Use of imipenem to detect KPC, NDM, OXA, IMP, and VIM carbapenemase activity from gram-negative rods in 75 minutes using liquid chromatography-tandem mass spectrometry.

Resistance to extended-spectrum ß-lactam antibiotics has led to a greater reliance upon carbapenems, but the expression of carbapenemases threatens to limit the utility of these drugs. Current methods to detect carbapenemase activity are suboptimal, requiring prolonged incubations during which ineffective therapy may be prescribed. We previously described a sensitive and specific assay for the detection of carbapenemase activity using ertapenem and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we assessed 402 Gram-negative rods, including both Enterobacteriaceae and non-Enterobacteriaceae expressing IMP, VIM, KPC, NDM, and/or OXA carbapenemases, by using imipenem, meropenem, and ertapenem with LC-MS/MS assays. LC-MS/MS methods for the detection of intact and hydrolyzed carbapenems from an enrichment broth were developed. No ion suppression was observed, and the limits of detection for all three drugs were below 0.04 µg/ml. The sensitivity and specificity of meropenem and ertapenem for carbapenemase activity among non-Enterobacteriaceae were low, but imipenem demonstrated a sensitivity and specificity of 96% and 95%, respectively, among all Gram-negative rods (GNR) tested, including both Enterobacteriaceae and non-Enterobacteriaceae. LC-MS/MS allows for the analysis of more complex matrices, and this LC-MS/MS assay could easily be adapted for use with primary specimens requiring growth enrichment.

EPR studies of iso-1-cytochrome c: effect of temperature on two-component spectra of spin label attached to cysteine at positions 102 and 47.

Wild-type iso-1-cytochrome c from Saccharomyces cerevisiae containing naturally occurring cysteine at position 102 and mutated protein S47C (derived from the protein in which C102 had been replaced by threonine) were labeled with cysteine-specific methanethiosulfonate spin label. Continuous wave (CW) electron paramagnetic resonance (EPR) was used to examine the effect of temperature on the behavior of the spin label in the oxidized and reduced forms of wild-type cytochrome c and in the oxidized form of the mutated protein. The computer simulations revealed that the CW EPR spectrum for each form of cytochrome c consists of at least two components [a fast (F) and a slow (S) component], which differ in the values of the rotational correlation times tauRparallel (longitudinal rotational correlation time) and tauRperpendicular (transverse rotational correlation time) and that the relative contributions of the F and S components of the spectra change with temperature. In addition, the values of the rotational correlation times (tauRparallel and tauRperpendicular) for the F component appear to change much more dramatically with the temperature than the respective values for the S component. A large difference between the behavior of the oxidized and reduced wild-type spin-labeled cytochromes c indicates that the temperature-induced unfolding of the protein in the region around C102 progresses more rapidly when cytochrome c is in the oxidized form.

Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques.

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.