A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis

Abstract

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...

A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata) Reveals Characteristics Departing from Classical Brucellae

“…With regard to Brucella , accurate identification by MALDI-TOF MS is currently limited because this genus is either not represented in the databases of the two main manufacturers, or only in a bioterrorism database with restricted availability (Cunningham and Patela, 2013 ). Using a safe inactivation protocol developed recently (Mesureur et al, 2016 ), we constructed an exhaustive MALDI-TOF MS Brucella database that can identify all Brucella spp., in most cases at the species level (J. Mesureur and A. Keriel, unpublished data). This database allowed us to clearly discriminate the frog isolates B13-0095 from O. anthropi and identified it as B. inopinata (this data will be presented in a separate publication).…”

Comparison of Stir Bar Sorptive Extraction and Solid Phase Microextraction of Volatile and Semi-Volatile Metabolite Profile of Staphylococcus Aureus

“…S. aureus ATCC 25,923 [37] was the reference strain used in this study. The strain was maintained as frozen stock on Microbank™ bead (Pro-Lab Diagnostic, Richmond Hill, ON, Canada) at −80 • C, and cultivated overnight onto LB medium (tryptone Fluka, Saint-Louis, MO, USA), yeast extract (Amresco, Solon, OH, USA), and sodium salt (Panreac, Barcelona, Spain)) at 37 • C with shaking at 210 rpm.…”

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Clinical Microbiology: What Are the Current Issues?

“…The challenge for such an inactivation procedure is to eliminate viability, while retaining the proteins largely intact to allow for MALDI-TOF MS-based identification. Several procedures have been suggested to this end [ 5 6 7 ] and applied with success [ 8 ]. The most important steps for inactivation include the use of organic solvents (70% ethanol, trifluoroacetic acid, and various other mixtures), mechanical lysis, and washing steps.…”