Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Analysis

All 39 taxon K isolates were grown on TSA (Oxoid CM0131) for 48 h at 28°C and were subcultivated twice prior to harvesting. Cell extracts were prepared as described previously (Wieme et al., 2014). For all isolates examined, both cell extracts (1 μl) and homogeneous cell smears of isolated colonies were transferred to the spot sites of a 96-well stainless steel target plate (Bruker Daltonics). Both cell smears and cell extracts were air-dried at room temperature. Each spot was overlaid with 1 μl of matrix solution (10 mg/ml α-cyano-4-hydroxycinnamic acid in acetonitrile:trifluoroacetic acid:MilliQ [50:2.5:47.5] water-solvent) and left to air-dry at room temperature. Cell smears were spotted in triplicate and cell extracts were spotted in duplicate to verify reproducibility. Prior to analysis, the mass spectrometer was externally calibrated using the Bacterial Test Standard (Bruker Daltonics) used for calibration. The target plate was measured twice automatically on a Bruker Microflex^TM LT/SH Smart platform (Bruker Daltonics). The spectra were obtained in linear, positive ion mode using FlexControl 3.4 software according to the manufacturer’s recommended settings (Bruker Daltonics).

The spectra of the cell smears and cell extracts were compared to those in the MALDI BioTyper in vitro diagnostics (MBT IVD) (DB-7712 MSP) library using the MBT Compass Explorer 4.1 software (Bruker Daltonics) according to the manufacturer’s default settings. The spectra were also compared with the combined spectra of the MBT IVD library and the LM-UGent in-house library, which consists of 298 main spectra (MSPs) representing 271 Burkholderia reference strains for which the identification has been verified through recA gene sequence analysis or MLST analysis, and which includes 17 taxon K strains (Supplementary Table 1). This in-house library was constructed by preparing a cell extract for each strain as described above, spotting each extract eight times on the target plate and measuring each spot four times to generate 32 spectra. FlexAnalysis 3.4 software (Bruker Daltonics) was used to assess the quality of the spectra generated. MBT Compass Explorer 4.1 software was used to generate MSPs based on a minimum of 24 spectra according to manufacturer’s instructions. Species-level identification was performed according to the Optimized Acceptance Criterium (OAC) (De Bel et al., 2011), meaning that the identification of the best matching spectrum was accepted in case of a log score ≥2.0 and when a 0.200 log difference was observed between the best matching spectrum and the highest-scoring spectrum of a different species.