Comparative study of Cronobacter identification according to phenotyping methods.

Stephen J Forsythe,Emily E Jackson

doi:10.1186/s12866-016-0768-6

Abstract

BackgroundMicrobiological criteria applied to powdered infant formula (PIF) require the absence of all Cronobacter spp. Consequently, misidentification of isolates from finished products can lead to significant financial losses for manufacturers and could increase the risk of neonatal infection. Biochemical identification of suspect isolates using commercially available test panels is recommended for use by PIF manufacturers by both the US FDA and ISO standard methods for Cronobacter species; however, phenotyping can be unreliable, particularly for a genus such as Cronobacter where the taxonomy has been subject to frequent changes. This study compared the predicted identification by commonly used phenotyping kits (API20E and ID32E) for over 240 strains of Cronobacter from diverse sources, which had been identified using DNA sequence analysis. In 2015, the databases associated with the API20E and ID32E biochemical test panels were updated, including the recognition of the Cronobacter genus. Thus, the identifications from multiple versions the databases were compared to each other and to identifications based on DNA sequencing methods.ResultsUsing previous versions of the API20E database, 90.0 % of strains (216/240) resulted in a match for the species identification; however, version 5.0 produced matches for only 82.3 % of strains (237/288). Similarly, the update to version 4.0 in the ID32E database caused the percentage of matches to drop from 88.9 % (240/270) to 43.2 % (139/322). A smaller study showed that the Vitek GN system identified all 14 strains, belonging all seven Cronobacter species, as members of the ‘C. sakazakii group,’ but also attributed three strains of Franconibacter helveticus and F. pulveris to this group. In silco analysis of a PCR-based method targeting ompA predicted that amplification would only occur with Cronobacter species and this method may be a feasible alternative to biochemical phenotyping.ConclusionsThese results indicate that commercially available biochemical test panels are not sufficiently reliable for speciation of Cronobacter isolates. Although DNA-sequence based methods would be the more reliable approach; however, this is not currently feasible for many food microbiology laboratories. Instead, a previously published PCR-based method targeting ompA is suggested as an alternative for identification of Cronobacter species based on in silico analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0768-6) contains supplementary material, which is available to authorized users.

Highlights

Microbiological criteria applied to powdered infant formula (PIF) require the absence of all Cronobacter spp
Since previous version(s) of the API20E database identified strains as “Enterobacter sakazakii,” this result was considered to be a match for all Cronobacter species
Though the Vitek GN cards identified all Cronobacter strains as members of the ‘C. sakazakii group,’ members of the Franconibacter were assigned to this group

Summary

Introduction

Microbiological criteria applied to powdered infant formula (PIF) require the absence of all Cronobacter spp. A false negative identification, in which a Cronobacter isolate is misidentified as a permitted organism, may result in neonatal infections, product recalls, and lost consumer confidence These losses can be significant for manufacturers as demonstrated in 2011 when a suspected outbreak of C. sakazakii in the United States led to product recalls and a subsequent 10 % drop in the manufacturers’ shares [2, 3]. This was despite the lack of laboratory evidence to linking their product to infant infections and deaths [2, 3]. Minor et al (2015) estimated the cost of C. sakazakii infections to be greater than $5 million per case [4]

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Results

Conclusion