In order to obtain a fast and accurate qualitative and quantitative AST for colistin of the 50 K. pneumoniae strains included in the study, CORE assay and colistin BMD susceptibility testing were performed in parallel including three biological and two technical replicates for each tested isolate. To optimize the CORE assay inoculum concentration, the time of the test incubation and to develop a classifying algorithm, preliminary experiments were performed on 10 well-characterized K. pneumoniae, 5 colistin resistant (colR) and 5 colistin susceptible (colS) strains (reported in bold in Table 2 ).
The CORE assay protocol was performed according to the steps reported inFigure 1 .
Briefly, all the K. pneumoniae strains were sub-cultured on MacConkey agar plates (bioMérieux) at 37° C for 24 h; a 0.5 McF suspension was made from grown colonies. Except for the inoculum size, ComASP (Compact Antimicrobial Susceptibility Panel) Colistin test was performed following the standard procedures.
After 3 h of incubation at 37°C, an aliquot of 1 μL of the bacterial suspension from each well was spotted in duplicate on the MALDI-TOF target plate (96 wells steel target slide, Autobio Diagnostics) by direct deposition with the addition of 1 μL of Autobio sample pretreatment reagent lysate 1 (formic acid). After drying, the spot was overlaid with 1 μL of Cyano 4 Hydroxycinnamic Acid CHCA AUTOF MS matrix (Autobio Diagnostics, CO., LTD, China) solubilized in lysate 2 (acetonitrile) and buffer (trifluoroacetic acid) as recommended.
The spectra acquisition was performed on an Autof 1,000 MS (Autobio Diagnostics, Zhengzhou, China) mass spectrometer in positive linear mode, at a laser frequency of 60 Hz, in the mass range 2–20 kDa, using the “microbe” automatic acquisition mode with an overall 240 laser-shot acquired by 40 shot steps for each spot. The mass spectrometer was calibrated with Autobio calibrating agent consisting of nine calibrating proteins, according to manufacturer’s instructions.
The protein spectra were analyzed using the Autof Acquirer version 1.0.55 software and the library v2.0.61 was used for the peaks matching. The identification results were interpreted according to the manufacturer criteria as following: Identification scores ≥9 were considered positive at the species level, scores between 6 and 9 as positive at the genus level, and scores <6 were defined as unreliable (no identification).
The CORE assay protocol was performed according to the steps reported in
Briefly, all the K. pneumoniae strains were sub-cultured on MacConkey agar plates (bioMérieux) at 37° C for 24 h; a 0.5 McF suspension was made from grown colonies. Except for the inoculum size, ComASP (Compact Antimicrobial Susceptibility Panel) Colistin test was performed following the standard procedures.
After 3 h of incubation at 37°C, an aliquot of 1 μL of the bacterial suspension from each well was spotted in duplicate on the MALDI-TOF target plate (96 wells steel target slide, Autobio Diagnostics) by direct deposition with the addition of 1 μL of Autobio sample pretreatment reagent lysate 1 (formic acid). After drying, the spot was overlaid with 1 μL of Cyano 4 Hydroxycinnamic Acid CHCA AUTOF MS matrix (Autobio Diagnostics, CO., LTD, China) solubilized in lysate 2 (acetonitrile) and buffer (trifluoroacetic acid) as recommended.
The spectra acquisition was performed on an Autof 1,000 MS (Autobio Diagnostics, Zhengzhou, China) mass spectrometer in positive linear mode, at a laser frequency of 60 Hz, in the mass range 2–20 kDa, using the “microbe” automatic acquisition mode with an overall 240 laser-shot acquired by 40 shot steps for each spot. The mass spectrometer was calibrated with Autobio calibrating agent consisting of nine calibrating proteins, according to manufacturer’s instructions.
The protein spectra were analyzed using the Autof Acquirer version 1.0.55 software and the library v2.0.61 was used for the peaks matching. The identification results were interpreted according to the manufacturer criteria as following: Identification scores ≥9 were considered positive at the species level, scores between 6 and 9 as positive at the genus level, and scores <6 were defined as unreliable (no identification).
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