Adaptive landscape of the metallo-β-lactamase NDM-1 to emerging β-lactam-based therapies.

Abstract

The New Delhi metallo-β-lactamase (NDM) is a major determinant of carbapenem resistance. This has prompted the development of novel therapeutic strategies to treat NDM producers. Since these therapies impose new selective pressures, their clinical deployment may favor NDM variants carrying escape mutations. To anticipate these events and inform therapeutic decisions, we explored the evolutionary landscape of NDM-1 under different selective constraints. To this end, we generated a diverse library of bla_NDM variants and challenged it with zinc starvation, antibiotics, or β-lactam/β-lactamase inhibitor combinations that represent novel and emerging therapies. Selection under zinc limitation and cefotaxime identified mutational trajectories that recapitulate clinical NDM evolution, validating the diversity of the library and its predictive nature. Drug-specific selections revealed sharply different evolutionary pathways. Mecillinam selected a narrow evolutionary pathway centered on residues N220 and M67, which enhance productive active-site interactions with this penicillin. Cefepime/taniborbactam selected multiple escape routes, dominated by substitutions at E152 and, secondarily, K211, that impair productive interaction with taniborbactam. In contrast, cefiderocol/xeruborbactam under iron-depleted conditions and aztreonam/avibactam did not select NDM variants conferring an improved resistance phenotype in the tested Escherichia coli background. These results show that NDM evolution is constrained by the chemistry of each therapeutic challenge. Substrate adaptation is possible for mecillinam, inhibitor escape is readily accessible for taniborbactam, whereas no NDM-centered escape was detected for the aztreonam- and xeruborbactam-based conditions tested. Mapping drug-specific NDM-centered landscapes can identify therapies that readily select enzyme-level escape and those for which resistance is more likely to require mechanisms beyond bla_NDM sequence variation.IMPORTANCECarbapenem-resistant infections by Enterobacterales are increasingly difficult to treat because bacteria can destroy some of the most powerful antibiotics used in the clinic. This study focuses on Escherichia coli carrying variants of the New Delhi metallo-β-lactamase (NDM), an enzyme that enables bacteria to resist carbapenem antibiotics. New therapies are being developed to overcome this problem, but bacteria may evolve again when exposed to these treatments. Here, we tested how New Delhi metallo-β-lactamase can adapt under the evolutionary pressure of last-resort therapies. The results show that not all treatments carry the same evolutionary risk. Some drugs allow the enzyme to adapt through specific mutations, whereas other combinations did not yield improved NDM variants in the tested bacterial background. This work identifies which treatments readily select changes within NDM and which may require resistance mechanisms elsewhere in the bacterial cell. These findings should be integrated with host species, permeability, drug targets, metal uptake, and co-produced enzymes when considering clinical resistance.