Membrane anchoring of New Delhi metallo‐β‐lactamase‐1 alters the fitness of <i>Escherichia coli</i> and increases its susceptibility to colistin by inducing outer membrane destabilization

New Delhi metallo-β-lactamase-1 (NDM-1)-producing bacteria are resistant to nearly all available β-lactam antibiotics and have become a public health threat. Antibiotic resistance often carries fitness costs, which typically manifest as a reduced bacterial growth rate. Here, we investigated the mechanism of fitness cost in NDM-1-producing bacteria. Our findings revealed that strains expressing bla_NDM-1 exhibited a significant growth defect under high osmotic stress. This fitness cost was attributed to the anchoring of NDM-1 to the bacterial outer membrane via its leader peptide, which destabilized the outer membrane. Replacing the membrane-anchoring residue Cys26 in the leader peptide with alanine not only restored outer membrane stability but also ameliorated the bacterial fitness cost. Furthermore, the anchoring of NDM-1 to the membrane increased bacterial susceptibility to the membrane-disrupting antibiotic colistin, both in vitro and in vivo, as confirmed in engineered and clinically isolated strains. In conclusion, membrane anchoring of NDM-1 increased the permeability of the bacterial outer membrane, thereby reducing the fitness of NDM-1-producing bacteria and enhancing their susceptibility to colistin. These results not only elucidate the mechanism of fitness cost associated with NDM-1 but also provide new insights into the rational use of colistin to combat infections caused by NDM-1-producing bacteria.