The mecillinam resistome in <i>Klebsiella pneumoniae</i> : how resistance to a one-target β-lactam triggers a diversity of responses

Antimicrobial resistance is a silent pandemic responsible for 1.14 million deaths worldwide in 2021, with a major contribution from <i>Klebsiella pneumoniae</i>. β-lactams are the most commonly used antibiotics in humans, and there is an urgent need to characterize the resistance mechanisms to these antibiotics in Enterobacterales other than <i>Escherichia coli</i>. Mecillinam is a narrow-spectrum β-lactam targeting a single penicillin-binding protein, PBP2. Pivmecillinam, its oral prodrug, is used as a first-line treatment for uncomplicated urinary tract infections. It has been used for decades in Europe but was only authorized by the US Food and Drug Administration in 2024. Here, we decipher mecillinam resistance mechanisms in <i>K. pneumoniae</i> by characterizing its resistome in a pan-susceptible strain. The chromosomally encoded SHV β-lactamase led to spontaneous mecillinam-resistant mutants appearing at a higher rate, growing faster and at higher mecillinam concentrations in <i>K. pneumoniae</i> than in <i>E. coli</i>. The most frequent genetic event was an unstable duplication leading to heteroresistance. The selected mutations leading to resistance affected a wide range of functions, with resistance being dependent or independent of the RelA (p)ppGpp synthetase. Through an in-depth characterization of six mutant strains, we showed that, in the presence of mecillinam, they all experienced different growth defects despite high minimal inhibitory concentrations. Overall, our results in <i>K. pneumoniae</i> suggest different mechanisms to escape the complex mode of action of β-lactams in synergy with the β-lactamase SHV.