Objectives: In order to find antimicrobial resistance gene(s) pre-dating the use of antibiotics through metagenomics, functional screening of a metagenomic library from the deep-seep sediments of Edison Seamount (ca. 10000 years old) was performed.
Methods: Among 60 antimicrobial-resistant clones, a single clone with the highest minimum inhibitory concentration (MIC) for ampicillin was selected. Sequence analysis revealed a new metallo-β-lactamase (MBL) gene, designated as bla. PNGM-1 retains a zinc ion-binding motif (HXHXDH, H and H), conserved in subclass B3 MBLs. The catalytic parameters of purified PNGM-1 and the MICs of β-lactams for Escherichia coli TOP10 transformants harbouring the bla gene were assessed.
Results: Antimicrobial susceptibility testing indicated reduced susceptibility to penicillins, narrow- and extended-spectrum cephalosporins, and carbapenems in E. coli TOP10 transformants harbouring the bla gene. In addition, kinetic analyses revealed that PNGM-1 hydrolysed almost all β-lactams.
Conclusions: The PNGM-1 enzyme is the first case of a subclass B3 MBL derived from a functional metagenomic library of a deep-sea sediment that pre-dates the antibiotic era.
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