Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro Validation of standard method EN ISO 22964:2017 — Microbiology of the food chain — Horizontal method for the detection of Cronobacter spp. Amparo de Benito a, ⁎ , Nathalie Gnanou Besse b , Isabelle Desforges c , Barbara Gerten d , Begoña Ruiz a , David Tomás e a Ainia, Centro Tecnológico, Parque Tecnológico de Valencia, E-46980 Paterna, Valencia, Spain b Université Paris Est, ANSES, Laboratory for Food Safety, F-94701 Maisons-Alfort, France c bioMérieux SA, F-69280 Marcy-l'Étoile, France d Merck KGaA, DE-64293 Darmstadt, Germany e Nestec Ltd. Nestlé Research Center, CH-1000 Lausanne 26, Switzerland ARTICLE INFO Keywords: Cronobacter spp. Detection method Validation Performance characteristics Food ABSTRACT The new version of the ISO standard method for detection of Cronobacter spp. (EN ISO 22964:2017) was vali- dated in the frame of the European Commission Mandate M381 to CEN. Seventeen laboratories from nine countries participated in the interlaboratory studies to determine the performance characteristics of the method. The performance of the method was evaluated using matrices for which the presence of Cronobacter spp. is considered to be of serious concern, such as infant formula and its ingredients and representatives of categories cited in the EC Regulation 2073/2005 on microbiological criteria for foodstuffs for Cronobacter spp. The five matrices included in the validation were: two types of powdered infant food formulas (with and without pro- biotics); lactose; starch and environmental samples (swabs). The samples were each tested at two different levels of contamination, plus a negative control. Inoculation levels ranged from 4 to 95 CFU/sample. Each participant examined eight replicates of each level of inoculation, a total of 24 samples per matrix type. Specificity was calculated for each matrix used in the validation, with results ranging between 99 and 100%. Sensitivity of the method was calculated for samples in which no fractional recovery was expected and the values that were obtained ranged between 65 and 100%, depending on the matrix, the inoculation level and the in- terfering microbiota present in the samples. LOD 50 value was calculated for three food items (the two powdered infant formulas and the starch) with values between 0.8 and 1.1 CFU/sample. 1. Introduction Cronobacter spp. are foodborne pathogens of special concern in in- fants, causing sepsis, meningitis, necrotising enterocolitis as a result of the consumption of contaminated infant formula powder (Farmer et al., 1980). Cronobacter spp. have the ability to persist in dry environments including powdered foods (Kalyantanda et al., 2015; Osaili and Forsythe, 2009) and can also affect immunocompromised adults. European Union regulation (EC) No. 2073/2005 as amended by regulation (EU) No. 365/2010, requires the absence of Cronobacter spp. in 30 units of 10 g test portions of dried infant formulae and dried dietary foods for special medical purposes intended for infants below six months of age. No further identification of Cronobacter to the level of species is necessary (Anonymous, 2007, 2010). European Regulation prescribes the revised Standard horizontal method ISO 22964:2017 (Anonymous, 2017a) for the detection of Cronobacter spp. to address the potential risk to infants and neonates who may consume dried in- fant formulae and dried dietary foods. The revision and validation of the standard has been prepared by the European Committee for Standardization (CEN), Technical Committee TC 275, Food Analysis, Working Group WG 6, Microbiology of the Food Chain, in collaboration with ISO/TC 34, Food products, Subcommittee SC 9. When compared with the previous version of this standard (ISO/TS 22964:2006) (IDF/RM 210:2006) “Milk and milk products - Detection of Enterobacter sakazakii” (Anonymous, 2006), the scope has been extended to the detection of Cronobacter spp. in food products for humans, feeding animals and environmental samples. Since the reclassification of E. sakazakii as the genus, “Cronobacter,” by Iversen et al., 2008a, there has been a need to change the scope of the former ISO/TS 22964:2006 to include the new genus that at present includes 7 species: Cronobacter sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C. universalis and C. condimenti (Forsythe et al., https://doi.org/10.1016/j.ijfoodmicro.2018.03.025 Received 30 August 2017; Received in revised form 29 January 2018; Accepted 23 March 2018 ⁎ Corresponding author. E-mail address: abenito@ainia.es (A. de Benito). International Journal of Food Microbiology xxx (xxxx) xxx–xxx 0168-1605/ © 2018 Elsevier B.V. All rights reserved. Please cite this article as: De Benito, A., International Journal of Food Microbiology (2018), https://doi.org/10.1016/j.ijfoodmicro.2018.03.025