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Front. Microbiol. | doi: 10.3389/fmicb.2019.00013

Rapid detection of extended-spectrum β-lactamases (ESBL) and AmpC β-lactamases in Enterobacterales: Development of a screening panel using the MALDI-TOF MS-based direct-on-target microdroplet growth assay

  • 1Institute of Medical Microbiology, University Hospital Münster, Germany
  • 2Bruker Daltonik GmbH, Germany
  • 3Institute of Medical Microbiology, University of Münster, Germany

Antibiotic resistant bacteria are a growing concern worldwide. Extended-spectrum β-lactamases (ESBL) represent the most common resistance mechanism of Gram-negative bacteria against β-lactams, underlining the need for adequate diagnostic methods that provide reliable information in the shortest time possible. AmpC, a less prevalent but increasingly relevant class of β-lactamases, pose an additional challenge as their detection is complex. Here, we present an ESBL and AmpC screening panel employing the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA).
Materials and Methods
Four reference strains recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to develop the panel, which was further validated on 50 clinical Enterobacterales isolates resistant to third generation cephalosporins. The panel relies on the synergistic effect between ESBL and/or AmpC β-lactamase inhibitors and cephalosporins, which indicates β-lactamase production. Microdroplets containing the tested microorganism, cephalosporins in different concentrations and inhibitors were pipetted onto an MBT Biotarget and incubated for 3 or 4 hours at 35 ± 1 °C. Afterwards, the liquid medium was removed and the material adhered to the spots was analyzed by MALDI-TOF MS. Synergy was detected by determining and comparing the minimum inhibitory concentrations of the tested cephalosporins with and without β-lactamase inhibitors. Data were interpreted following a diagnostic algorithm proposed by EUCAST in order to establish a final diagnosis. In comparison, PCR, broth microdilution (BMD) and combination disk tests (CDT) were performed.
Compared to the PCR results, the following positive and negative percent agreement values (PPA/NPA) were obtained for each resistance mechanism: ESBL, 94.44%/100%; AmpC, 94.44%/93.75% and ESBL+AmpC, 100%/100%. These results, obtained after 4 hours of incubation, were comparable with those of BMD and showed a higher accuracy than CDT.
We propose a novel phenotypic method for detection of ESBL and AmpC β-lactamases in Enterobacterales that provides reliable results in a short time, representing a promising alternative to the diagnostic techniques currently available. This easy-to-perform approach has potential for being implemented in routine laboratories, contributing to the further diversification of mass spectrometry technology into other fields such as antibiotic resistance testing.

Keywords: extended spectrum beta lactamase, AmpC beta lactamase, MALDI - TOF, Rapid diagnostic assays, multiresistance, Enterobacterales , Enterobacteriaceae, Hafniaceae, Morganellaceae, Yersiniaceae, minimum inhibitory concentration (MIC), Broth microdilution, combination disc method, PCR, Mass Spectrometry

Received: 27 Sep 2018; Accepted: 07 Jan 2019.

Edited by:

Miklos Fuzi, Semmelweis University, Hungary

Reviewed by:

Filipa Grosso, Faculdade de Farmácia, Universidade do Porto, Portugal
Armand Paauw, Netherlands Organisation for Applied Scientific Research (TNO), Netherlands  

Copyright: © 2019 Correa-Martínez, Idelevich, Sparbier, Kostrzewa and Becker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Karsten Becker, University of Münster, Institute of Medical Microbiology, Münster, Germany,