Effect of amino acid substitution in New Delhi metallo-β-lactamase on carbapenem susceptibility

The aim of this work was investigation of clinically important amino acid substitutions of NDM-1 variants. A blaNDM-1 gene was cloned into expression vector pET100/D-TOPO. The sequence of NDM-1 variants with substituted amino acids was determined by ClustalW program. A pET100/D-TOPO + blaNDM-1 was used to generate the alanine mutations at different positions, such as NDM-2 (P28A), NDM-3 (D95A), NDM-4 (M154A), NDM-5 (V88A), NDM-7 (D130A), and NDM-9 (E152A). The mutant variants were transformed into Escherichia coli DH5α. Changes in the activities of alanine mutation variants were determined by E-test. All samples had 32 μg/ml MIC values against ampicillin. The 28th amino acid mutation sample had the highest MIC value against ceftazidime, whereas decreased MIC value for piperacillin. It was observed that the resistance to imipenem was increased in mutant variants D95A, M154A, D130A, and E152A, comparing with P28A and V88A. It was found that NDM-1 has 0.64 μg/ml and the 130th amino acid mutation sample has 0.75 μg/ml meropenem MIC value.