A new variant of the New Delhi metallo-enzyme (NDM) carbapenemase was identified in a multidrugresistant Escherichia coli ST648 isolate recovered from the perineum and throat of a patient in the United Kingdom with a recent history of hospitalization in India. NDM-5 differed from existing enzymes due to substitutions at positions 88 (Val3Leu) and 154 (Met3Leu) and reduced the susceptibility of E. coli TOP10 transformants to expanded-spectrum cephalosporins and carbapenems when expressed under its native promoter.

ObjectivesMetallo-β-lactamase (MBL)-based resistance is a threat to the use of most β-lactam antibiotics. Multiple variants of the New Delhi MBL (NDM) have recently been reported. Previous reports indicate that the substitutions affect NDM activity despite being located outside the active site. This study compares the biochemical properties of seven clinically reported NDM variants.MethodsNDM variants were generated by site-directed mutagenesis; recombinant proteins were purified to near homogeneity. Thermal stability and secondary structures of the variants were investigated using differential scanning fluorimetry and circular dichroism; kinetic parameters and MIC values were investigated for representative carbapenem, cephalosporin and penicillin substrates.ResultsThe substitutions did not affect the overall folds of the NDM variants, within limits of detection; however, differences in thermal stabilities were observed. NDM-8 was the most stable variant with a melting temperature of 72°C compared with 60°C for NDM-1. In contrast to some previous studies, kcat/KM values were similar for carbapenem and penicillin substrates for NDM variants, but differences in kinetics were observed for cephalosporin substrates. Apparent substrate inhibition was observed with nitrocefin for variants containing the M154L substitution. In all cases, cefoxitin and ceftazidime were poorly hydrolysed with kcat/KM values <1 s−1 μM−1.ConclusionsThese results do not define major differences in the catalytic efficiencies of the studied NDM variants and carbapenem or penicillin substrates. Differences in the kinetics of cephalosporin hydrolysis were observed. The results do reveal that the clinically observed substitutions can make substantial differences in thermodynamic stability, suggesting that this may be a factor in MBL evolution.

CHROMagar COL-APSE is a sensitive and specific medium for the growth of COL-resistant bacterial pathogens. Due to the low limit of detection (10 c.f.u.), it may be useful as a primary isolation medium in the surveillance and recovery of COL-resistant bacteria from complex human, veterinary and environmental samples, especially those with plasmid-mediated MCR-1 or novel mechanisms of polymyxin resistance.

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