Efficient Detection of Carbapenemase Activity in Enterobacteriaceae by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Less Than 30 Minutes.
The recognition of carbapenemase-producing Enterobacteriaceae (CPE) isolates is a major laboratory challenge, and their inappropriate or delayed detection may have negative impacts on patient management and on the implementation of infection control measures. We describe here a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based method to detect carbapenemase activity in Enterobacteriaceae. After a 20-min incubation of the isolate with 0.5 mg/ml imipenem at 37°C, supernatants were analyzed by MALDI-TOF in order to identify peaks corresponding to imipenem (300 Da) and an imipenem metabolite (254 Da). A total of 223 strains, 77 CPE (OXA-48 variants, KPC, NDM, VIM, IMI, IMP, and NMC-A) and 146 non-CPE (cephalosporinases, extended-spectrum β-lactamases [ESBLs], and porin defects), were tested and used to calculate a ratio of imipenem hydrolysis: mass spectrometry [MS] ratio = metabolite/(imipenem + metabolite). An MS ratio cutoff was statistically determined to classify strains as carbapenemase producers (MS ratio of ≥0.82). We validated this method first by testing 30 of our 223 isolates (15 CPE and 15 non-CPE) 10 times to calculate an intraclass correlation coefficient (ICC of 0.98), showing the excellent repeatability of the method. Second, 43 strains (25 CPE and 18 non-CPE) different from the 223 strains used to calculate the ratio cutoff were used as external controls and blind tested. They yielded sensitivity and specificity of 100%. The total cost per test is <0.10 U.S. dollars (USD). This easy-to-perform assay is time-saving, cost-efficient, and highly reliable and might be used in any routine laboratory, given the availability of mass spectrometry, to detect CPE.
Service de Bactériologie, CHU La Cavale Blanche, Brest, France.
Camille
C
Lasserre
Service de Médecine Interne, CHU La Cavale Blanche, Brest, France.
Luc
L
De Saint Martin
Service de Bactériologie, APHP, LabEx LERMIT, University of Paris-Sud, Orsay, France.
Gaelle
G
Cuzon
Laboratoire de Bactériologie, CHU Dinant-Godinne UCL Namur, Yvoir, Belgium.
Pierre
P
Bogaerts
Service de Bactériologie, CHU La Cavale Blanche, Brest, France.
Estelle
E
Lamar
Laboratoire de Bactériologie, CHU Dinant-Godinne UCL Namur, Yvoir, Belgium.
Youri
Y
Glupczynski
Service de Bactériologie, APHP, LabEx LERMIT, University of Paris-Sud, Orsay, France.
Thierry
T
Naas
Service de Bactériologie, CHU La Cavale Blanche, Brest, France didier.tande@chu-brest.fr.
Didier
D
Tandé
D000900:Anti-Bacterial Agents / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis*; D001431:Bacteriological Techniques / Q000379:methods*; D004755:Enterobacteriaceae / Q000201:enzymology*; D006801:Humans; D006868:Hydrolysis; D015378:Imipenem / Q000378:metabolism; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors; D001618:beta-Lactamases / Q000032:analysis*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015378:Imipenem; D001618:beta-Lactamases; C063912:carbapenemase