{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T05:36:49Z","timestamp":1783661809905,"version":"3.55.0"},"reference-count":47,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2019,12,10]],"date-time":"2019-12-10T00:00:00Z","timestamp":1575936000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Burkholderia (B.) mallei, the causative agent of glanders, and B. pseudomallei, the causative agent of melioidosis in humans and animals, are genetically closely related. The high infectious potential of both organisms, their serological cross-reactivity, and similar clinical symptoms in human and animals make the differentiation from each other and other Burkholderia species challenging. The increased resistance against many antibiotics implies the need for fast and robust identification methods. The use of Raman microspectroscopy in microbial diagnostic has the potential for rapid and reliable identification. Single bacterial cells are directly probed and a broad range of phenotypic information is recorded, which is subsequently analyzed by machine learning methods. Burkholderia were handled under biosafety level 1 (BSL 1) conditions after heat inactivation. The clusters of the spectral phenotypes and the diagnostic relevance of the Burkholderia spp. were considered for an advanced hierarchical machine learning approach. The strain panel for training involved 12 B. mallei, 13 B. pseudomallei and 11 other Burkholderia spp. type strains. The combination of top- and sub-level classifier identified the mallei-complex with high sensitivities (&gt;95%). The reliable identification of unknown B. mallei and B. pseudomallei strains highlighted the robustness of the machine learning-based Raman spectroscopic assay.<\/jats:p>","DOI":"10.3390\/molecules24244516","type":"journal-article","created":{"date-parts":[[2019,12,10]],"date-time":"2019-12-10T10:52:41Z","timestamp":1575975161000},"page":"4516","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":34,"title":["A Machine Learning-Based Raman Spectroscopic Assay for the Identification of Burkholderia mallei and Related Species"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5105-4637","authenticated-orcid":false,"given":"Amira A.","family":"Moawad","sequence":"first","affiliation":[{"name":"Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena, Germany"},{"name":"Animal Health Research Institute, Agricultural Research Center, 12618 Dokki-Giza, Egypt"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anja","family":"Silge","sequence":"additional","affiliation":[{"name":"Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University, Helmholtzweg 4, 07743 Jena, Germany"},{"name":"InfectoGnostics Research Campus Jena, Center of Applied Research, Philosophenweg 7, 07743 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Bocklitz","sequence":"additional","affiliation":[{"name":"Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University, Helmholtzweg 4, 07743 Jena, Germany"},{"name":"Leibniz-Institute of Photonic Technology, Member of the Leibniz Research Alliance \u2013 Leibniz Health Technologies, Albert-Einstein-Str. 9, 07745 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katja","family":"Fischer","sequence":"additional","affiliation":[{"name":"Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petra","family":"R\u00f6sch","sequence":"additional","affiliation":[{"name":"Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University, Helmholtzweg 4, 07743 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Uwe","family":"Roesler","sequence":"additional","affiliation":[{"name":"Institute for Animal Hygiene and Environmental Health, Free University Berlin, Robert-von Ostertag-Str. 7\u201313, 14163 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mandy C.","family":"Elschner","sequence":"additional","affiliation":[{"name":"Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4257-593X","authenticated-orcid":false,"given":"J\u00fcrgen","family":"Popp","sequence":"additional","affiliation":[{"name":"Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University, Helmholtzweg 4, 07743 Jena, Germany"},{"name":"InfectoGnostics Research Campus Jena, Center of Applied Research, Philosophenweg 7, 07743 Jena, Germany"},{"name":"Leibniz-Institute of Photonic Technology, Member of the Leibniz Research Alliance \u2013 Leibniz Health Technologies, Albert-Einstein-Str. 9, 07745 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heinrich","family":"Neubauer","sequence":"additional","affiliation":[{"name":"Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07743 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,12,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1016","DOI":"10.1111\/tbed.13126","article-title":"Development of a rapid and sensitive recombinase polymerase amplification-lateral flow assay for detection of Burkholderia mallei","volume":"66","author":"Saxena","year":"2019","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e32","DOI":"10.1111\/tbed.12665","article-title":"Development of a real-time loop-mediated isothermal amplification assay for detection of Burkholderia mallei","volume":"65","author":"Pal","year":"2018","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e000129","DOI":"10.1136\/vetreco-2015-000129","article-title":"Incidence of Burkholderia mallei infection among indigenous equines in India","volume":"2","author":"Malik","year":"2015","journal-title":"Vet. Rec. Open"},{"key":"ref_4","first-page":"3","article-title":"Glanders outbreak at Tehran Zoo, Iran","volume":"4","author":"Khaki","year":"2012","journal-title":"Iran. J. Microbiol."},{"key":"ref_5","unstructured":"Dedie\u2019, K., Bockemqhl, J., Kqhn, H., Volkmer, K.J., and Weinke, T. (1993). Rotz und Melioidose. Bakterielle Zoonosen bei Tier und Menschen. Stuttgart7Ferdinand Enke Verlag."