Screening for carbapenemase-associated biomarkers in Klebsiella oxytoca using matrixassisted laser desorption/ionization time of flight mass spectrom

Abstract: Antibiotic resistant bacteria are threatening human health, and the resistance is progressingfaster than the development of new antimicrobial compounds. Antibiotic resistant infections cost enormous sums of money and resources, but most importantly human lives. Therefore, early prediction and detection of antibiotic resistance in bacteria are research areas of high priority. The use of analytical instruments such as matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) is a powerful tool for research in antibiotic resistant bacteria, both as quick microbe identification and for other areas in the field.Due to its relatively easy interpretable spectra provided by the soft ionization technique, and ability to ionize macromolecules without compromising sample integrity, it has also been used for biomarker screening for detection of antimicrobial resistance. Although these studies have shown promising results, the area is still progressing and needs further method development and standardized protocols. This study aimed to use MALDI-TOF MS for carbapenemaseassociated screening in Klebsiella oxytoca. The presumed key spectral peaks would derive from presence of the enzyme Verona integron-encoded metallo-β-lactamase, type 1 (VIM-1), and would not be found if VIM-1 were absent. The isolates, carrying the enzyme, used in the study were isolated from wastewater and river water in Örebro, Sweden. Bacteria genus and species was determined by MALDI-TOF identification, whereupon the microbes were tested for antibiotic susceptibility using the disk diffusion method. Carbapenem hydrolysis assay was used to confirm the presence/absence of functional carbapenemase. A genotypic confirmation was performed by polymerase chain reaction and gel electrophoresis. Mass spectra from MALDI-TOF were compared for identification of possible biomarker peaks that could indicate carbapenem resistance. Nine key mass spectral peaks were found that could potentially be used as biomarkers in future studies. The peaks differentiated two groups of Klebsiella oxytocaisolates, one group producing functional carbapenemase and one group that did not, consistent with the aim of this study.