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Background   Enterobacter sakazakii  is a foodborne pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. The current FDA detection method includes two enrichment steps, the subculturing of the second enrichment broth on a selective agar (VRBG), a further subculturing of selected grown colonies on TSA and the subsequent biochemical identification of yellow-pigmented colonies by API20E. However, there is a strong need for simplified methods for isolation and identification of  E. sakazakii . In this study, two chromogenic media, which allow to indicate presumptive  E. sakazakii  colonies by the alpha glucosidase activity, as well as a newly developed 1,6-alpha-glucosidase based conventional PCR assay and a rRNA oligonucleotide probe based commercial test system for identification of presumptive  E. sakazakii  were evaluated on 98 target and non-target strains. The methods were compared with respect to specificity aspects.    Results  A total of 75 presumptive  E. sakazakii  and 23 non-target strains were analysed by using chromogenic media, alpha-glucosidase based PCR assay, and the VIT assay. For most presumptive  E. sakazakii  strains on the chromogenic media, the PCR and VIT assay confirmed the identification. However, for a number of presumptive  E. sakazakii  isolates from fruit powder, the alpha-glucosidase PCR and VIT assay did not correspond to the typical  E. sakazakii  colonies on DFI and ESIA. Further characterization by API32E identification, phylogenetic analysis of partial 16S rRNA sequences and ribotyping strongly suggested, that these strains did not belong to the species  E. sakazakii . The newly developed alpha-glucosidase based PCR assay as well as the commercially available VIT  Enterobacter sakazakii  identification test showed an excellent correlation with the 16S rRNA data, and are thus well suited for identification of  E. sakazakii .    Conclusion  The results indicate that presumptive colonies on ESIA and DFI media need further species identification. Both evaluated molecular methods, the alpha-glucosidase PCR and the 16S RNA in situ hybridisation test (VIT), although based on completely different target regions and methodologies performed equally well in terms of specificity.

Comparison of two chromogenic media and evaluation of two molecular based identification systems for Enterobacter sakazakii detection