Abstract
BackgroundEnterobacter 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.ResultsA 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.ConclusionThe 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.
Highlights
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 Food and Drug Administration (FDA) method for detection of E. sakazakii includes a pre-enrichment procedure in buffered peptone water (BPW), enrichment in Enterobacteriaceae enrichment (EE) broth, plating on violet red bile glucose agar (VRBG) and picking of five grown colonies onto tryptone soy agar (TSA) plates, which are incubated at 25°C for 48–72 hours
The results for the 63 E. sakazakii strains were in good concordance, blue-green coloured colonies on DFI agar and turquoise on ESIA media, except for one E. sakazakii strain (FSM 322), which gave grey-white colonies on DFI agar (Figure 1A) and was considered negative on this medium
Summary
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. The current Food and Drug Administration (FDA) method for detection of E. sakazakii includes a pre-enrichment procedure in buffered peptone water (BPW), enrichment in Enterobacteriaceae enrichment (EE) broth, plating on violet red bile glucose agar (VRBG) and picking of five grown colonies onto tryptone soy agar (TSA) plates, which are incubated at 25°C for 48–72 hours. A feature, which can be used for this is the α-glucosidase activity, which was demonstrated to be present in all E. sakazakii strains and not in most other members of the Enterobacteriaceae [5] Based on this biochemical property several differential media were developed recently [6,7,8,9]. Two of these media the Oxoid Chromogenic Enterobacter sakazakii Agar (Oxoid CM1055, Oxoid, UK) – known as the Druggan-Forsythe-Iversen (DFI) formulation [7] –
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