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/S0001-706X(99)00066-2","article-title":"Ecology of Burkholderia pseudomallei and the interactions between environmental Burkholderia spp. and human-animal hosts","volume":"74","author":"Dance","year":"2000","journal-title":"Acta Trop."},{"key":"ref_7","first-page":"2763","article-title":"Melioidosis: A review","volume":"14","author":"Foong","year":"2014","journal-title":"Rural Remote Health"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1097\/00001432-200204000-00005","article-title":"Melioidosis","volume":"15","author":"Dance","year":"2002","journal-title":"Curr. Opin. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1586\/eri.10.4","article-title":"Present and future therapeutic strategies for melioidosis and glanders","volume":"8","author":"Estes","year":"2010","journal-title":"Expert Rev. Anti Infect. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"108","DOI":"10.4103\/sjmms.sjmms_118_16","article-title":"Melioidosis: Can Tropical Infections Present in Nonendemic Areas? A Case Report and Review of the Literature","volume":"6","author":"Alwarthan","year":"2018","journal-title":"Saudi J. Med. Med. Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"15008","DOI":"10.1038\/nmicrobiol.2015.8","article-title":"Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis","volume":"1","author":"Limmathurotsakul","year":"2016","journal-title":"Nat. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"554","DOI":"10.1097\/QCO.0b013e32833fb88c","article-title":"Melioidosis: Advances in diagnosis and treatment","volume":"23","author":"Cheng","year":"2010","journal-title":"Curr. Opin. Infect. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1016\/j.mcp.2009.04.001","article-title":"DNA microarray-based detection and identification of Burkholderia mallei, Burkholderia pseudomallei and Burkholderia spp.","volume":"23","author":"Schmoock","year":"2009","journal-title":"Mol. Cell. Probes"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Karger, A., Stock, R., Ziller, M., Elschner, M.C., Bettin, B., Melzer, F., Maier, T., Kostrzewa, M., Scholz, H.C., and Neubauer, H. (2012). Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption\/Ionisation mass spectrometric typing. BMC Microbiol., 12.","DOI":"10.1186\/1471-2180-12-229"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1046\/j.1462-2920.2003.00471.x","article-title":"Diversity and significance of Burkholderia species occupying diverse ecological niches","volume":"5","author":"Coenye","year":"2003","journal-title":"Environ. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"120","DOI":"10.3389\/fpls.2013.00120","article-title":"Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants","volume":"4","author":"Mitter","year":"2013","journal-title":"Front. Plant Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1187","DOI":"10.1099\/ijs.0.63149-0","article-title":"Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plant-beneficial properties","volume":"55","author":"Sessitsch","year":"2005","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1038\/ismej.2015.73","article-title":"Molecular mechanisms underlying the close association between soil Burkholderia and fungi","volume":"10","author":"Stopnisek","year":"2016","journal-title":"ISME J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.diagmicrobio.2013.06.011","article-title":"Performance of MALDI-ToF MS for species identification of Burkholderia cepacia complex clinical isolates","volume":"77","author":"Fehlberg","year":"2013","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00081-19","DOI":"10.1128\/JCM.00081-19","article-title":"Identification of Burkholderia pseudomallei by Use of the Vitek Mass Spectrometer","volume":"57","author":"Gassiep","year":"2019","journal-title":"J. Clin. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1016\/j.tim.2017.01.002","article-title":"Cultivation-Free Raman Spectroscopic Investigations of Bacteria","volume":"25","author":"Lorenz","year":"2017","journal-title":"Trends Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.mimet.2014.10.002","article-title":"Comparison of Raman spectroscopy and two molecular diagnostic methods for Burkholderia cepacia complex species identification","volume":"107","author":"Orelio","year":"2014","journal-title":"J. Microbiol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1016\/j.addr.2015.04.006","article-title":"Isolation and identification of bacteria by means of Raman spectroscopy","volume":"89","author":"Pahlow","year":"2015","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_24","first-page":"89","article-title":"The application of Raman spectroscopy for the detection and identification of microorganisms","volume":"47","author":"Kirchhoff","year":"2015","journal-title":"J. Raman Spectrosc."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1007\/s00216-014-7906-5","article-title":"Raman spectroscopic detection and identification of Burkholderia mallei and Burkholderia pseudomallei in feedstuff","volume":"407","author":"Meisel","year":"2015","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1002\/cphc.200390004","article-title":"Raman spectroscopy\u2014A prospective tool in the life sciences","volume":"4","author":"Petry","year":"2003","journal-title":"ChemPhysChem Eur. J. Chem. Phys. Phys. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4410","DOI":"10.1039\/C7AY01363A","article-title":"Common mistakes in cross-validating classification models","volume":"9","author":"Guo","year":"2017","journal-title":"Anal. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1543","DOI":"10.1039\/b507715j","article-title":"Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions","volume":"130","author":"Harz","year":"2005","journal-title":"Analyst"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"8333","DOI":"10.1007\/s00216-015-8743-x","article-title":"Destruction-free procedure for the isolation of bacteria from sputum samples for Raman spectroscopic analysis","volume":"407","author":"Lorenz","year":"2015","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4997","DOI":"10.1039\/c1an15761b","article-title":"Assessment of two isolation techniques for bacteria in milk towards their compatibility with Raman spectroscopy","volume":"136","author":"Meisel","year":"2011","journal-title":"Analyst"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1002\/bip.20447","article-title":"Characterization of bacterial growth and the influence of antibiotics by means of UV resonance Raman spectroscopy","volume":"82","author":"Neugebauer","year":"2006","journal-title":"Biopolymers"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1002\/cphc.200600507","article-title":"Towards a detailed understanding of bacterial metabolism\u2014Spectroscopic characterization of Staphylococcus epidermidis","volume":"8","author":"Neugebauer","year":"2007","journal-title":"ChemPhysChem Eur. J. Chem. Phys. Phys. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"9610","DOI":"10.1021\/ac401806f","article-title":"Culture independent Raman spectroscopic identification of urinary tract infection pathogens: A proof of principle study","volume":"85","author":"Kampe","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_34","first-page":"S97","article-title":"Micro-Raman Spectroscopy: A novel method for identification of Sepsis pathogens","volume":"39","author":"Munchberg","year":"2011","journal-title":"Infection"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"5839","DOI":"10.1007\/s00216-018-1196-2","article-title":"The application of UV resonance Raman spectroscopy for the differentiation of clinically relevant Candida species","volume":"410","author":"Silge","year":"2018","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1016\/j.syapm.2014.05.007","article-title":"Identification of water-conditioned Pseudomonas aeruginosa by Raman microspectroscopy on a single cell level","volume":"37","author":"Silge","year":"2014","journal-title":"Syst. Appl. Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"9873","DOI":"10.1021\/ac302250t","article-title":"Identification of Bacillus anthracis via Raman Spectroscopy and Chemometric Approaches","volume":"84","author":"Meisel","year":"2012","journal-title":"Anal. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"5339","DOI":"10.1002\/anie.201201266","article-title":"Raman Spectroscopic Detection of Anthrax Endospores in Powder Samples","volume":"51","author":"Meisel","year":"2012","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2895","DOI":"10.1128\/AEM.02481-09","article-title":"Raman Spectroscopy-Compatible Inactivation Method for Pathogenic Endospores","volume":"76","author":"Schumacher","year":"2010","journal-title":"Appl. Environ. Microb."},{"key":"ref_40","unstructured":"Team, R.C. (2019, November 10). R: A Language and Environment for Statistical Computing Vienna, Austria: R Foundation for Statistical Computing. Available online: http:\/\/www.R-project.org\/."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.aca.2011.06.043","article-title":"How to pre-process Raman spectra for reliable and stable models?","volume":"704","author":"Bocklitz","year":"2011","journal-title":"Anal. Chim. Acta"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.chemolab.2016.03.024","article-title":"Automatization of spike correction in Raman spectra of biological samples","volume":"155","author":"Ryabchykov","year":"2016","journal-title":"Chemom. Intell. Lab. Syst."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1524\/zpch.2011.0077","article-title":"Checking and Improving Calibration of Raman Spectra using Chemometric Approaches","volume":"225","author":"Dorfer","year":"2011","journal-title":"Z. Phys. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1016\/0168-583X(88)90063-8","article-title":"SNIP, a statistics-sensitive background treatment for the quantitative analysis of PIXE spectra in geoscience applications","volume":"34","author":"Ryan","year":"1988","journal-title":"Nucl. Instrum. Methods Phys. Res. Sect. B Beam Interact. Mater. Atoms"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2929","DOI":"10.1007\/s00216-010-3895-1","article-title":"The influence of intracellular storage material on bacterial identification by means of Raman spectroscopy","volume":"397","author":"Burkhardt","year":"2010","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_46","first-page":"499","article-title":"Burkholderia mallei and Burkholderia pseudomallei as bloterrorism agents: National aspects of emergency preparedness","volume":"9","author":"Gilad","year":"2007","journal-title":"IMAJ RAMAT GAN"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Elschner, M.C., Thomas, P., El-Adawy, H., Mertens, K., Melzer, F., Hnizdo, J., and Stamm, I. (2017). Complete Genome Sequence of a Burkholderia pseudomallei Strain Isolated from a Pet Green Iguana in Prague, Czech Republic. Genome Announc., 5.","DOI":"10.1128\/genomeA.01761-16"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/24\/24\/4516\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:41:00Z","timestamp":1760190060000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/24\/24\/4516"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,12,10]]},"references-count":47,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2019,12]]}},"alternative-id":["molecules24244516"],"URL":"https:\/\/doi.org\/10.3390\/molecules24244516","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,12,10]]}}}