Rapid detection of carbapenemase activity through monitoring ertapenem hydrolysis in Enterobacteriaceae with LC-MS/MS.

ABSTRACT:

Background

Bacteria are increasingly resistant to antibiotics used to treat life-threatening infections in critically ill patients. The carbapenems represent the last line of defense against Gram-negative rods that are increasingly resistant to all other classes of ?-lactam antibiotics used to treat life-threatening infections in critically ill patients. Carbapenem resistance in Gram-negative rods is most commonly caused by expression of carbapenemases, enzymes that hydrolyze the ?-lactam ring of carbapenem antibiotics rendering them inactive. All of the available diagnostic tests rely on bacterial growth rendering them time consuming; therefore, rapid diagnostic tests are needed to identify multidrug (including carbapenem)-resistant bacteria.

Results

We report the development of a novel LC-MS/MS method that detects carbapenemase activity from bacterial isolates. Incubation of a bacterial suspension with physiological levels of ertapenem leads to carbapenemase-mediated drug hydrolysis that produces a specific metabolite with an 18 Da increase in m/z within 1 h. Using the ratio of metabolite:parent, detected by LC-MS/MS from the culture, the sensitivity, specificity and a threshold cutoff for carbapenemase production (interpretive criteria) have been determined.

Conclusion

A 100% correlation of our LC-MS/MS assay with the modified Hodge test (functional test for carbapenemase production) and PCR emphasizes the robust nature of this assay. The assay requires minimal hands-on time and a straightforward protocol allowing convenient implementation into clinical laboratories. Inclusion of stable isotope-labeled standard will further increase the robustness of the assay. This assay offers several advantages over other similar assays that use MALDI-TOF MS analysis.

SUBMITTER: Peaper DR 

PROVIDER: S-EPMC5753620 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature