Microflex maldi tof ms system

All isolates were grown on Columbia blood agar plates (bioMérieux, Nürtingen, Germany) for 18 h at 37 °C. Then, a single colony was picked and plated on a 96-spot steel target (Bruker Daltonik, Bremen, Germany) and superimposed by α-Cyano-4-hydroxycinnamic acid (Bruker Daltonik, Bremen, Germany), which was used as an analytical matrix. The spectra were analyzed using a microflex MALDI-TOF MS system (Bruker Daltonik, Bremen, Germany) applying the flexControl software 3.1 (Bruker Daltonik, Bremen, Germany). This method has been described in more detail in a previous publication by our group [16 (link)]. For the MALDI-TOF MS experiments, calibration and positive control were performed with the BRUKER bacterial test standard (BTS).

A total of 165 animal isolates were collected between 2002 and 2021 from dogs (n = 134), cats (n = 3), and a horse (n = 1) at the Veterinary Microbiological Diagnostic Centre of Utrecht University and from humans (n = 27) at the University Medical Centre Utrecht and Certe Medical Microbiology Friesland and Noordoostpolder in the Netherlands. An additional set of isolates (n = 33) were selected from public databases (NCBI and the Sequence Read Archive) and included in the genomic analysis. The complete set of genomes (n = 198) represented 149 isolates from dogs, 35 from humans, and 14 from other animals. Isolates from the current study were identified as S. schleiferi using MALDI TOF-MS with a Bruker Microflex MALDI TOF-MS system, Biotyper software, and MBT-BDAL-5627 MSP library 5267 (Bruker, Germany). S. schleiferi isolates with MALDI identification scores of ≥2.0 were included in the study.

The bacterial cell pellet of the second aliquot was dissolved in 50 μL of formic acid 70% by vortexing; then, 50 μL of pure acetonitrile was incorporated into the solution, vortexed, and centrifuged (10,000 g for 2 min) for the following direct MALDI-TOF MS identification assay. In brief, 1 μL supernatant was spotted onto a target plate, and 1 μL of an α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (Bruker Daltonik GmbH, Bremen, Germany) was added and air dried at room temperature. Identifications were performed by the Bruker microflex MALDI-TOF MS system using the MALDI Biotyper 3.0 RTC database (Bruker Daltonik GmbH, Bremen, Germany). According to the manufacturer, a score of ≥2.0 indicates reliable identification at the species level, while a score between 1.7 and 1.99 indicates a reliable identification at the genus level, and a score below 1.7 indicates a NRI. Thus, these were the criteria used when comparing the results of the direct MALDI-TOF MS identification assay with those from the conventional culture-dependent identification for the evaluation of direct identification performance.

All isolates were characterized using the Bruker microflex MALDI-ToF MS system and Biotyper 3.0 software (Bruker Daltonik GmbH, Bremen, Germany). Samples were analyzed in duplicate using the direct transfer method as follows: bacterial cells from a single colony were transferred onto a stainless steel target, the spot was air dried at ambient temperature, was overlaid with 1 μl matrix HCCA (α-cyano-4-hydroxy-cinnamic acid) diluted in a solution of 50% acetonitrile and 2.5% trifluoroacetic acid, and allowed to air dry a second time at ambient temperature. The target plate was subsequently introduced into the microflex MALDI-ToF mass spectrometer for automated measurement of mass spectra and comparison to the reference database (Biotyper v3.0). Identifications scores ≥2.0 were required for confident species identification; scores <2.0 and ≥1.7 were considered confident genus identification; isolates with scores <1.7 were re-analyzed in duplicate, and if they failed to achieve a score ≥1.7 on the second round, they were classified as unidentified.

Initially, the positive blood cultures were subjected to Gram staining to determine the presence of gram-positive or gram-negative bacteria. Based on these results, appropriate agar plates, including blood, Maconkey, chocolate, and anaerobic blood agar, were used for further culturing. The plates were grown in an incubator (Thermo Fisher Scientific, USA) at 35°C in a 5% CO₂ or anaerobic atmosphere until visible colonies appeared. Identification was then performed with the Bruker microflex MALDI-TOF MS system using the MALDI Biotyper 3.0 Realtime classification (RTC) database (Bruker Daltonics, Bremen, Germany) as described previously (Chen et al., 2013 (link); Tian et al., 2016 (link); Zhou et al., 2017 (link)). In brief, a pure bacterial colony was smeared onto a steel target plate with a wood toothpick, and 70% formic acid solution was added to lyse the bacterial cells. Once the formic acid solution dried, 1 μL of α-cyano-4-hydroxycinnamic acid (HCCA) matrix (Bruker Daltonics) solution was added for subsequent MALDI-TOF MS (Bruker Daltonics) identification. The calibration and validation of MALDI-TOF MS was carried out once a week with a bacterial test standard (BTS) according to the manufacturer's instructions (Bruker Daltonics).