Verification of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens

Abstract

bWe examined the utility of a single matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry method for the identification of security-sensitive biological agents (risk group 3 bacterial pathogens). The goal was 2-fold: to verify a method for inclusion into our scope of accreditation, and to assess the biological safety of extractions. We developed our sample flow to include a tube-based chemical extraction, followed by filtration, before processing on MALDI-TOF MS instruments in a containment level 2 laboratory. Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) is a rapid, robust, and cost-effective technology, which is emerging as a routine bacterial identification method in clinical diagnostic laboratories (1-3). Despite its utility, there are concerns with the safety of sample processing and the quality of spectral databases for the identification of security-sensitive biological agents (SSBAs). As our laboratory predominately works with SSBA bacterial pathogens (the risk group 3 [RG-3] pathogens: Francisella tularensis, Yersinia pestis, Bacillus anthracis, Brucella species, and Burkholderia pseudomallei), our goal was to verify the usefulness and safety of a MALDI-TOF MS method for inclusion into our scope of accreditation (ISO 17025). We chose to pursue a full-tube-based protein extraction method involving cellular disruption and filtration, rather than the direct-colony plate (smear) method (4, 5), in an effort to mitigate the risk of viable organisms.Over a period of 1 year, 146 bacterial samples (including 57 SSBA samples; Table 1) were processed as previously described (6). The bacteria were previously identified by standard genotypic and phenotypic methods. In brief, all cultures were chemically extracted in a microcentrifuge tube using 70% ethanol, 70% formic acid, and acetonitrile, and all SSBA bacteria were extracted in a biosafety level 3 (BSL-3) laboratory. SSBA extracts from sporeforming bacteria were filtered through a 0.1 M microcentrifuge filtration unit (EMD Millipore, Etobicoke, Ontario, Canada), as recommended by Dauphin and Bowen (7). The viability of the extract was performed by inoculating 10% of the extract onto both sheep's blood agar (SBA) (or cysteine heart agar [CHA] for F. tularensis) and into 2 ml of tryptic soy broth (TSB), and incubating at 35°C and 5% CO 2 for 48 h. After 48 h, the TSB was further subcultured to SBA or CHA and monitored for an additional 72 h. Any bacterial growth was tested with agent-specific assays to rule out the SSBA, indicating a contaminant. After a lack of growth was confirmed, extracts were removed from the BSL-3 laboratory, and 1.5 l of the extract was applied (at a minimum of four replicate spots) to an MSP-96 MALDI target plate (Bruker Daltonics, East Milton, Ontario, Canada), with an overlay of 1.5 l of ␣-cyano-4-hydroxycinnamic acid (CHCA) matrix solution (50% acetonitrile and 2.5% trifluoroacetic acid). Mass spectra were acquired with the FlexControl software (version 3.0...

The Brief Case: Misidentification of Brucella melitensis as Ochrobactrum anthropi by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)

“…The (10,11). Interestingly, B. melitensis was not misidentified as O. anthropi by either system using any of the available databases (9).…”

“…The use of Bruker's select relevant (SR) library using 6 strains of B. melitensis and 3 strains of B. suis identified all 9 to the genus level and 4 to the species level and misidentified B. suis as B. melitensis (14). A larger study examining 30 organisms belonging to 5 Brucella species using the Bruker system was unable to identify these organisms to the species level; however, a custom database was able to accurately identify all strains to the species level (10).…”

Elucidation of protein biomarkers for verification of selected biological warfare agents using tandem mass spectrometry

“…However, the single MALDI-MS approach suffers several limitations due to lack of peptide sequence information, need of pure cultures to produce consistent results, and effect of culture conditions on spectral quality and ensuing identifications [23][24][25] . The inability to differentiate high-consequence pathogens from related bacterial species can be a serious issue for clinical diagnostic laboratories and more so in a biothreat perspective 28,50,54 . Incorrect identifications with single MALDI-TOF MS methods have resulted in laboratory exposures with Francisella tularensis 55 and B. pseudomallei 56 .…”

“…The multiplexing capability and suitability for analyzing complex samples such as air, water, culture medium, bodily fluids, and food are of immense value for the utility of MS based techniques in biothreat scenario. MS platforms such as Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS) are gaining popularity in microbiological research, largely for diagnostic applications, using mass fingerprinting or peptide sequencing approaches 18 .MALDI-TOF MS is emerging as a rapid, robust, and cost-effective technology for a routine bacterial identification method, largely in clinical diagnostic laboratories [19][20][21][22] . For example, MALDI Biotyper and VITEK MS have been approved by the U.S. Food and Drug Administration for bacterial identification in clinical diagnostic laboratories [23][24][25][26] .…”

“…MALDI-TOF MS is emerging as a rapid, robust, and cost-effective technology for a routine bacterial identification method, largely in clinical diagnostic laboratories [19][20][21][22] . For example, MALDI Biotyper and VITEK MS have been approved by the U.S. Food and Drug Administration for bacterial identification in clinical diagnostic laboratories [23][24][25][26] .…”