Direct Detection of CMY-2 by MALDI-TOF Mass Spectrometry

FIGUEROA ESPINOSA ROQUE A; RUMI MARÍA VALERIA; MARCHISIO MARTÍN; BARRIOS RUBEN; VAY CARLOS; ALMUZARA MARISA; GUTKIND GABRIEL; DI CONZA JOSÉ

New Orleans

Congreso; ASM MICROBE 2017; 2017

American Society for Microbiology

Background: Resistance to different b-lactams mediated by different b-lactamase families is one of the most relevant problems in current antimicrobial therapy; fast characterization of the involved mechanisms is critical for effective antimicrobial therapy. At present, MALDI-TOF mass spectrometry (MS) is routinely used for rapid species identification. Also, it has been proposed for identifying b-lactam hydrolysis products, after incubating the microorganism with the antibiotics for at least 1 hour. However, the potential of these methods for discriminating specific resistance markers has not yet been fully investigated. In this study, very simple and direct MALDI-TOF MS protocols were applied in order to find a distinctive protein profile in CMY-2 producing E. coli.Methods: 28 E. coli strains were included in this study. They were previously analyzed by PCR and sequencing and grouped according to the presence of b-lactamase genes: 15 were CMY-2 producers from clinical sample (n=13) or transconjugants (n=2), 7 were ESBL-producers (no CMY-producers) and 6 were susceptible to TGC. All were processed by MALDI-TOF MS (Bruker Daltonics, Germany) after protein extraction with organic solvents at different mass ranges. MALDI-TOF spectra were acquired with a Microflex LT under the control of Flexcontrol software (version 3.0). Peaks compatible with the theoretical mass of mature CMY-2 enzyme were detected employing mMass 5.5.0.Results: Two groups of isolates could be differentiated after peak analysis: a 39,854 Da MS peak corresponding to the mature protein could be detected in CMY-2-producers when the instrument operated at a mass range of 17,000 to 50,000 Da. This distinctive peak was present in the spectra for all CMY-2-producing E. coli (wild type and transconjugants), and consistently absent in the control groups (ESBL producers and susceptible strains). Besides, when these strains were analyzed in the identification range (2,000 to 20,000 Da), no distinctive peak was identified. Conclusions: Although our results are preliminary, MALDI-TOF spectra contain information that allows distinguishing CMY-2 producers on a rapid and reliable way. The time required for this test is substantially less than that needed for hydrolysis products, and it does not require additional reagents to those used for the microorganism identification. Overall, if the method can be extended to other b-lactamases, or even other resistance markers, may prove a significant improvement in lab abilities for their detection with extensive clinical implications.