Resistome characterization of spontaneous mecillinam resistant mutants from Klebsiella pneumoniae strain

Klebsiella pneumoniae is feared as a nosocomial pathogen. It is a major actor of the antimicrobial resistance crisis, becoming rapidly highly resistant to multiple antibiotics and contributing to the dissemination of antibiotic resistance. ß-lactams are the most used antibiotics in humans and to treat K. pneumoniae infections. In Enterobacterales, resistance to ß-lactams results of both the expression of ß-lactamases and mutations in the core genome. It is an urgent need to understand the mode of action and the resistance mechanisms in other Enterobacterales than the model organism E. coli. Among ß-lactams, mecillinam targets a single penicillin binding protein, PBP2. Pivmecillincam, the oral prodrug of mecillinam, is used in first line in uncomplicated urinary tract infections. It has been used for decades in Europe, but has only been authorized in 2024 by the US Food and Drug Administration. However, little is known on resistance mechanisms and mode of action in K. pneumoniae. To address these issues, we investigated the mecillinam resistome in a pan-susceptible hospital strain of K. pneumoniae. We showed that K. pneumoniae SHV ß-lactamase led to spontaneous mecillinam resistant mutant appearing at a higher rate that grow faster and at higher mecillinam concentrations than in E. coli. A 720 genes-duplication was the most frequent mechanism. Other selected mutations affected a large diversity of functions with resistance being RelA-independent or dependent for mutations in most genes from 'amino-acid metabolism' and 'translation' categories. Through an in-depth characterization of six mecillinam resistant mutants we showed that, in the presence of mecillinam, they all experienced different growth defects despite high minimal inhibitory concentrations. Overall, our results in K. pneumoniae suggest different mechanisms to escape the complex mode of action of ß-lactam in synergy with the chromosomally encoded ß-lactamase SHV.