Evaluation of different pretreatment protocols to detect accurately clinical carbapenemase-producing Enterobacteriaceae by MALDI-TOF.
Carbapenemase-resistant bacteria are increasingly spreading worldwide causing public concern due to their ability to elude antimicrobial treatment. Early identification of these bacteria is therefore of high importance. Here, we describe the development of a simple and robust protocol for the detection of carbapenemase activity in clinical isolates of Enterobacteriaceae, suitable for routine and clinical applications.



The final protocol involves cellular lysis and enzyme extraction from a defined amount of bacterial cells followed by the addition of a benchmark drug (e.g. the carbapenem antibiotic imipenem or ertapenem). Carbapenem inactivation is mediated by enzymatic hydrolysis (cleavage) of the β-lactam common structural motif, which can be detected using MALDI-TOF MS.



A total of 260 strains were studied (208 carbapenemase producers and 52 non-carbapenemase producers) resulting in 100% sensitivity and 100% specificity for the KPC, NDM and OXA-48-like PCR-confirmed positive isolates using imipenem as benchmark. Differences between the benchmark (indicator) antibiotics imipenem and ertapenem, buffer constituents and sample preparation methods have been investigated. Carbapenemase activity was further characterized by performing specific inhibitor experiments. Intraday and interday reproducibility (coefficient of variation) of the observed hydrolysis results were 15% and 30%, respectively. A comparative study of our extraction method and a recently published method using whole bacterial cells is presented and differences are discussed.



Using this method, an existing carbapenemase activity can be directly read from the mass spectrum as a ratio of hydrolysed product and substrate, setting an important step towards routine application in clinical laboratories.
Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam, The Netherlands.
Carmine G
CG
Monteferrante
Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam, The Netherlands.
Sadaf
S
Sultan
Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam, The Netherlands.
Marian T
MT
Ten Kate
Department of Neurology, Erasmus University Medical Center, Rotterdam, The Netherlands.
Lennard J M
LJ
Dekker
Bruker Daltonics, Bremen, Germany.
Katrin
K
Sparbier
Bruker Daltonics, Bremen, Germany.
Markus
M
Peer
Bruker Daltonics, Bremen, Germany.
Markus
M
Kostzrewa
Department of Neurology, Erasmus University Medical Center, Rotterdam, The Netherlands.
Theo M
TM
Luider
Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam, The Netherlands w.goessens@erasmusmc.nl.
Wil H F
WH
Goessens
Department of Neurology, Erasmus University Medical Center, Rotterdam, The Netherlands TI-COAST, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Peter C
PC
Burgers
D000150:Acinetobacter / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D015780:Carbapenems / Q000494:pharmacology; D019411:Clinical Laboratory Techniques / Q000379:methods; D021902:Enterobacter aerogenes / Q000187:drug effects / Q000201:enzymology; D004755:Enterobacteriaceae / Q000201:enzymology*; D004756:Enterobacteriaceae Infections / Q000188:drug therapy / Q000382:microbiology*; D000077727:Ertapenem; D006801:Humans; D006868:Hydrolysis; D015378:Imipenem / Q000494:pharmacology; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology*; D008826:Microbial Sensitivity Tests; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D001618:beta-Lactamases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D047090:beta-Lactams / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D047090:beta-Lactams; D015378:Imipenem; D001618:beta-Lactamases; C063912:carbapenemase; D000077727:Ertapenem