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 ␤-lactam antibiotics is a growing challenge for managing severe bacterial infections. The rapid and costefficient determination of ␤-lactam resistance is an important prerequisite for the choice of an adequate antibiotic therapy. ␤-Lactam resistance is based mainly on the expression/overexpression of ␤-lactamases, which destroy the central ␤-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 ␤-lactam antibiotics: ampicillin, piperacillin, cefotaxime, ceftazidime, ertapenem, imipenem, and meropenem. ␤-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.

La espectrometría de masas MALDI-TOF en el laboratorio de microbiología clínica

“…Sparbier y colaboradores 52 , empleando un espectrómetro de masas Microflex LT (Bruker Daltonics Inc.), demostraron que la hidrólisis enzimática del anillo de β-lactámico resulta en la desaparición del pico original en el espectro, a través de un desplazamiento de la masa molecular de +18 Da del agente antibiótico intacto. Esta hidrólisis es seguida por una descarboxilación del producto hidrolizado, lo que resulta en un desplazamiento del pico de -44 Da más de la forma hidrolizada, cambios en los espectros que son fácilmente detectados por MALDI-TOF MS. Mediante este principio, y empleando diferentes agentes inhibidores de β-lactamasas como ácido clavulánico, tazobactam o ácido 3-aminofenilborónico (APBA), los mismos autores describieron la detección de resistencia a ampicilina en E. coli, de cefotaxima y ceftazidima en E. coli y K. pneumoniae, así como la hidrolisis de carbapenémicos por K. pneumoniae a través del uso de la tecnología MALDI-TOF MS. Adicional a los procedimientos basados en la hidrólisis, recientemente se ha reportado la utilidad de esta tecnología para la detección de cepas de E. coli pertenecientes al grupo clonal ST131, productoras de Betalactamasas de Espectro Extendido (BLEE), mediante la visualización de un pico específi-co en el espectro de masas 53,54 .…”

Abstracts of the 75th Scientific Conference of the University of Latvia

“…Sparbier et al (2012) separated appeared peaks belonging to sensitive or resistant bacteria. Mentioned peaks in her work were also observed, but they could not be strictly classified for resistant or sensitive strains only, they appeared in both type of strains that could be explained with different MALDI-TOF instrument usage.…”

“…Antibiotic resistance is usually checked on the phenotypic level by the Etest, MIC or disc diffusion that requires another day before reporting results. Lately MALDI-TOF is widely used for fast bacteria identification and now for analyses of β-lactamase activity (Bradford 2012;Sparbier et al 2012).…”

“…They were further centrifuged for 2 min at 13 000 rpm and 1 µL of supernatant placed onto polished steel MALDI target plate and covered with α-cyano-4-hydroxy-cinnamic acid (50% acetonitrile, 1% trifluoroacetic acid; Bruker Daltonik, Germany), done twice with two replicates. Spectra were obtained in mass range from 100 to 1000 Da with MALDI-TOF and processed with FlexAnalyses program (Sparbier et al 2012). Bacteria CTX resistance was verified with disc diffusion method.…”

Evaluation of MALDI-TOF MS in the microbiology laboratory

“…MALDI-TOF MS technique is able to detect antibiotic mass alteration due to the chemical modification in the antimicrobial molecule. For this test, it is necessary to incubate the bacterial protein extract with the antibiotic for a period of 1-4 hours so that it is possible to detect the presence of β-lactamase with hydrolytic activity on penicillins, cephalosporins and/or carbapenems (4,27) .…”