Rapid detection of the plasmid-mediated quinolone resistance determinant AAC(6′)-Ib-cr in Enterobacteriaceae by MALDI-TOF MS analysis

[Objectives] Rapid detection of the plasmid-mediated quinolone resistance determinant AAC(6′)-Ib-cr in Enterobacteriaceae by measuring acetyltransferase activity against fluoroquinolones by MALDI-TOF MS analysis.[Methods] The presence of the AAC(6′)-Ib-cr enzyme was determined by MS by measuring the acetyltransferase activity of a collection of 81 isogenic Escherichia coli control strains [10 carrying the AAC(6′)-Ib-cr enzyme during exposure to ciprofloxacin, norfloxacin and levofloxacin] and further analysis of 36 clinical isolates [25 carrying the AAC(6′)-Ib-cr enzyme in addition to different combinations of quinolone resistance mechanisms]. The effect of acetylation yields an increase of 43 Da in the mass of ciprofloxacin and norfloxacin, but not of levofloxacin, that can be observed by visual inspection of the mass peaks in the spectra.[Results] Based on the characteristic peak pattern for the acetylated and non-acetylated forms of ciprofloxacin and norfloxacin, a clear differentiation between AAC(6′)-Ib-cr-producing isolates and non-AAC(6′)-Ib-cr-producing isolates was detected after an incubation time of 30 min, both in the isogenic control strains and in the clinical isolates. Levofloxacin was found intact. A 100% agreement was found between the MALDI-TOF-MS-based assay and the results of the molecular characterization of the tested isolates.[Conclusions] MALDI-TOF MS is an outstanding method for detection of the AAC(6′)-Ib-cr enzyme in clinical samples. The method is easy to perform and not time consuming, as analytical results can be obtained within minutes. For the first time, MALDI-TOF MS has been used to detect resistance promoted by enzymatic modification of antibiotics aside from β-lactamases, expanding the capacity of analysis into new families of antibiotics.This work was supported by the Fondo de Investigación Sanitaria (grant numbers PI12/00552 and PI15/00860 to G. B. and PI11-00934 to J. M. R.-M.) integrated in the Plan Nacional de I + D and funded by the Instituto de Salud Carlos III (ISCIII). We also thank the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0014 to G. B.), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad and co-financed by European Development Regional Fund ‘A Way to Achieve Europe’ ERDF. M. O. is financially supported by the Río Hortega Programme of the ISCIII (CM15/00155).Peer reviewe