Minimum Inhibitory Concentration (MIC)
Genes encoding the NDM variants NDM-1, -2, -4, -5, -6 and -7 were amplified using DNA templates prepared from NDM-producing clinical isolates or E. coli transconjugants.(22–24) Both full coding sequences and the NDM gene with the native ISAb125 promoter (22) were amplified and cloned in the pCR-Blunt II TOPO vector (Invitrogen, Paisley, UK) and transformed in E. coli TOP10. The susceptibility of the transformants containing pCR2.1 NDM and pCR2.1 NDM P+ plasmids to ampicillin, cefalotin, cefoxitin, ceftazidime, ertapenem, imipenem, meropenem and doripenem was determined using the Etest method (bioMérieux, Basingstoke, UK) on Mueller–Hinton agar.
ISAb125 promoter genes included in this upload. For enzymes with MIC Absolute with a > symbol, the next higher multiplier was used for MIC Relative calculation. >256 ug/mL for example used 512 divided by the negative control plasmid's MIC.
22 Hornsey M Phee L Wareham DW A novel variant, NDM-5, of the New Delhi metallo-β-lactamase in a multidrug-resistant Escherichia coli ST648 isolate recovered from a patient in the United Kingdom Antimicrob Agents Chemother 2011 55 5952 4
23 Göttig S Hamprecht AG Christ S et al. Detection of NDM-7 in Germany, a new variant of the New Delhi metallo-β-lactamase with increased carbapenemase activity J Antimicrob Chemother 2013 68 1737 40
24 Tada T Miyoshi-Akiyama T Dahal RK et al. NDM-8 metallo-β-lactamase in a multidrug-resistant Escherichia coli strain isolated in Nepal Antimicrob Agents Chemother 2013 57 2394 6
MICs are expressed as x-fold relative to the negative control (cells with empty pCR2.1 plasmid without ISAba125 promoter).
NDM-3 (D95N) and NDM-8 (M154L+D130G) variants were transformed in E. coli DH5α instead of E. coli TOP10
The absolute MICs for the negative control were as follows:
Ampicillin (AM): 4 ug/ml
Cefalotin (CE): 0.008 ug/ml
Cefoxitin (FO): 0.008 ug/ml
Ceftazidime (CA): 0.125 ug/ml
Ertapenem (ER): 0.008 ug/ml
Imipenem (IP): 0.25 ug/ml
Doripenem (DO): 0.032 ug/ml
Meropenem (ME): 0.047 ug/ml