Comparative study of Cronobacter identification according to phenotyping methods

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.

Novel Method for Reliable Identification of Siccibacter and Franconibacter Strains: from “Pseudo-Cronobacter” to New Enterobacteriaceae Genera

“…Methods Together with the fact that Franconibacter and Siccibacter bacteria have biochemical properties very similar to those of Cronobacter bacteria, this all means that it is quite possible that they could be misidentified in the analysis of food or powdered formula samples. In this study, similarly to Jackson et al (29), we too have shown that ID32E misidentifies Franconibacter and Siccibacter strains, predominantly as Cronobacter strains. However, we have also shown that the use of certain biochemical tube tests enables a "pseudo-Cronobacter" strain misidentified by ID32E to be either differentiated from an actual Cronobacter strain or identified as an actual Franconibacter or Siccibacter strain.…”

“…However, we have also shown that the use of certain biochemical tube tests enables a "pseudo-Cronobacter" strain misidentified by ID32E to be either differentiated from an actual Cronobacter strain or identified as an actual Franconibacter or Siccibacter strain. All of the above data support Jackson's view that these tests should be replaced by a more reliable identification technique, such as PCR or another molecular biology method (29).…”

Examining the Presence of <i>Cronobacter</i> spp. in Ready-to-eat Edible Insects

“…22) . However, they are not always feasible, due to factors such as cost and time 22,23) . Other PCR-based methods are therefore an alternative.…”

Invasive Cronobacter species infection in infants and children admitted to a rural Kenyan hospital with a high prevalence of malnutrition

“…It is possible that some Cronobacter isolates were misidentified as Enterobacter or other related species because the API20E and ID32E assays are not fully sensitive for the detection of Cronobacter species [15]. Similarly, sub-culturing isolates with a specific growth medium for pigmentation has also been reported to have a sensitivity of only 80%, and hence sub-culturing was used for further characterisation of the samples but not for isolate selection.…”

“…It is possible that other samples were missed as Cronobacter species including the two further samples that were API20E positive but ID32E negative. Other Enterobacter species such as E. hormaechei can cause infections from ingested food and have culture profiles similar to those of Cronobacter [15]. Thus, the true burden of Cronobacter disease could be slightly higher, but the plausible number remains very low and it is very unlikely to be a burden of public health significance.…”