Version 2 (20170523) of the MALDI-TOF Mass Spectrometry Database for Identification and Classification of Highly Pathogenic Microorganisms from the Robert Koch-Institute (RKI)

(Version 20170523) Edit (Nov 30, 2018): New database version (v.3 - 20181130) - available: 10.5281/zenodo.1880975 Version 2 (20170523) of the RKI’s MALDI-TOF mass spectral database is an update of the original database (version 20161027, https://doi.org/10.5281/zenodo.163517). The RKI database contains mass spectral entries from highly pathogenic (biosafety level 3, BSL-3) bacteria such as Bacillus anthracis, Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei and Francisella tularensis as well as a selection of spectra from their close and more distant relatives. The database can be used as a reference for the diagnostics of BSL-3 bacteria using proprietary and free software packages for MALDI-TOF MS-based microbial identification. Spectral data are distributed as a 7-zip archive that contains the original mass spectra in its native data format (Bruker Daltonics). Please refer to the pdf file (170523-ZENODO-Metadata.pdf) to obtain information on the metadata of the spectra. Do not try to print this document (~1100 pages!) The pkf-file (170523_ZENODO_Peaklist_30Peaks_1.6.pkf) contains the MS peak list data in a Matlab compatible format. The latter data file can be imported into MicrobeMS, a Matlab-based free-of-charge software solution developed at RKI. MicrobeMS is available from http://www.microbe-ms.com. The RKI mass spectral database will be updated on a regular basis. The author's grateful thanks are given to the following persons for providing microbial strains and species. Without their help this work would not be possible. Wolfgang Beyer - University of Hohenheim, Faculty of Agricultural Sciences, Stuttgart, Germany Guido Werner - Robert Koch-Institute, Nosocomial Pathogens and Antibiotic Resistances (FG13), Wernigerode, Germany Alejandra Bosch - CINDEFI, CONICET-CCT La Plata, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina Michal Drevinek - National Institute for Nuclear, Biological and Chemical Protection, Milin, Czech Republic Roland Grunow - Robert Koch-Institute, Highly Pathogenic Microorganisms (ZBS2), Berlin, Germany Daniela Jacob - Robert Koch-Institute, Highly Pathogenic Microorganisms (ZBS2), Berlin, Germany Silke Klee - Robert Koch-Institute, Highly Pathogenic Microorganisms (ZBS2), Berlin, Germany Jörg Rau - Chemisches und Veterinäruntersuchungsamt Stuttgart, Fellbach, Germany Jens Jacob - Robert Koch-Institute, Hospital Hygiene, Infection Prevention and Control (FG14), Berlin, Germany Martin Mielke - Robert Koch-Institute, Department 1 - Infectious Diseases, Berlin, Germany Monika Ehling-Schulz - Functional Microbiology, Institute of Microbiology, University of Veterinary Medicine, Vienna, Austria Armand Paauw - Department of Medical Microbiology, CBRN protection, Universitair Medisch Centrum Utrecht, TNO, Rijswijk, The Netherland

Multiplex detection of microbial and plant toxins by immunoaffinity enrichment and matrix-assisted laser desorption/ionization mass spectrometry

Plant and microbial toxins such as ricin, staphylococcal enterotoxin B (SEB), and the botulinum neurotoxins (BoNT) are considered as potential biological warfare agents. Specific screening methods are, therefore, required that enable unambiguous and sensitive identification of these biohazards, particularly for the occurrence of the toxins in complex sample matrixes. The present study describes a combination of a multiplex-immunoaffinity purification approach, followed by matrix-assisted laser desorption/ionization (MALDI)-based detection for the simultaneous identification of ricin, SEB, BoNT/A, and BoNT/B. The method comprises an affinity enrichment step, using specific monoclonal antibodies for each of the four toxins which have been selected from a pool of antibodies. The selected antibodies allow for specific and simultaneous capture of ricin, SEB, BoNT/A, BoNT/B, and the corresponding BoNT complexes. These were subsequently identified by MALDI time-of-flight (TOF) mass spectrometry (MS), following tryptic digest. The sensitivity of the technique was approximately 500 fmol for each of the toxins. These toxins were detectable within 8 h, even when present in complex matrixes such as milk or juice. Furthermore, the MALDI-based multiplex assay allowed for the discrimination of closely related BoNT sero- and subtypes, including a real case of food-borne botulism in Germany

<i>Burkholderia puraquae</i> sp. nov., a novel species of the <i>Burkholderia cepacia</i> complex isolated from hospital settings and agricultural soils

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3% from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 ºC, aesculin hydrolysis, and lysine decarboxylase and β-galactosidase activities.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Fermentaciones IndustrialesInstituto de Biotecnologia y Biologia Molecula