ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018  1595

Each exclusivity organism in the Cronobacter study was 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 stock culture tubes. 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 TSAwas incubated for 18–24 h at 35–37 ± 1°C. Following incubation, if growth was present on an agar plate, an isolated colony was analyzed by the Bruker MALDI Biotyper using the DT technique. When needed, the eDT procedure was used or the 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 . Salmonella species .—The Salmonella organisms were streaked to XLD (Bio-Rad or equivalent), RSA (Bio-Rad), and TSA (Bio-Rad or equivalent). All plates were incubated at 34–38ºC. The XLD and RSA were incubated for 20–26 h, and the TSA was incubated for 18–24 h. Following incubation, if growth was present on an agar plate, an isolated colony was analyzed by the Bruker MALDI Biotyper using the DT technique. When needed, the eDT procedure was used or the EXT if necessary to obtain a reliable result. Growth from each TSA plate was also analyzed by conducting a Gram stain, spot oxidase test (Thermo Fisher Scientific), Poly O and H antisera agglutinations (Bio-Rad or equivalent), and biochemical analysis using either API 20E (bioMérieux), AOAC Official Method 978.24 , or GN VITEK 2 (bioMérieux), AOAC Official Method 2011.17 . Exclusivity organisms: Salmonella .—The exclusivity organisms consisted of non- Salmonella Gram-negative organisms that were closely related to the target organisms. Each exclusivity organism in the Salmonella study was subcultured onto three agars: XLD (Bio-Rad or equivalent), RSA (Bio-Rad), and TSA (Bio-Rad or equivalent). All plates were incubated at 34–38ºC. The XLD and RSA were incubated for 20–26 h, and the TSAwas incubated for 18–24 h. Following incubation, if growth was present on an agar plate, an isolated colony was analyzed by the Bruker MALDI Biotyper using the DT technique. When needed, the eDT procedure was used or the EXT if necessary to obtain a reliable result. Growth from each TSA plate was also analyzed by conducting a Gram stain, spot oxidase test (Thermo Fisher Scientific), Poly O and H antisera agglutinations (Bio-Rad or equivalent), and biochemical analysis using either API 20E (bioMérieux), AOAC Official Method 978.24 , or GN VITEK 2 (bioMérieux), AOAC Official Method 2011.17 .

AOAC Official Method 2017.09 Confirmation and Identification of Salmonella Species, Cronobacter Species, and Other Gram-Negative Organisms Bruker MALDI Biotyper Method First Action 2017 [Applicable to confirmation and identification of Salmonella species, Cronobacter species, and other Gram-negative organisms from select media types.] See Tables 2017.09A – H for a summary of results of the interlaboratory study. See Tables 2017.09I – P for detailed results of the interlaboratory study. A. Principle The Bruker MALDI Biotyper Method is intended to be used for the automated identification and confirmation of bacteria. Classification and identifications are based on proteomic fingerprinting using MALDI-TOF MS. First, the MALDI process transforms the proteins and peptides from the isolated microorganisms into positively charged ions. This is achieved by irradiating the matrix–sample composite with a UV laser. The matrix absorbs laser energy and transfers protons to the intact proteins or peptides in the gas phase. These ions are electrostatically accelerated and arrive in the flight tube at a mass-dependent speed. Because different proteins/peptides have different masses, ions arrive at the detector at different times (TOF). The MALDI Biotyper System measures the time (in the nanosecond range) between pulsed acceleration and the corresponding detector signal of the ions, and the time is converted into an exact molecular mass. The highly abundant microbial ribosomal proteins result in a mass spectrum with a characteristic mass and intensity distribution pattern. This pattern is species-specific for many bacteria, yeasts, and molds and can be used as a “molecular fingerprint” to identify a test organism. The mass spectra are transformed into peak lists by the MALDI Biotyper software and are compared to the patterns in the reference library.

B. Apparatus and Reagents

Items available from Bruker Daltonik GnbH (Bremen, Germany) . ( a )  Microflex LT/SH MALDI-MS System (Cat. No. 8269956) or MALDI Biotyper Smart System (Cat. No. 1853665).

( b )  MBT library .—Version MSP 6903 or higher. ( c )  MBT Compass software .—Cat. No. 1843241. ( d )  MBT Explorer module .—Cat. No. 1828476. ( e )  MBT Compass software .—Cat. No. 1829023. ( f )  Barcode scanner .—Cat. No. 8268821, optional.

Statistical Analysis

( g )  Holder for barcode scanner .—Cat. No. 8276754, optional. ( h )  MSP 96 target polished steel BC (Cat. No. 8280800) or MSP 48 target polished steel BC (Cat. No. 8281817), or MBT Biotarget 96 (Cat. No. 1840375) with the MSP adapter MALDI Biotarget (Cat. No. 8267615). ( i )  HCCA (α-cyano-4 hydroxycinnamic acid) Matrix, portioned (Cat. No. 8255344) or MBT Galaxy HCCA Matrix GPR (Cat. No. 1823405).

Each collaborating laboratory reported bacterial identification results for each organism on the data sheets provided. The data sheets were submitted to the study director at the end of testing for statistical analysis. Data for each organism was analyzed to determine the percentage of accurate identification and percentage of misidentification by the Bruker MALDI Biotyper Method and the reference method.