ERP Micro December 2019

1596  B astin et al . : J ournal of AOAC I nternational V ol . 102, N o . 5, 2019 Biotyper ® method, the time-to-result for accurate and reliable confirmations and identifications from isolated colonies on selective or nonselective culture plates can be reduced to 30 min to 2 h to investigate a full 96-spot target plate (9).

methods to extend the method to include the confirmation and identification of Campylobacter species and the identification of other nonpathogenic Gram-negative bacteria.

The Bruker MALDI Biotyper method is designed to rapidly confirm, meaning to confirm the presumptive result of an alternative or a reference method, and identify, meaning to determine the identity of an analyte from a variety of agar plates. The method was validated for use with common nonselective, selective, and chromogenic agars following isolation of the organisms per the appropriate reference or proprietary method. Samples are analyzed using matrix-assisted laser desorption/ ionization time-of-flight (MALDI–TOF) MS. The highly abundant microbial ribosomal proteins result in a mass spectrum with a characteristic mass and intensity distribution pattern. The generated spectrum pattern of these proteins is used to reliably and accurately identify a particular microorganism by matching against a library [version unsupervisedMain Spectra (MSP) 6903] containing, currently, 7311 reference strains for 2509 species. Prior to the collaborative study, the Bruker MALDI Biotyper method was validated according to the current AOAC INTERNATIONAL Appendix J Guidelines (10) in a pre- collaborative study as well as a First Action Official Method SM approved for Salmonella spp., Cronobacter spp., and other Gram-negative organisms. To fulfill the requirements at the genus and species level, 150 Salmonella spp., 150 Cronobacter spp., and 150 Campylobacter spp. strains were evaluated along with 100 nontarget organisms. Each Salmonella species target strain was evaluated from one nonselective agar [Tryptic Soy Agar (TSA)] and from five selective agars [Xylose Lysine Deoxycholate (XLD), standard formulation; Brilliant Green Sulfa Agar (BGA), standard formulation; RAPID’ Salmonella Agar (RSA; Bio-Rad); Brilliance ™ Salmonella Agar (BSA; Thermo Fisher Scientific); and AES Salmonella Agar Plate (ASAP ® ; bioMérieux)]. Each Cronobacter target strain was evaluated from one nonselective agar, TSA, and from up to two selective agars, Enterobacter sakazakii Isolation Agar (ESIA) and Cronobacter Chromogenic Isolation Agar (CCI). Each Campylobacter target strain was evaluation from one nonselective agar, Columbia Blood Agar (CBA), and the fol­ lowing four selective agars: modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA, standard formulation), Campy­ Food Agar (CFA), RAPID’ Campylobacter Agar (RCA; Bio-Rad), and Campy CefexAgar (CCA, standard formulation). The nontarget strains were tested on the same nonselective and selective agar plates. The MALDI Biotyper demonstrated correct confirmation for the 150 Salmonella spp. strains, the 150 Cronobacter spp. strains, and the 150 Campylobacter strains from the following species ( C. coli, C. hyointestinalis, C. jejuni, C. lari, C. lari subspecies concheus, subantarcticus, and upsaliensis ) for all the culture media. For each exclusivity panel (non- Salmonella, non- Cronobacter , and non- Campylobacter ), no misidentification was observed. All isolates evaluated were well characterized prior to analysis using conventional procedures, additional molecular tests, or up to 500 base pairs 16S rDNA sequencing (Accugenix or GenBank). This method was granted First Action Official Method of Analysis SM (OMA) status in December 2017 (OMA 2017.09 ). The purpose of this collaborative method extension was to compare the reproducibility of the Bruker Biotyper method to the FDA/BAM, USDA/FSIS MLG, and ISO reference

Collaborative Study

Study Design In this collaborative study, three sets of 24 isolates were evaluated. The three sets consisted of 16 target organisms each [inclusivity panel: Cronobacter , Salmonella , and Campylobacter (Tables  2017.09A , 2017.09E , and 2017.09I )] and 8 non- Cronobacter, non- Salmonella , or non- Campylobacter organisms (exclusivity panel: Tables 2017.09C , 2017.09G , and 2017.09J ). For the analysis of the Cronobacter species and Campylobacter species, Q Laboratories, Inc. (Cincinnati, OH) served as the coordinating laboratory. For the analysis of the Salmonella species, ADRIA Développement (Quimper, France) served as the coordinating laboratory. A total of 48 isolates, including 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 the method extension to include Campylobacter species, one set of 24 isolates was evaluated per participant. The set consisted of 16 target organisms each (inclusivity panel: Campylobacter spp.) and 8 non- Campylobacter organisms (exclusivity panel). The Salmonella strains were previously identified using the FDA/BAM Chapter 5, USDA/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 up to 500 base pairs (Accugenix or GenBank). The Campylobacter strains were previously identified using the FDA/BAM Chapter 7, USDA/MLG 41.04, and ISO 10272 reference methods (6–8) as well as by 16S rDNA sequencing on up to 500 base pairs (Accugenix or GenBank). The other isolates were identified using biochemical galleries and 16S rDNA sequencing on up to 500 base pairs (Accugenix or GenBank). 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. The strains used in this evaluation were propagated onto TSA with 5% sheep blood from a Q Laboratories or an ADRIA Développement frozen stock culture collection stored at –70°C. 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 at ADRIA Développement and incubated under proper conditions for optimal growth prior to being shipped to each site. For the method extension, the strains (both target and nontarget) used in this evaluation were propagated onto CBA (Part No. A16BX; Hardy Diagnostics) from a Q Laboratories frozen stock culture collection stored at –70°C. Each organism Preparation of Isolates