Most phenotypic methods routinely employed for the detection of ESBL and AmpC producing Enterobacteriaceae require 18 hours of incubation. Aiming to offer this clinically relevant information in a shorter time, we developed a MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA) as a one-step screening and confirmation panel in accordance with the EUCAST guidelines.
DOT-MGA was performed on 12 clinical Enterobacteriaceae strains displaying resistance against third-generation cephalosporins plus 4 control strains recommended by EUCAST for detection of ESBL production. Microdroplets (6 µl) containing bacterial suspension and antibiotics (cefpodoxime, cefotaxime, ceftazidime, cefepime with or without clavulanic acid and/or cloxacillin) in cation-adjusted Mueller-Hinton broth (CA-MHB) were spotted directly onto MBT Biotargets 96 (Bruker Daltonics, Germany). Targets were incubated for 4 hours at 36 °C in plastic transport boxes in order to avoid evaporation. Subsequently, culture medium was removed and MALDI-TOF MS of the cells adhered to the target’s surface was performed. The minimum inhibitory concentration (MIC) was considered to be the lowest concentration at which the MALDI Biotyper software (Bruker) provided no species identification. ESBL/AmpC production was defined as an 8-fold or greater decrease of the cephalosporin MIC when combined with clavulanic acid and/or cloxacillin, respectively. A computer-based algorithm was established to interpret the results, which were compared with those of broth microdilution (reference method). Resistance mechanisms of the clinical isolates were assessed by PCR.
The method correctly identified the ESBL and AmpC production of both control and clinical strains (positive agreement, 100%; negative agreement, 100%), allowing the differentiation of each mechanism even in isolates displaying combined resistance. The results agreed with the characterization of the strains by PCR.
We present a rapid method for phenotypic detection of ESBL and AmpC that yields reliable results in a significantly shorter time, thus representing a potentially valuable tool in the routine detection of multidrug resistant pathogens.
K. Sparbier, Bruker Daltonik: Employee , Salary .
M. Kostrzewa, Bruker Daltonik: Employee , Salary .
K. Becker, Bruker Daltonik: Co-inventor of a pending patent , Licensing agreement or royalty and Speaker honorarium .