Detection of acetyltransferase modification of kanamycin, an aminoglycoside antibiotic, in bacteria using ultrahigh-performance liquid chromatography tandem mass spectrometry.

Abstract

The occurrence of antibiotic-resistant bacteria is a worldwide issue that has the potential, if not addressed, to eliminate classes of antibiotics that have extended life expectancy in the last century. An approach to confront this threat is the development of technologies that greatly accelerate the detection of antibiotic resistance to minimize unnecessary treatment involving antibiotics. Development of an analytical method for rapid detection of aminoglycoside resistance using liquid chromatography/mass spectrometry (LC/MS) has not been reported in the literature and is described here. A strain of Escherichia coli carrying a plasmid encoding an aminoglycoside-modifide enzyme (N-acetyltransferase) was incubated with kanamycin, an aminoglycoside. The antibiotic and its modified form were observed using LC/MS. An ABSciex QTrap 6500+ was used for kinetic and quantitative analysis and high-resolution structural elucidation was performed using a Thermo Fisher Q-Exactive hybrid quadrupole-orbitrap mass spectrometer. Detection of kanamycin modification was achieved in less than an hour of incubation. Calibration curves for both modified and unmodified kanamycin from 0.5 to 50 μg mL-1 were obtained. Generation and depletion of modified and unmodified kanamycin as a function of time were performed. High-resolution mass spectrometry was employed for confirmation and structural elucidation of the novel precursor and product ion biomarkers with high mass accuracy (≤7 ppm). A newly developed analytical method is able to determine bacterial resistance to aminoglycosides (via acetylation of kanamycin), qualitatively and quantitatively, within 30minutes and 6hours of incubation with kanamycin, respectively. High-resolution data support the placement of an acetyl group on kanamycin confirming aminoglycoside resistance and its mechanism. Quantification was achieved for both forms of the antibiotic 50- to 100-fold lower than the minimum inhibitory concentration for the resistant bacteria and can be used to replace conventional antimicrobial susceptibility tests.