Raman spectroscopic detection and identification of Burkholderia mallei and Burkholderia pseudomallei in feedstuff
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Burkholderia mallei (the etiologic agent of glanders in equines and rarely humans) and Burkholderia pseudomallei, causing melioidosis in humans and animals, are designated category B biothreat agents. The intrinsically high resistance of both agents to many antibiotics, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Current methods to identify these organisms may require up to 1 week, as they rely on phenotypic characteristics and an extensive set of biochemical reactions. In this study, Raman microspectroscopy, a cultivation-independent typing technique for single bacterial cells with the potential for being a rapid point-of-care analysis system, is evaluated to identify and differentiate B. mallei and B. pseudomallei within hours. Here, not only broth-cultured microbes but also bacteria isolated out of pelleted animal feedstuff were taken into account. A database of Raman spectra allowed a calculation of classification functions, which were trained to differentiate Raman spectra of not only both pathogens but also of five further Burkholderia spp. and four species of the closely related genus Pseudomonas. The developed two-stage classification system comprising two support vector machine (SVM) classifiers was then challenged by a test set of 11 samples to simulate the case of a real-world-scenario, when “unknown samples” are to be identified. In the end, all test set samples were identified correctly, even if the contained bacterial strains were not incorporated in the database before or were isolated out of animal feedstuff. Specifically, the five test samples bearing B. mallei and B. pseudomallei were correctly identified on species level with accuracies between 93.9 and 98.7 %. The sample analysis itself requires no biomass enrichment step prior to the analysis and can be performed under biosafety level 1 (BSL 1) conditions after inactivating the bacteria with formaldehyde.
KeywordsAnimal feedstuff Burkholderia mallei Burkholderia pseudomallei Pseudomonas Raman spectroscopy
Funding of the research projects “Pathosafe” (FKZ 13N9547 and FKZ 13N9549) and “RamaDek” (FKZ 13N11168) from the Federal Ministry of Education and Research, Germany (BMBF) as well as funding of “EQADeBa” by the EU, EAHC Agreement no. 2007 204 is gratefully acknowledged. We also thank Katja Fischer (Friedrich Loeffler Institute, Germany) for doing the sample preparation and inactivation experiments. We highly appreciate the help of Dr. Holger Scholz, Institute of Microbiology, Federal Armed Forces, Munich, Germany, and Dr. Ulrich Wernery, Central Veterinary Research Institute, Dubai, UAE for providing B. mallei and B. pseudomallei strains.
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