Brucella are highly infectious bacterial pathogens responsible for a severely debilitating zoonosis called brucellosis. Half of the human population worldwide is considered to live at risk of exposure, mostly in the poorest rural areas of the world. Prompt diagnosis of brucellosis is essential to prevent complications and to control epidemiology outbreaks, but identification of Brucella isolates may be hampered by the lack of rapid and cost-effective methods. Nowadays, many clinical microbiology laboratories use Matrix-Assisted Laser Desorption Ionization–Time Of Flight mass spectrometry (MALDI-TOF MS) for routine identification. However, lack of reference spectra in the currently commercialized databases does not allow the identification of Brucella isolates. In this work, we constructed a Brucella MALDI-TOF MS reference database using VITEK MS. We generated 590 spectra from 84 different strains (including rare or atypical isolates) to cover this bacterial genus. We then applied a novel biomathematical approach to discriminate different species. This allowed accurate identification of Brucella isolates at the genus level with no misidentifications, in particular as the closely related and less pathogenic Ochrobactrum genus. The main zoonotic species (B. melitensis, B. abortus and B. suis) could also be identified at the species level with an accuracy of 100%, 92.9% and 100%, respectively. This MALDI-TOF reference database will be the first Brucella database validated for diagnostic and accessible to all VITEK MS users in routine. This will improve the diagnosis and control of brucellosis by allowing a rapid identification of these pathogens.

dWe describe a simple protocol to inactivate the biosafety level 3 (BSL3) pathogens Brucella prior to their analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. This method is also effective for several other bacterial pathogens and allows storage, and eventually shipping, of inactivated samples; therefore, it might be routinely applied to unidentified bacteria, for the safety of laboratory workers. Brucellosis is a zoonosis caused by bacteria of the genus Brucella, which are transmitted to humans through direct contact, ingestion of contaminated animal products, or aerosolization (1, 2). Brucellosis is endemic in several regions of the world, with more than 500,000 new cases each year (2-5). To date, 10 recognized species of Brucella have been described, with the most pathogenic for humans being Brucella melitensis (2). Diagnoses of clinical brucellosis are made initially using serological tests and are confirmed by isolation of the agent (6, 7). The recent introduction of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has revolutionized the identification of bacteria and yeasts (8, 9). However, the procedures recommended by the manufacturers for non-biosafety level 3 (BSL3) organisms are not adequate for the manipulation of Brucella strains, which are classified as BSL3 (potential bioterrorism pathogens) and represent potential health hazards for laboratory workers (1, 10). Any inactivation procedure must allow safe sample handling outside the BSL3 environment and must avoid destruction of the biomarkers used for identification. Therefore, we developed a simple, safe, and efficient sample preparation method for Brucella isolates that is compatible with their analysis by the current MALDI-TOF MS platforms.Solvent inactivation of Brucella. We first applied the direct transfer procedure recommended by the manufacturer to a panel of Brucella strains, working in class II microbiological safety cabinets in a BSL3 laboratory (Table 1). The target plate (Vitek MS DS slide; bioMérieux) was inoculated by picking a portion of a colony from a plate with a 1-l disposable loop (Sarsted), and the deposit was overlaid with 1 l of ␣-cyano-4-hydroxycinnamic acid (CHCA) matrix solution (saturated solution of CHCA in a solvent mixture composed of 33% acetonitrile, 33% absolute ethanol, 3% trifluoroacetic acid, and 31% water) and then air dried for 2 min at room temperature. The dried material was recovered with a sterile swab and spread on culture plates, which were then incubated for up to 3 weeks under the optimal growth conditions for the corresponding bacteria indicated in Table 1. Viable bacteria were recovered for most strains, showing that this protocol does not kill all Brucella strains, possibly because the bacterial deposit was not completely covered with the matrix solution. This risk is not acceptable for BSL3 pathogens. To overcome this problem, different liquid-phase inactivation protocols were tested in 1.5-ml Eppendorf tubes (see F...

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