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

## TL;DR

This study explores how Klebsiella pneumoniae develops resistance to mecillinam, a beta-lactam antibiotic, revealing diverse resistance mechanisms and growth patterns.

## Contribution

The study identifies novel resistance mechanisms in K. pneumoniae, including chromosomal duplication and varied growth responses to mecillinam.

## Key findings

- Spontaneous mecillinam-resistant mutants in K. pneumoniae arise more frequently and at higher concentrations than in E. coli.
- Resistance mechanisms include an unstable duplication and mutations affecting diverse functions, some dependent on RelA (p)ppGpp synthetase.
- Mutant strains show varied growth defects despite high resistance levels, indicating complex interactions with mecillinam.

## Abstract

Antimicrobial resistance is a silent pandemic responsible for 1.14 million deaths worldwide in 2021, with a major contribution from Klebsiella pneumoniae. 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 Escherichia coli. 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 K. pneumoniae 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 K. pneumoniae than in E. coli. 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 K. pneumoniae suggest different mechanisms to escape the complex mode of action of β-lactams in synergy with the β-lactamase SHV.