Stefan Zimmermann (Presenter)
Department of Infectious Diseases
Bio: Senior Physician, Clinical Microbiology and ID specialist Head of Division Bacteriology at the Department of Infectious Diseases, University Hospital heidelberg
Authorship: Stefan Zimmermann and Irene Burckhardt
Department of Infectious Diseases, University Hospital Heidelberg, Heidelberg, Germany
| Short Abstract Multi-resistant Gram-negative bacteria are one of the biggest threads of global health. Mass spec-trometry opens up new possibilities for rapid and reliable detection of the underlying mechanisms. If such bugs are detected in the hospital, effective infection control is urgently necessary, which includes rapid in-house tools for outbreak analysis. New approaches show that this kind of analysis can also be done by mass spectrometry technologies. |
Long Abstract
Introduction
Various methods for detection of carbapenemase activity in Gram-negative bacteria have been suggested, but they do not detect all types of carbapenem resistance (e.g. PCR) or a time consuming (e.g. commercial susceptibility tests) . A recently described quantitative assay using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) lacks this disadvantage. We investigated rapid and reliable detection of carbapenem resistance as an exemplar of a novel easy-to-perform variant of antimicrobial susceptibility testing (AST).
Carbapenem resistant Gram-negative bacteria are the main thread for nosocomial outbreaks in hospitals. MALDI-TOF MS provides not only rapid and reliable test results, but is also able to deliver fast subtyping results to distinguish those outbreaks.
Methods
We used MALDI-TOF MS (matrix assisted laser desorption ionization time of flight mass spectrome-try) to determine carbapenem resistance by monitoring the degradation of ertapenem through strains carrying the following carbapenemases: IMP-1, IMP-2, KPC-2, VIM-1, VIM-2 and NDM-1.
Various approaches were performed to further optimize the technology and the workflow for routine use. Three different outbreaks with K. pneumoniae, P. aeruginosa and A. baumannii were investigated using MALDI-TOF (Bruker, microflex). Suspicious isolates were collected, grown on blood agar, extracts were prepared from each strain and 24 spectra of each strain were recorded. Spectra were quality controlled, calibrated and a dendrogram as well as a composite correlation index (CCI) were calculated using software provided by the manufacturer. Results were compared to pulsed field gel electrophoresis. Additionally the MALDI-TOF technique was evaluated in terms of reliability, handling and cost.
Results
Degradation of ertapenem was clearly visible within one hour for NDM-1 and IMP-1. IMP-2, KPC-2 and VIM-1 needed about 1.5 hours for degradation and VIM-2 needed 2.5 hours. All controls were negative (e.g. ESBL+ strains, K1+ strains). Also imipenem or meropenem can be used for the assay. In an optimized protocol the assay can be done “on target”, working for Enterobacteriaceae and non-fermenters. In all three outbreaks results were in good accordance with the pulsed-field electrophoresis results. However, it is mandatory that all strains are grown under the exact same conditions (i.e. together). This precludes creating a reference dendrogram or CCI and adding data of suspicious strains later. Due to the mathematical algorithms the dendrogram as well as the CCI can change substantially after addition of new data to the dendrogram or CCI file.
Conclusions & Discussion
We recommend this method as a fast confirmation test for carbapenem resistant bacterial isolates. This method is especially helpful in an outbreak situation. In addition, MALDI-TOF is a promising technique for the investigation of minor outbreaks. It is fast, cost effective and reliable. No special hardware is necessary (e.g. a pulsed-field gel electrophoresis chamber). However, spectra of all strains have to be grown simultaneously to give reliable results. Improved software tools must be developed to establish MALDI-TOF as an outbreak investigation tool.
References & Acknowledgements:
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