Detection of Extended-Spectrum β-Lactamase and Carbapenemase Activity in Gram-Negative Bacilli Using Liquid Chromatography – Tandem Mass Spectrometry

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Vlad Serafim,1,2 Ajit J Shah,3 Monica Licker,4,5 Florin George Horhat,4 Silvana Vulpie,5 Corina Musuroi,4,5 Delia Muntean4,5 1Genetics Discipline, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania; 2The National Institute of Research and Development for Biological Sciences, Bucharest 060031, Romania; 3Department of Natural Sciences, Middlesex University, London NW4 4BT, UK; 4Department of Microbiology, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania; 5“Pius Brînzeu” Emergency Clinical County Hospital, Timișoara 300723, RomaniaCorrespondence: Monica LickerDepartment of Microbiology, “Victor Babes” University of Medicine and Pharmacy, 2 E. Murgu, Timisoara 300041, RomaniaTel +40 0256-707513Email licker.monica@umft.roPurpose: Several mass spectrometry-based methods for antimicrobial sensitivity testing have been described in recent years. They offer an alternative to commercially available testing systems which were considered to have disadvantages in terms of cost- and time-efficiency. The aim of this study was to develop an LC-MS/MS-based antibiotic hydrolysis assay for evaluating antimicrobial resistance (AMR) of Gram-negative bacteria.Materials and Methods: Four species of Gram-negative bacilli (Klebsiella pneumoniae, Escherichia coli, Providencia stuartii and Acinetobacter baumannii) were tested against six antibiotics from three different classes: ampicillin, meropenem, imipenem, ceftazidime, ceftriaxone and cefepime. Bacterial suspensions from each species were incubated with a mixture of the six antibiotics. Any remaining antibiotic following incubation was measured using LC-MS/MS. The results were interpreted using measurements obtained for an E. coli strain sensitive to all antibiotics and expressed as percentage of hydrolyzed antibiotic. These were subsequently compared to commercially-available system for the bacteria identification and susceptibility testing.Results: Overall, LC-MS/MS assay and commercial antimicrobial susceptibility platform results showed good agreement in terms of an organism being resistant/sensitive to an antibiotic. The time required to complete the LC-MS/MS-based hydrolysis test was under 5 h, significantly shorter that commercially available susceptibility testing platforms.Conclusion: By using a sensitive strain for results interpretation and simultaneous use of multiple antibiotics, the proposed protocol offers improved robustness and multiplexing over previously described methods for antibiotic sensitivity testing. Nevertheless, further research is needed before routine assimilation of the method, especially for strains with intermediate resistance.Keywords: antimicrobial, resistance, β-lactamases, LC-MS/MS, Gram-negative, mass spectrometry

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