Microflex lt maldi biotyper system

The bacterial isolates were identified with MALDI-TOF MS, using both the Microflex LT-MALDI Biotyper System with the Biotyper spectral database ver.11 (Bruker Daltonics, Bremen, Germany) and the Autof MS2000 System with the current online spectral library (Autobio Diagnostics, Zhengzhou, China). The standard methods of direct colony transfer and rapid (on-target) extraction with formic acid were used for sample preparation prior to the acquisition of mass spectra, as described previously [79 (link),80 ]. The criteria for “confident species-level identification” were as follows: log scores ≥ 2.0 for the Bruker Biotyper System and scores ≥ 9.0 for the Autobio Autof System. Only the highest-match score values (1st of the 10 best hits) of the clinical isolates against the corresponding reference libraries were reported in the Results section. To generate the reference spectra of the selected Acinetobacter strains for the Autobio library, the full (in-tube) extraction method was used; the extracts from each culture were spotted four times onto a ground steel target and each spot was measured and processed three times according to the manufacturer’s instruction.

In the central surveillance laboratory, the species identity of the isolates was confirmed by MALDI-TOF mass spectrometry with the Microflex LT-MALDI Biotyper System (Bruker Daltonics, Bremen, Germany).
The susceptibility to a range of antibiotics, including ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, ceftazidime, ceftazidime-avibactam, cefepime, aztreonam, cefotaxime, ertapenem, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, doxycycline, tigecycline, chloramphenicol, trimethoprim-sulfamethoxazole, fosfomycin, and colistin, was determined by a reference broth microdilution method according to ISO 20776-2:2021 [17 ] and European committee on antimicrobial susceptibility testing (EUCAST) [18 ] methodology. Susceptibility testing results were interpreted according to EUCAST v.12.0 Clinical breakpoints [19 ].
All isolates with colistin minimum inhibitory concentration (MIC) >0.5 mg/L (1105 E. coli and 1483 K. pneumoniae) were screened for the presence of mcr genes by means of real-time PCR as described elsewhere [20 (link)].

Fungi were identified using Matrix Assisted Laser Desorption/ionization Time-Of-Flight (MALDI-TOF) mass spectrometry (BrukerDaltonics, Germany). A standard method was used for protein extraction, according to the manufacturer’s recommendations^{27 (link),29 (link)}. One μl of each protein extraction supernatant and the matrix HCCA (a-cyano-4-hydroxycinnamic acid) were spotted on the MSP96 target (Bruker Daltonics, Germany) with a positive control (Candida albicans) and a negative control (the matrix). The data acquisition was performed on the MALDI Biotyper (Microflex LT system; Bruker Daltonics GmbH, Bremen, Germany) using Flex Control^TM software and MALDI BioTyper RTC identification software (Bruker Daltonics).