A Novel Metallo-β-Lactamase AMM-1 From Alteromonas mangrovi Reveals a Cryptic Environmental Reservoir of Carbapenem Resistance

Abstract

Carbapenem resistance driven by metallo-β-lactamases (MBLs) poses a formidable global challenge as these enzymes can degrade a wide range of β-lactam antibiotics, including last-line carbapenems. Despite extensive documentation of MBL-producing pathogens, their evolutionary origins and ecological reservoirs are still poorly understood. Here, we report the discovery and in-depth characterisation of AMM-1, a previously unrecognised B1.2 MBL identified within a metagenome-assembled genome of Alteromonas mangrovi obtained from the Yangtze River Estuary. Comparative sequence analyses and phylogenetics reveal that AMM-1 clusters closely with clinically significant MBLs, underscoring its potential impact to human health. Structural modelling confirms the presence of a conserved di-zinc binding site critical for β-lactam hydrolysis, while heterologous expression in Escherichia coli (E. coli) demonstrates a marked increase in resistance against multiple β-lactam classes, including carbapenems. Phylogenetic depth analysis and ancestral reconstruction delineate AMM-1's distinct evolutionary path, placing it deeper than IMP-1 and SPM-1 but shallower than NDM-1. Flexibility simulations reveal unique active-site loop dynamics (L3 and L10), with reduced mobility in key regions that shape substrate binding stability and spectrum. Notably, AMM-1 is stably located on the host chromosome without flanking mobile genetic elements, suggesting that it may have persisted as a vertically inherited trait rather than a recently acquired component of a mobile resistome. These findings highlight the capacity of environmental microbes to serve as long-standing, cryptic reservoirs of potent resistance determinants, emphasising the need for integrated environmental surveillance and preemptive stewardship strategies. By unveiling the molecular and functional properties of AMM-1, this work provides critical insights into how resistance elements can reside, evolve and potentially mobilise within natural habitats, ultimately informing efforts to predict and mitigate the future emergence of carbapenem-resistant bacterial pathogens.