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A rapid and functional assay for detection of resistance against lactam antibiotics by MALDI-TOF mass spectrometry

Abstract number: P758

Sparbier K., Weller U., Schubert S., Boogen C., Kostrzewa M.

Objective: The growing number of antibiotic resistant microorganisms is an increasing health care problem. b-lactam-resistant bacteria express b-lactamases which destroy the b-lactam ring of b-lactam antibiotics by hydrolysis. A rapid MALDI-TOF MS based assay was set up to analyze hydrolysis of different b-lactam antibiotics by certain bacteria.

Methods: Ampicillin and cephalosporins were tested with ESBL E. coli strains and DH5a as negative control. Ertapenem, Imipenem and Meropenem were tested with carbapenemase positive Klebsiella pneumoniae strains and a sensitive strain as negative control. Antibiotics were dissolved in water. 10 ml of this solution were inoculated with 5 colonies of the corresponding bacteria and incubated for 3 h at 37°C under agitation. After centrifugation, 1 ml of the supernatant was directly applied to a MALDI sample carrier plate. Dried spots were overlaid with MALDI matrix. MALDI-TOF MS spectra were acquired on a microflex LT.

Results: The MS spectrum corresponding to DH5a revealed the molecular peak of ampicillin at [M+H]+ 350 and the sodium adducts [M+Na]+ at 372 and [M+2Na]+ at 394 Da. In contrast, the ESBL derived spectra revealed clearly decreased peaks for ampicillin and it's adducts. Additional peaks at 368, 394, 412 and 324 Da appeared corresponding to the hydrolyzed form of ampicillin, its sodium adducts and the hydrolyzed, decarboxylated form of ampicillin, respectively. A slight spontaneous hydrolysis of ampicillin was observed for DH5a.

Comparable results were achieved for Klebsiella pneumoniae and carbapenems. Carbapenems did not tend to form sodium adducts. Additionally, the hydrolyzed form of carbapenems is very labil and decarboxylated immediately. A clear difference was observed between the carbapenem sensitive strain and the carbapenamase positive strains.

For evaluation, the ratio of the sum of the areas of the non-hydrolyzed forms and the sum of the enzymatically converted forms of the antibiotics was calculated. The quotient is less than 1 for resistant bacteria and exceeds 1 for antibiotics sensitive strains facilitating a simple and easy detection of resistant bacterial strains.

The analysis of cephalosporin resistant bacteria resulted in the disappearance of the antibiotic molecular peak without the appearance of the corresponding hydrolysis products.

Conclusions: The developed approach provides a rapid method for the detection of penicillin and carbapenem resistant bacteria within 4 h.

Session Details

Date: 07/05/2011
Time: 00:00-00:00
Session name: Abstracts of 21st ECCMID / 27th ICC
Subject:
Location: Milan, Italy, 7 - 10 May 2011
Presentation type:
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