Identification of CMY-2-Type Cephalosporinases in Clinical Isolates of Enterobacteriaceae by MALDI-TOF MS

Abstract

This study exploited the possibility to detect Citrobacter freundii-derived CMY-2-like cephalosporinases in Enterobacteriaceae clinical isolates using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Periplasmic proteins were prepared using a modified sucrose method and analyzed by MALDI-TOF MS. A ca. 39,850-m/z peak, confirmed to represent a C. freundii-like ␤-lactamase by in-gel tryptic digestion followed by MALDI-TOF/TOF MS, was observed only in CMY-producing isolates. We have also shown the potential of the assay to detect ACC-and DHA-like AmpC-type ␤-lactamases. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is increasingly used as a procedure for identification of pathogenic bacteria and fungi due to its time-and cost-effectiveness (1, 2). Recently, further applications of MALDI-TOF MS focusing on antimicrobial resistance mechanisms, including detection of carbapenemase activity in Enterobacteriaceae, Pseudomonas spp., and Acinetobacter spp., have been described (3-7).In 2007, Camara and Hays described for the first time the use of MALDI-TOF MS for differentiating wild-type Escherichia coli from ampicillin-resistant (Amp r ) plasmid-transformed E. coli strains by the direct visualization of a ␤-lactamase (8). In a recent MALDI-TOF MS study, Schaumann et al. were not able to distinguish Enterobacteriaceae and P. aeruginosa isolates producing extended-spectrum ␤-lactamases (ESBLs) or metallo-␤-lactamases (MBLs) from nonproducers (9). Consequently, so far the attempts to visualize native ␤-lactamases by MALDI-TOF MS in wild-type bacteria have been mostly unsuccessful.We describe here a new assay for the identification of CMY-2-like ␤-lactamases in clinical enterobacterial isolates by MALDI-TOF MS. These enzymes are the most prevalent acquired AmpCtype cephalosporinases in Enterobacteriaceae (10). The method is based on the extraction of periplasmic proteins and the detection of CMY-2-like ␤-lactamases by MALDI-TOF MS according to their molecular weight.Thirty-eight characterized Enterobacteriaceae strains from collections of the Faculty of Medicine and University Hospital in Plzen, Czech Republic, the Hellenic Pasteur Institute in Athens, Greece, and the National Medicines Institute in Warsaw, Poland, were used (Table 1) (11,12). The group included 29 CMY-2-positive clinical isolates, two E. coli transconjugants/transformants with CMY-2-like enzymes (E. coli A15 or DH5␣), and seven non-CMY-producing isolates (13-21). E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 13883 were used as negative controls. Purified CMY-2 enzyme (13) was used as a positive control for MALDI-TOF MS measurements.Isolates were inoculated into 50 ml of Mueller-Hinton broth (Oxoid Ltd.) and incubated at 35°C for 16 h. Cultures were centrifuged at 5,000 ϫ g for 20 min, and the cell pellet was used for the extraction of the periplasmic proteins, performed essentially as described by Naglak and Wang (22). Briefly, the pellet was resuspended in 360 l ...

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for the Rapid Detection of Antimicrobial Resistance Mechanisms and Beyond

“…Gaibani et al demonstrated that the 11,109-Da peak was detected in 88.2% of the KPC producers, confirming previous results (94). Papagiannitsis et al described the identification of CMY-2 ␤-lactamases by detection of the mass peak at 39.850 Da by MALDI-TOF MS (95). The preparation of the sample involved a labor-intensive method with extraction of the periplasmic proteins performed by a modified sucrose method that lasts around 22 h. This group also performed some preliminary analyses for detection of ACC and DHA enzymes.…”

Rapid detection of enterobacteriaceae producing extended spectrum beta-lactamases directly from positive blood cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry

“…If bacteria are sensitive, the antibiotic spectra will not change. Appearance of the mass peak corresponding to the β ‐lactamase enzyme can be monitored, but it has only been described for ampicillin‐resistant Escherichia coli and for the identification of CMY‐2 β ‐lactamases . Besides, these methods that focus on the enzyme identification are much slower and more tedious to carry out in a clinical laboratory.…”

Evaluation of a Semiquantitative Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Rapid Antimicrobial Susceptibility Testing of Positive Blood Cultures

“…One approach to shorten the time to result for antibiotic susceptibility testing (AST) is the use of matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) for identification and resistance testing. The most common approach using mass spectrometry is the detection of ␤-lactamases with a functional assay that measures their hydrolysis products (4)(5)(6)(7)(8)(9). This test provides results within 1 to 3 h, but, by its very nature, it is limited to the detection of ␤-lactamase-mediated resistance.…”