Genomic approach for the identification of resistance mechanisms to new antibiotics and inhibitors of resistance mechanisms in clinically relevant Gram-negative bacteria

In the last decades, genomics has revolutionized microbiology through advances in sequencing technologies and computational tools, enabling vast bacterial genome analyses. These developments have deepened understanding of bacterial evolution, diversity, and molecular basis of antimicrobial resistance. Over the past three years, this PhD project explored how Klebsiella pneumoniae rapidly evolves under antibiotic pressure, leveraging advances in whole-genome sequencing and bioinformatics to uncover the genetic basis of antimicrobial resistance. Whole-genome sequencing has been used in the different projects, based on clinical isolates obtained by the Policlinico Umberto I of Rome, Italy, as a key method for identifying resistance mechanisms known and novel to perform comparative genomics, and trace resistance evolution along the years in high-risk clones of K. pneumoniae. Early investigations revisited K. pneumoniae sequence type (ST) 37 isolates, initially detected in 2006 and thought to have disappeared after 2010. Genomic analysis of new ceftazidime-avibactam (CZA)-resistant ST37 isolates revealed their close relation to historical strains, suggesting long-term persistence in the hospital environment and re-emergence under selective antibiotic pressure. Subsequent studies focused on K. pneumoniae ST307, a globally disseminated high-risk clone. Genomic and plasmid analyses of CZA- and carbapenem-resistant strains revealed complex plasmid rearrangements and recombination events that explained diverse resistance profiles, demonstrating the remarkable genetic plasticity of ST307. A clinical case involving ST512 highlighted in vivo evolution of resistance during prolonged antimicrobial therapy, including plasmid loss, porin mutations, and a cirA nonsense mutation conferring cefiderocol resistance. Further work identified a novel mechanism of cefiderocol resistance involving interaction between two plasmids pKpQIL (carrying blaKPC variants) and pKPN (harboring the ferric citrate fec system) which was shown to reduce drug susceptibility in both E. coli and K. pneumoniae. In the final phase, a CRISPR-Cas9 conjugative system was engineered to selectively eliminate plasmid-borne blaKPC genes. Overall, the research of this thesis highlights how K. pneumoniae adapts rapidly to new antibiotics, with sequence type-specific trajectories shaped by plasmid dynamics and their acquisition of antimicrobial resistance determinants.