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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 <jats:italic>B. pseudomallei<\/jats:italic> and <jats:italic>B. mallei<\/jats:italic> and to build up a reliable reference database for both organisms.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>A collection of ten <jats:italic>B. pseudomallei<\/jats:italic> and seventeen <jats:italic>B. mallei<\/jats:italic> 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 <jats:italic>B. mallei<\/jats:italic>, higher genetic diversity among <jats:italic>B. pseudomallei<\/jats:italic> 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 <jats:italic>B. pseudomallei<\/jats:italic> (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\u2009Da specifically and reproducibly found in the spectra of this strain.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats: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 <jats:italic>B. mallei<\/jats:italic> than in <jats:italic>B. pseudomallei<\/jats:italic> 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.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1471-2180-12-229","type":"journal-article","created":{"date-parts":[[2012,10,10]],"date-time":"2012-10-10T06:14:12Z","timestamp":1349849652000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":53,"title":["Rapid identification of Burkholderia mallei and Burkholderia pseudomalleiby intact cell Matrix-assisted Laser Desorption\/Ionisation mass spectrometric typing"],"prefix":"10.1186","volume":"12","author":[{"given":"Axel","family":"Karger","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R\u00fcdiger","family":"Stock","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario","family":"Ziller","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mandy C","family":"Elschner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Barbara","family":"Bettin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Falk","family":"Melzer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Maier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Markus","family":"Kostrzewa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Holger C","family":"Scholz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heinrich","family":"Neubauer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Herbert","family":"Tomaso","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2012,10,10]]},"reference":[{"issue":"1","key":"1798_CR1","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1128\/CMR.4.1.52","volume":"4","author":"DA Dance","year":"1991","unstructured":"Dance DA: Melioidosis: the tip of the iceberg?. 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