Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based functional assay for rapid detection of resistance against β-lactam antibiotics

Abstract

Resistance against β-lactam antibiotics is a growing challenge for managing severe bacterial infections. The rapid and cost-efficient determination of β-lactam resistance is an important prerequisite for the choice of an adequate antibiotic therapy. β-Lactam resistance is based mainly on the expression/overexpression of β-lactamases, which destroy the central β-lactam ring of these drugs by hydrolysis. Hydrolysis corresponds to a mass shift of +18 Da, which can be easily detected by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Therefore, a MALDI-TOF MS-based assay was set up to investigate different enterobacteria for resistance against different β-lactam antibiotics: ampicillin, piperacillin, cefotaxime, ceftazidime, ertapenem, imipenem, and meropenem. β-Lactamases are enzymes that have a high turnover rate. Therefore, hydrolysis can be detected by MALDI-TOF MS already after a few hours of incubation of the bacteria to be tested with the given antibiotic. The comparison of the MS-derived data with the data from the routine procedure revealed identical classification of the bacteria according to sensitivity and resistance. The MALDI-TOF MS-based assay delivers the results on the same day. The approved routine procedures require at least an additional overnight incubation.

Fig 1

(A and B) MALDI-TOF MS spectra of ampicillin after incubation with the β-lactamase-negative E. coli strain DH5α (A) and a β-lactamase-producing strain (B). (C) Inhibition of hydrolysis by a β-lactamase-producing strains was performed in the presence of clavulanic acid. Peaks corresponding to the nonhydrolyzed form of ampicillin are highlighted in gray. Peaks corresponding to the hydrolyzed form of ampicillin are indicated with an arrow.

Fig 2

(A and B) MALDI-TOF MS spectra of piperacillin after incubation with the β-lactamase-negative E. coli strain DH5α (A) and a β-lactamase-producing strain (B). (C) Inhibition of hydrolysis by a β-lactamase-producing strains was performed in the presence of tazobactam. Peaks corresponding to the nonhydrolyzed form of piperacillin are highlighted in gray. Peaks corresponding to the hydrolyzed form of piperacillin are indicated with an arrow.

Fig 3

MALDI-TOF MS spectra of cefotaxime (A to C) and ceftazidime (D to F) after incubation with the β-lactamase-negative E. coli strain DH5α (A and D) and a β-lactamase producing strain (B and E). Inhibition of hydrolysis by a β-lactamase-producing strains was performed in the presence of clavulanic acid (C and F). Peaks corresponding to the nonhydrolyzed form of the respective antibiotic are highlighted in gray. Peaks corresponding to the hydrolyzed form of the respective antibiotic are indicated with an arrow.

Fig 4

MALDI-TOF MS analysis of carbapenems after incubation with a carbapenem-sensitive K. pneumoniae strain (A, D, and G), with a carbapenem-resistant K. pneumoniae strain (B, E, and H), and with a carbapenem-resistant strain in the presence of the carbapenemase inhibitor APBA (C, F, and I). (A, B, and C) Conversion of ertapenem. (D, E, and F) Reaction of meropenem. (G, H, and I) Disappearance of the molecular peak of imipenem after incubation with a carbapenemase-resistant K. pneumoniae strain and the conservation of this peak in the presence of APBA. Incubation times were 20 min for ertapenem, 15 min for meropenem, and 10 min for imipenem. Peaks corresponding to the nonhydrolyzed form of the respective antibiotic are highlighted in gray. Peaks corresponding to the hydrolyzed forms of the respective antibiotic are indicated with an arrow.

Fig 5

MALDI-TOF MS spectra after incubation of ertapenem with different K. pneumoniae strains isolated from fresh blood cultures. (A) Carbapenem-sensitive strain. (B and C) Carbapenem-resistant strains. Peaks representing the nonhydrolyzed form of the respective antibiotic are highlighted in gray. Peaks corresponding to the hydrolyzed forms of the respective antibiotic are indicated with an arrow.