title: 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 β-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 β-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.
  
  
  CONCLUSIONS
  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.
authors: Department of Laboratory Medicine, Yale School of Medicine, 15 York Street, New Haven, CT 06510, USA.
authors: David R
authors: DR
authors: Peaper
authors: Mandar V
authors: MV
authors: Kulkarni
authors: Alexandra N
authors: AN
authors: Tichy
authors: Michael
authors: M
authors: Jarvis
authors: Thomas S
authors: TS
authors: Murray
authors: Michael E
authors: ME
authors: Hodsdon
mesh_terms: D000900:Anti-Bacterial Agents / Q000032:analysis / Q000493:pharmacokinetics; D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D001431:Bacteriological Techniques / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism; D000077727:Ertapenem; D008826:Microbial Sensitivity Tests; D053719:Tandem Mass Spectrometry / Q000379:methods*; D001618:beta-Lactamases / Q000032:analysis* / Q000378:metabolism; D047090:beta-Lactams / Q000032:analysis* / Q000493:pharmacokinetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D047090:beta-Lactams; D001618:beta-Lactamases; C063912:carbapenemase; D000077727:Ertapenem