| Summary: | <p>Abstract</p> <p>Background</p> <p><it>Burkholderia (B.) pseudomallei</it> and <it>B. mallei</it> are genetically closely related species. <it>B. pseudomallei</it> causes melioidosis in humans and animals, whereas <it>B. mallei</it> is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of <it>B. pseudomallei</it> and <it>B. mallei</it> and to build up a reliable reference database for both organisms.</p> <p>Results</p> <p>A collection of ten <it>B. pseudomallei</it> and seventeen <it>B. mallei</it> strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing <it>B. mallei</it>, higher genetic diversity among <it>B. pseudomallei</it> was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of <it>B. pseudomallei</it> (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain.</p> <p>Conclusions</p> <p>Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in <it>B. mallei</it> than in <it>B. pseudomallei</it> isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.</p>
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