Research Article

LC-MS/MS: a rapid and simple new method for the determination of carbapenem β-lactamases

Published: November 19, 2015
Genet. Mol. Res. 14 (4) : 14457-14468 DOI: 10.4238/2015.November.18.8

Abstract

We investigated the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pan-drug-resistant Acinetobacter baumannii (PDR-AB) with carbapenem resistance. Eight strains were randomly selected from 84 clinical isolates of PDR-AB strains obtained by the Kirby-Bauer and agar dilution methods. An efflux pump inhibition test was used to screen for the efflux pump phenotype. An ethylenediaminetetraacetic acid (EDTA) synergy test was used to screen for the β-lactamase phenotype, and a three-dimensional test was used to detect extended spectrum β-lactamase (ESBL) and ampicillin C, KPC, and carbapenemase. ESBL genes were amplified by polymerase chain reaction and sequenced. Outer membrane proteins were extracted from a sensitive strain and the PDR-AB strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected to LC-MS/MS. Peptide mass fingerprinting data were retrieved, and proteins with differential expression were identified. Results of the efflux pump inhibition tests showed that the minimum inhibitory concentrations for meropenem were decreased in 4 of the 8 strains by at least 25% of the original value. The results of the EDTA synergy test were negative, and the modified Hodge’s tests were positive for all strains. PCR and sequencing confirmed that seven, five, and all eight of the PDR-AB strains contained blaOXA-23, blaTEM-1, and KPC-2, respectively. OXA-23 and CsuC proteins were differentially expressed between the drug-resistant and -sensitive strains. Production of blaOXA-23 enzyme and pilus molecular chaperone to guide synthesis of CsuC protein may be involved in the resistance of A. baumannii to carbapenems. LC-MS/ MS provides a quick and easy method for carbapenemase detection.

We investigated the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pan-drug-resistant Acinetobacter baumannii (PDR-AB) with carbapenem resistance. Eight strains were randomly selected from 84 clinical isolates of PDR-AB strains obtained by the Kirby-Bauer and agar dilution methods. An efflux pump inhibition test was used to screen for the efflux pump phenotype. An ethylenediaminetetraacetic acid (EDTA) synergy test was used to screen for the β-lactamase phenotype, and a three-dimensional test was used to detect extended spectrum β-lactamase (ESBL) and ampicillin C, KPC, and carbapenemase. ESBL genes were amplified by polymerase chain reaction and sequenced. Outer membrane proteins were extracted from a sensitive strain and the PDR-AB strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected to LC-MS/MS. Peptide mass fingerprinting data were retrieved, and proteins with differential expression were identified. Results of the efflux pump inhibition tests showed that the minimum inhibitory concentrations for meropenem were decreased in 4 of the 8 strains by at least 25% of the original value. The results of the EDTA synergy test were negative, and the modified Hodge’s tests were positive for all strains. PCR and sequencing confirmed that seven, five, and all eight of the PDR-AB strains contained blaOXA-23, blaTEM-1, and KPC-2, respectively. OXA-23 and CsuC proteins were differentially expressed between the drug-resistant and -sensitive strains. Production of blaOXA-23 enzyme and pilus molecular chaperone to guide synthesis of CsuC protein may be involved in the resistance of A. baumannii to carbapenems. LC-MS/ MS provides a quick and easy method for carbapenemase detection.

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