ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018  1621

is clearly highlighted in the MALDI Biotyper result report. One collaborator misidentified B. thuringiensis and B. pumilus as L. monocytogenes . Confirmatory reference methods results.— When the non- Listeria organisms were confirmed following the reference method, 116 out of 127 isolates were correctly confirmed and identified as non- Listeria to produce a correct identification rate of 91.3%. One participant did not obtain growth for Lactococcus lactis Ad425 on TSA/YE, therefore there was no confirmation conducted. There were 11 instances in which a Gram-positive non- Listeria (exclusivity) organism was incorrectly identified as a Listeria species. Two collaborators incorrectly identified B. thuringiensis Ad2067 as either L. grayi or L. ivanovii . There were five collaborators that obtained a result of L. grayi for B. pumilus Ad733. One collaborator incorrectly identified Leuconostoc pseudomesenteroides Ad835, L. lactis Ad425, and S. hominis Ad849 as L. seeligeri . Enterococcus mundtii Ad1365 was incorrectly identified as L. welshimeri for one collaborator. For one of the misidentifications, the L. monocytogenes isolate was misidentified as S. hominis, one of the exclusivity organisms. This misidentification may be the result of the collaborator streaking the incorrect sample because an inclusivity organism was identified as one of the exclusivity organisms used in the collaborative study design. Out of 1732 total Listeria isolates analyzed, only 23 were not able to be identified. These identifications to the genus level only were obtained from two collaborators, and in many cases appear to be due to the collaborator failing to apply the complete MALDI Biotyper workflow. If the workflow is not completely followed, the results can be less reliable. The smear of the isolate on the target plate is technique driven. The proteins from the agar can cause a misleading result; therefore, it is critical to select a pure colony not containing any of the agar. Other techniques that need to be strictly followed are the BTS spotting, HCCA addition, and complete drying of the formic acid during the preparation of the target plate. Additionally, these isolates, when analyzed from other agar plates by these collaborators, did produce identification to the species level. A total of 1973 out of 1997 Gram-positive bacteria isolates were correctly identified at the species level (or group level in the case of B. cereus group) with the MALDI Biotyper, resulting in a correct identification rate of 98.8%, and 1994 out of 1997 isolates were correctly identified to the genus level, resulting in a correct identification rate of 99.9%. In a very few cases (less than 15), the extraction protocol was required to enable an identification result at the species level, except for one collaborator who proceeded directly to the extraction procedure after a single direct transfer. The subtyping module improves the MALDI Biotyper workflow for the identification of the Listeria species by avoiding the extraction procedure in most of the cases where it was previously required to be performed to obtain a correct Discussion No negative feedbackwas provided regarding the performance of the Bruker MALDI Biotyper method by the participants of the collaborative study, which included academia, industry, and regulatory agencies located within the European Union.

species identification. In most of the cases, the easy and fast DT or eDT procedures provided reliable identification for most of the Gram-positive isolates, and the MALDI Biotyper method proved to be robust, as various growth temperatures, media, culture age, and different operators had no notable impact on the bacterial identification rate. Overall, the data generated during this evaluation demonstrates the reproducibility of this method using both disposable and reusable targets. The candidate method produced a higher percent positive agreement when compared with the confirmation procedures of the reference methods (FDA/BAM, USDA-FSIS/MLG, ISO) for the confirmation and identification of L. monocytogenes and Listeria species. All non- Listeria Gram-positive organisms were compared by following the confirmation procedures and to an appropriate 16S database using up to 500 base pairs (Accugenix or GenBank). The data obtained during the precollaborative evaluation indicated that all Listeria species, both common and newly identified, were identified to the genus level. The six common Listeria species were identified to the species level as demonstrated during the precollaborative evaluation and the Official Methods of Analysis SM evaluation. Regardless of the platform (Biotyper or Smart System), software, and target being used, the method produced robust and reliable results (13).

Recommendations

It is recommended that the Bruker MALDI Biotyper method be adopted as Official First Action status for the confirmation and identification of L. monocytogenes, non- monocytogenes Listeria species, and other Gram-positive organisms from selected agars.

Acknowledgments

The authors would like to thank the Coordinating Laboratory, ADRIA Développement, Quimper, France. We would like to extend a sincere thank you to the following collaborators for their dedicated participation in this study: Maryse Rannou, Claudie Le Doeuff, and Sarah Peron, ADRIA Développement (Quimper, France) Thomas Charrier, Eurofins (Nantes, France) Alexander Leclerc, Institut Pasteur, CNR & CCOMS Listeria (Paris, France) Benoit Thuillier, LABOCEA (Quimper, France) Michael Treilles, LASAT (Champdeniers, France) Elodie Cauvin, LABEO (Saint Lo, France) Alice Marthino, Groupe CARSO (Venissieux, France) Florence Crombe, Institut Scientifique de Sante Publique (Brussels, Belgium) Annet Heuvelink, GDAnimal Health (Deventer, Netherlands) Greetje Castelijn, Nederlandse Voedsel- en Warenautoriteit (Wageningen, Netherlands) Roy Betts, CCFRA (Chipping Campden, United Kingdom) Matteo Capocefalo, ALS (Rotherham, United Kingdom) Catherine Cockcroft, Eurofins (Wolverhampton, United Kingdom) Thomas Koch, Eurofins CLF Specialized Nutrition Testing Services (Friedrichsdorf, Germany)