ERP Micro December 2019

1594  B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018

Laboratories (Cincinnati, OH) or an ADRIA Développement (Quimper, France) frozen stock culture collection stored at –70°C. The Salmonella strains were previously identified using the FDA/BAM Chapter 5, USDA-FSIS/MLG 4.09, and ISO 6579 reference methods (2, 3, 5) and serotyped using the Kauffmann–White scheme. The Cronobacter strains were previously identified using the FDA/BAM Chapter 29 and ISO 22964 reference methods (1, 4), as well as by 16S rDNA sequencing (Accugenix ® or GenBank). The other isolates were identified using biochemical galleries and 16S rDNA using up to 500 base pairs (Accugenix or GenBank). Each organism was incubated for 24–48 h at temperatures and atmospheric conditions most appropriate for organism growth. Isolated colonies were streaked to TSA slants at Q-Laboratories and stock culture tubes (TSA, Bio-Rad or equivalent) at ADRIA Développement and incubated under proper conditions for optimal growth prior to being shipped to each site. All samples were labeled with a randomized, blind-coded, three-digit number affixed to the TSA slant tube. 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 collaborator at refrigerated temperature (2–8°C) until the following Monday, when analysis was initiated. For the Cronobacter evaluation, 14 collaborators from seven laboratories participated. For the Salmonella evaluation, 15 collaborators from 15 laboratories participated. Isolate Distribution Collaborators were instructed to follow the appropriate preparation and analysis as outlined in the study protocol. One set of 24 isolates (16 target Cronobacter or Salmonella organisms and 8 non- Cronobacter or non- Salmonella organisms) were analyzed in each collaborative study by each participant. Cronobacter species .—The Cronobacter organisms were subcultured onto three agars: CCI (Thermo Fisher Scientific or equivalent), ESIA (Bio-Rad or equivalent), and TSA (Bio-Rad or equivalent) from the blind-coded TSA slants. The CCI plates were incubated for 20–26 h at 44 ± 2°C, the ESIA plates were incubated for 20–26 h at 41.5 ± 2°C, and the TSA was incubated for 18–24 h at 35–37 ± 1°C. Following incubation, growth present on each plate was analyzed by the Bruker MALDI Biotyper using the direct transfer (DT) technique. When needed, the extended direct transfer (eDT) procedure was used or the tube extraction (EXT) if necessary to obtain a reliable result. Growth from the TSA plate was also analyzed by conducting a spot oxidase test (Thermo Fisher Scientific) and by conducting biochemical confirmation analysis using either API 20E (bioMérieux) or GN VITEK 2 (bioMérieux), AOAC Official Method 2011.17 (8). Exclusivity organisms: Cronobacter .—The exclusivity organisms consisted of non- Cronobacter Gram-negative organisms that were closely related to the target organisms. Isolate Analysis

by matching against a library (version MSP 6903 or higher) currently containing 7311 reference strains for 2509 species. Prior to the collaborative study, the Bruker MALDI Biotyper method was validated according to the current AOAC Appendix J Guidelines in a precollaborative study (7). To fulfill the requirements at the genus and species level, 150 Salmonella spp. and 150 Cronobacter spp. strains were evaluated, along with 100 nontarget organisms. Each Salmonella target strain was evaluated from one nonselective agar, Tryptic Soy Agar (TSA, standard formulation), and from up to five selective agars: Xylose Lysine Deoxycholate (XLD, standard formulation), Brilliant Green Sulfa Agar (BGSA, standard formulation), RAPID’ Salmonella agar (RSA, Bio-Rad), Brilliance TM Salmonella Agar (BSA, Thermo Fisher Scientific), and ASAP ® (ASAP, bioMérieux). Each Cronobacter target strain was evaluated from one nonselective agar, TSA (standard formulation), and from up to two selective agars: Enterobacter sakazakii Isolation Agar (ESIA, standard formulation) and Chromogenic Cronobacter Isolation Agar (CCI, standard formulation). The nontarget strains were evaluated from the same nonselective and selective agar plates. The MALDI Biotyper demonstrated correct confirmation for the 150 Salmonella spp. strains and the 150 Cronobacter spp. strains for all the culture media. For each exclusivity panel (non- Salmonella and non- Cronobacter ), no misidentification to, respectively, Salmonella spp. or Cronobacter spp. was observed. All isolates evaluated were well characterized prior to analysis using conventional procedures, additional molecular tests, or 16S ribosomal deoxyribonucleic acid (rDNA) sequencing. 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 Salmonella spp. and Cronobacter spp. and identifying other nonpathogenic Gram-negative bacteria. Study Design In this collaborative study, two sets of 24 isolates were evaluated. The two sets consisted of 16 target organisms each (inclusivity panel: Cronobacter and Salmonella ) and 8 non- Cronobacter or non- Salmonella organisms (exclusivity panel). For the analysis of the Cronobacter species, Q Laboratories, Inc. served as the coordinating laboratory. For the analysis of the Salmonella species, ADRIA Développement served as the coordinating laboratory. A total of 48 isolates consisting of 32 target organisms (inclusivity panels for the Cronobacter species and Salmonella species) and 16 non- Cronobacter or non- Salmonella organisms (exclusivity panels for each target strain) were sent to each participating collaborator for evaluation. 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 Tryptic Soy Agar with 5% Sheep Blood (SBA) from a Q