Characterization of β-lactamase enzyme activity in bacterial lysates using MALDI-mass spectrometry.
Plasmid-encoded β-lactamases are a major reason for antibiotic resistance in gram negative bacteria. These enzymes hydrolyze the β-lactam ring structure of certain β-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of β-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine β-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the β-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible β-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of β-lactamase activity in various bacterial strains by mass spectrometry.
Department of Neurology, Laboratory of Neuro-Oncology and Clinical and Cancer Proteomics, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands. g.hooff@erasmusmc.nl
Gero P
GP
Hooff
Jeroen J A
JJ
van Kampen
Roland J W
RJ
Meesters
Alex
A
van Belkum
Wil H F
WH
Goessens
Theo M
TM
Luider
D001426:Bacterial Proteins / Q000737:chemistry*; D002138:Calibration; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D010400:Penicillin G / Q000031:analogs & derivatives / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000592:standards; D001618:beta-Lactamases / Q000737:chemistry*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; C030990:benzylpenicilloic acid; D010400:Penicillin G