ERP Micro December 2019

1610  B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018

MICROBIOLOGICAL Methods

Confirmation and Identification of Listeria monocytogenes, Listeria spp. and Other Gram-Positive Organisms by the Bruker MALDI Biotyper Method: Collaborative Study, First Action 2017.10 B enjamin B astin , P atrick B ird , E rin C rowley , M. J oseph B enzinger J r , J ames A gin , and D avid G oins Q Laboratories, Inc., 1400 Harrison Ave, Cincinnati, OH 45214 D aniele S ohier 1 , M arkus T imke , M arian A wad , and M arkus K ostrzewa Bruker Daltonik GmbH, Fahrenheitstraße 4, 28359 Bremen, Germany

O ver the past decade, Listeria monocytogenes has been linked as the cause of several fatal foodborne outbreaks (1). The organism can survive in extreme conditions, which causes various problems for food manufacturers (2). When analyzing commodities for contamination, it can take up to 7 days to confirm and identify an organism following the various traditional reference methods for the detection, or enumeration, of Listeria species or L. monocytogenes in food products: U.S. Food and Drug Administration (FDA)/ BacteriologicalAnalyticalManual (BAM)Chapter10(3), U.S. Department of Agriculture (USDA)-Food Safety and Inspection Service (FSIS)/ Microbiology Laboratory Guidebook (MLG) 8.10 (4), International Organization for Standardization (ISO) 11290-1 and 11290-2 (5, 6). This extended time can lead to delays in recalls and result in widespread outbreaks. With the Bruker MALDI Biotyper ® method, the time to result for accurate and reliable confirmations and identifications from isolated colonies on selective and nonselective culture plates can be reduced to from 30 min to 2 h for investigation of a full 96-spot target plate (7). The Bruker MALDI Biotyper method is designed to rapidly confirm (meaning to confirm the preliminary presumptive result of an alternative or a reference method) and identify (meaning to determine the identity of an analyte; 8) select Gram-positive organisms from select agar plates. The method was validated to be used with the most common nonselective, selective, and chromogenic agar plates following isolation of the organisms per the appropriate regulatory method. The technology uses matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, which identifies the unique protein fingerprint of an organism. The generated spectrum pattern of these proteins is used to reliably and accurately identify a particular microorganism by matching against a library (version MSP 6903 or higher) currently containing 7311 reference strains for 2509 species. For Listeria identification, a subtyping software is used to differentiate the closely related common Listeria species: L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri. This module starts automatically when a characteristic mass spectrum of these species is obtained; L. grayi does not require the use of the subtyping module. As described by Elbehiry et al. (7), approximatively 30 min per isolate are required from target plate to the final results, and nearly 2 h are required to investigate a full 96-spot target plate (9). However, running the extraction procedure when needed required less than 40 min and enabled 100.0% accuracy for the identification of

Submitted for publication January 10, 2018. This method was approved by the Expert Review Panel for Microbiology Methods for Food and Environmental Surfaces as First Action. The Expert Review Panel for Microbiology Methods for Food and Environmental Surfaces invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and is critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. Supplemental Information is available on the J. AOAC Int . website, http://aoac.publisher.ingentaconnect.com/content/aoac/jaoac 1 Corresponding author’s email: daniele.sohier@bruker.com The Bruker MALDI Biotyper ® method utilizes matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) MS for the rapid and accurate confirmation and identification of Gram-positive bacteria from select media types. This alternative method was evaluated using nonselective and selective agar plates to identify and confirm Listeria monocytogenes, Listeria species, and select Gram-positive bacteria. Results obtained by the Bruker MALDI Biotyper were compared with the traditional biochemical methods as prescribed in the appropriate reference method standards. Sixteen collaborators from 16 different laboratories located within the European Union participated in the collaborative study. A total of 36 blind-coded isolates were evaluated by each collaborator. In each set of 36 organisms, there were 16 L. monocytogenes strains, 12 non- monocytogenes Listeria species strains, and 8 additional Gram-positive exclusivity strains. After testing was completed, the total percentage of correct identifications (to both genus and species level) and confirmation from each agar type for each strain was determined at a percentage of 99.9% to the genus level and 98.8% to the species level. The results indicated that the alternative method produced equivalent results when compared with the confirmatory procedures specified by each reference method.

DOI: https://doi.org/10.5740/jaoacint.18-0013