ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018  1611

L. monocytogenes, L. innocua, L. ivanovii, L. fleischmannii, L. grayi, L. seeligeri, L. weihenstephanensis , and L. welshimeri, as shown on a set of 1201 strains received in the context of a Listeria national surveillance program in France (10). Prior to the collaborative study, the Bruker MALDI Biotyper method was validated according to the current AOAC Official Methods of Analysis SM Appendix J Guidelines in a precollaborative study (8). To fulfill the requirements at the genus and species level, 100 L. monocytogenes strains, 120 Listeria spp. strains different from L. monocytogenes , and 100 relevant non- Listeria spp. strains were evaluated. Each target strain was evaluated from one nonselective agar plate, Tryptic Soy Agar with Yeast (TSA/YE, standard formulation), and five selective agars: Ottaviani & Agosti (O&A, standard formulation), PALCAM (standard formulation), Oxford Agar (OXA, standard formulation), Modified Oxford Agar (MOX, standard formulation), and a chromogenic agar, RAPID’L. mono TM (RLM, Bio-Rad). The MALDI Biotyper demonstrated correct confirmation for the 110 strains belonging to L. grayi, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri species from all the culture media. The 100 L. monocytogenes strains were also correctly identified from all the culture media, except in one case: one wild-type strain of L. monocytogenes was identified to L. innocua on RLM. Because of the limited availability of the 10 newly described Listeria species, i.e., L. coloradensis, L. fleischmannii, L. floridensis, L. cornellensis, L. grandensis, L. riparia, L. londoniensis, L. marthii, L. rocourtiae , and L. weihenstephenensis, only a few reference spectra are present in the library (version MSP 6903). The library may not contain all organisms, but the library can be updated by adding newly identified organisms. In some cases, the tested strains are identified at the species or genus level, but in some other cases there is no identification possible. As described by Bruker, the test L. marthii strain was misidentified as L. monocytogenes . The 100 non- Listeria spp. strains were evaluated from the same nonselective and selective agar plates, and no misidentification of these strains as Listeria species was observed. All isolates were wild-type strains. Table 2 of the Supplemental Information provides the origins of each strain used in the collaborative study. Prior to testing, all isolates were well characterized by either 16S ribosomal deoxyribonucleic acid (rDNA) sequencing using up to 500 base pairs (Accugenix ® or Genbank) or by biochemical analysis. The purpose of this collaborative study was to compare the reproducibility of the Bruker Biotyper method to the FDA/BAM, USDA-FSIS/MLG, and ISO reference methods for confirming and identifying L. monocytogenes , Listeria species, and select Gram-positive organisms. Study Design In this collaborative study, one set of 36 isolates was evaluated. The set consisted of 16 L. monocytogenes isolates, 12 non- monocytogenes Listeria isolates, and 8 non- Listeria Gram-positive organisms. A detailed collaborative study packet outlining all necessary information related to the study, including isolate preparation and documentation of results, was sent to each collaborating laboratory prior to the initiation of the study. Collaborative Study

Preparation of Isolates

The strains used in this evaluation were propagated onto TSA/YE (Thermo Fisher) from an ADRIA Développement (Quimper, France) frozen stock culture collection stored at –70°C. All Listeria strains were previously identified following the FDA/BAM Chapter 10, USDA-FSIS/MLG 8.10, and ISO 11290 parts 1 and 2 (4–7), as well as by 16S rDNA sequencing with up to 500 base pairs (Accugenix or GenBank). Each organism was incubated for 24–48 h at 35–37°C. Isolated colonies were streaked to culture stock tubes (TSA/YE; Bio-Rad) and incubated for 24–48 h at 35–37°C prior to being shipped to each laboratory. All samples were labeled with a randomized, blind-coded, three-digit number. Isolates were shipped on a Wednesday via overnight delivery according to the Category B Dangerous Goods shipment regulations set forth by the International Air Transport Association. Upon receipt, samples were held by the collaborating laboratory at refrigerated temperature (2–8°C) until the following Monday when analysis was initiated. Collaborators were instructed to follow the appropriate preparation and analysis as outlined in the study protocol. One set of 36 isolates (16 L. monocytogenes target organisms, 12 Listeria species target organisms, and 8 non- Listeria Gram-positive organisms) were analyzed in each collaborative study by each participant. Collaborators were instructed to follow the appropriate preparation and analysis as outlined in the study protocol. L. monocytogenes and Listeria species .—The 16 L.monocytogenes and12 Listeria species target organismswere subcultured to four agars: O&A (Bio-Rad or equivalent), OXA (bioMérieux or equivalent), RLM (Bio-Rad), and TSA/YE (Bio-Rad or equivalent) from the blind-coded stock culture tubes. The agar plates were incubated for 24–48 h at 34–38ºC (following the specific temperature and incubation time as specified by manufacturer or reference method). Following incubation, growth present on each plate was analyzed by the Bruker MALDI Biotyper using the direct transfer (DT) technique and by the extended direct transfer (eDT) or the tube extraction (EXT) if necessary to obtain a reliable result. Growth from each TSA/YE plate was also analyzed by conducting a catalase test, determining hemolysis reaction from a blood agar plate, conducting a motility test, and by conducting biochemical confirmation using API Listeria (bioMérieux). Non-Listeria Gram-positive organisms .— Exclusivity .—The exclusivity organisms consisted of non- Listeria Gram-positive organisms that were closely related to the target organisms. Each exclusivity organism was subcultured to four agars: O&A (Bio-Rad or equivalent), OXA (bioMérieux or equivalent), RLM (Bio-Rad), and TSA/YE (Bio-Rad or equivalent) from the blind-coded stock culture tubes. The agar plates were incubated for 24–48 h at 34–38ºC (following the specific temperature and incubation time as specified by manufacturer or reference method). Following incubation, growth present on each plate Isolate Distribution Isolate Analysis