recently been employed for plasmid curing in model strains of Escherichia coli. During this thesis, this system was further exploited by targeting the blaTEM-1 resistance gene located on a high copy plasmid (i.e. > 100 copies/cell) and by directly tackling the blaTEM-1-and blaKPC genes from clinical isolates, both prevalent in multidrug-resistant bacteria. It is known that clinical Enterobacteriaceae strains possess multiple resistance mechanisms which could impair the efficiency of the system. In addition, the resistance levels are directly dependent of the plasmid copy number. Hence, the aim of this thesis was to explore further the CRISPR-Cas9 technology in challenging conditions, especially when facing high copy plasmids and when targeting clinical bacteria. This thesis aims at contributing to clarify all the possible scenarios obtained with the application of the CRISPR-Cas9 system in resistance reduction in order to establish this tool as an alternative method to counteract AMR. By using independent techniques as fluorescence-activated cell sorting (FACS), qPCR, and fluorescence microscopy, low levels of plasmid maintenance were detected in the cells upon CRISPR-Cas9 insertion. However, even though plasmid integrity could be detected, sequence alterations in the blaTEM-1 gene were observed, resulting in a dysfunction of the gene product and, therefore, in an antibiotic sensitive strain. In a clinical isolate of E. coli, plasmid clearance and re-sensitization to five beta-lactams were achieved. Reusability of antibiotics could be confirmed by infecting larvae of Galleria mellonella with CRISPR-Cas9-treated E. coli, as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of Enterobacter cloacae and to a lesser extent in Klebsiella variicola, both of which harboured also the blaCTX-M gene. When changing target to the blaKPC gene, resistance reduction to the intermediate level of imipenem was achieved in 63% of the retrieved clones of Klebsiella oxytoca clinical isolate. Interestingly, both still resistant and intermediate sensitive clones had the plasmid copy number and the blaKPC gene expression reduced. Moreover, fitness levels were also significantly decreased when either intermediate or still resistant clones were compared to the CRISPR-Cas9 untreated control. However here, CRISPR-Cas9 did not contribute to increase larvae survival after challenge with different clones of K. oxytoca. Finally, a functional CRISPR-Cas9 delivery system via bacteriophages was developed, expanding the possibilities of applicability of the technique. To conclude, our data demonstrated that the targeted strain was not able to sufficiently evade the CRISPR-Cas9-based manipulation and maintain resistance phenotype by means of plasmid amplification. Despite the versatile challenges imposed by clinical isolates, the interference with the resistance gene led after all from minor to clear resistance reductions. Overexpression of efflux pumps or alterations in porins in the clinical isolates, if having occurred, did not prevent resistance reduction. Moreover, all possible CRISPR-Cas9-based outcomes of targeting a resistance gene, i.e. plasmid clearance; resistance gene disruption; or reduction of plasmid copy number impacted the horizontal gene transfer of resistant plasmids. In light of the fact that antimicrobial resistance has spread worldwide with serious impact on human lives, the findings of this study provides relevant details regarding the possible outcomes when using the CRISPR-Cas9 as an alternative tool to reduce resistance in clinically relevant pathogens