Cronobacter (formerly known as Enterobacter sakazakii) has been recognized as a genus of Enterobacteriaceae since 2007 (Iversen et al., 2007), which consists of seven species including C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C. universalis, and C. condimenti (Joseph et al., 2012a; Joseph et al., 2012b). Cronobacter species are opportunistic foodborne pathogens that can cause severe infections such as meningitis, septicemia, necrotizing enterocolitis, and pneumonia in neonates and in immuno-compromised individuals (Iversen and Forsythe, 2003; World Health Organization [WHO], 2007). C. sakazakii, C. malonaticus, and C. turicensis are considered the pathogenic Cronobacter species that cause the majority of severe illnesses (Jaradat et al., 2014).
Although the reservoirs of Cronobacter spp. and their modes of transmission are still unclear, contaminated foods, especially powdered infant formulae (PIF), have been suggested as the primary vehicle of transmission for Cronobacter spp., with the importance of dusts and airborne contaminations serving as a secondary route of transmission (Jung and Park, 2006; World Health Organization [WHO], 2007). It was reported that Cronobacter spp. were isolated from 20% of powdered infant formulae in Korea (Lee et al, 2012). Understanding the transmission routes (e.g., raw materials, or environmental) and vehicles (e.g., powdered infant formula, or cereals) of sporadic Cronobacter outbreaks is of public health importance (Friedemann, 2007).
Currently, culture-dependent isolation and morphological/biochemical methods for the detection and identification of Cronobacter spp. from PIF are recommended by the International Organization for Standardization and the International Dairy Federation. In general, ISO 22964:2017 is reinforced as the regional standard method. In China, GB 4789.40–2016 is specified as the official standard test method for the detection of Cronobacter spp. from PIF (China National food safety standards, 2016). For the approved standard methods of qualitative testing, the common features and procedures of these traditional culture methods often involve pre-enrichment and selective enrichment procedures followed by biochemical and/or serological confirmation (Norberg et al., 2012; United States Food and Drug Administration, 2002).
Despite sharing common test principles, many of the standard methods have different procedures. Therefore, standard method harmonization has become necessitated by a growing demand on international cooperation and exchange of data, particularly during food-borne outbreaks (Espeillac, 2015). From a regulatory perspective, microbiological criteria are often defined by public authorities at different levels (e.g., national, regional, and international). Each level adopting a different standard method and establishing microbiological criteria based on their own testing methodology could lead to discrepancies and confusion in the interpretation of the testing results, in addition to an increase in costs for testing and holding products. Particularly in the global food trade, if different national standard methods are used by exporting and importing countries, analyses are duplicated and delays in trade become inevitable (Espeillac, 2015). Thus, evaluating and establishing the equivalence between standard methods is of major technical significance to ensure a uniform application and control of legislation for the global food trade. On the other hand, from the perspective of R&D and practical users, method comparison studies continue through the method development pipeline in the validation of alternative methods. A misuse mistake made by the food industry is the misuse or arbitrary adoption of rapid methods without validation, which renders testing results unreliable. Therefore, before adopting a new rapid method or a different standard method in routine analysis, it is important to carry out a scientific and systematic evaluation to ensure that the performance characteristics meet the requirements for a specified intended use. A key component of this validation process involves comparison of the alternative method to a reference method and establishing objective evidence that the alternative method performs equivalently or superior to the reference method (Vicky et al., 2010).
Establishing equivalence between an alternative method and the standard method by comparison studies is an essential part of method validation. In official method validation processes, regulatory or accredited third party organizations conduct studies to evaluate the performance of a new or an updated method according to internationally or regionally recognized validation guidelines. Up to now, there is no international standard nor guideline that specifically addresses equivalence evaluations between two established standard methods. However, most of these studies are conducted following two major validation guidelines that are internationally recognized, namely the AOAC International Methods Committee Guidelines for Validation of Qualitative and Quantitative Food Microbiological Official Methods of Analysis (Feldsine, et al., 2012) (hereafter referred to as “AOAC Guidelines”) and the ISO 16140 series, Microbiology of the Food Chain-Method Validation (International Organization for Standardization, 2016a&2016b) (hereafter referred to as “ISO 16140”), sometimes with modifications (Amparo et al., 2019). In China, the Certification and Accreditation Administration of China has recently published a national industry standard to guide the evaluation of the equivalence of food microbiological testing standard methods (RB/T 037-2020) (Certification and Accreditation Administration of China, 2020). The designed plan of this study is based on the principles of ISO 16140.
The objective of this study was to conduct an interlaboratory cross-validation between two standard methods, GB 4789.40–2016 (reference method) and ISO 22964:2017 (alternative method) for the detection of Cronobacter spp. from PIF(Anonymous, 2017). The results submitted by multiple laboratories were used to estimate the difference in performance parameters between GB 4789.40–2016 and ISO 22964:2017.