Culture media for the isolation of Cronobacter spp.

Enterobacter sakazakii is a member of the Enterobacteriaceae and has been implicated in causing necrotising enterocolitis, as well as bacteraemia and meningitis in infants. In some cases, the infection has been linked to ingestion of infant formula milk (IFM) that has not been terminally sterilised. The nomenclature of E. sakazakii has been clarified and it has now been accepted as a group of six species comprising a novel genus, Cronobacter. Outbreaks in neonatal intensive care units resulting in relatively high case fatality rates and the recognition of IFM as a significant route of infection prompted the development of culture-based detection methods. Development of enrichment broths specific for Cronobacter spp., coupled to the use of fluorogenic and chromogenic substrates in culture media has significantly improved the sensitivity and specificity of methods. This review presents the history and rationale behind the currently available methods, and gives an overview of the principles involved in designing these microbiological media.

Improving the enrichment procedure for Enterobacteriaceae detection.

The current ISO standard method for detection of Enterobacteriaceae (21528-1:2004) includes enrichment in EE broth which has been shown to be inhibitory to some members of this family, notably Cronobacter spp. A shortened procedure omitting the EE broth has been proposed, however competition from Gram-positive flora may be detrimental to the effective recovery of low levels of target organisms in some sample matrices. In this study we investigated novel cost effective modifications, designed to improve ISO 21528-1:2004 for the detection of Enterobacteriaceae. Initial experiments used a worse-case scenario involving stressed Enterobacteriaceae strains known to grow poorly in laboratory media as well as representative background competitors from powdered milk. The interaction between the Enterobacteriaceae and their competitors was characterised and additives to enhance the growth of target strains over non-target strains were investigated. Supplementation of BPW with 40 microM 8-hydroxyquinoline, 0.5 gL(-1) ammonium iron(III) citrate, 0.1 gL(-1) sodium deoxycholate and 0.1 gL(-1) sodium pyruvate (BPW-S) improved the recovery of Enterobacteriaceae from artificially and naturally contaminated samples. This improvement of the pre-enrichment broth may also be of interest for methods designed to detect specific foodborne pathogens belonging to the Enterobacteriaceae (e.g. Salmonella spp., Cronobacter spp.) that require a pre-enrichment step in BPW.

Development of a novel screening method for the isolation of "Cronobacter" spp. (Enterobacter sakazakii).

A differential medium, "Cronobacter" screening broth, has been designed to complement agars based on hydrolysis of chromogenic alpha-glucopyranoside substrates. The broth was evaluated using 329 Enterobacteriaceae strains (229 target isolates), spiked/naturally contaminated samples, and a parallel comparison with current methods for raw materials, line/end products, and factory environment samples.

A selective differential medium for Enterobacter sakazakii, a preliminary study.

Enterobacter sakazakii can cause fatal invasive infection of neonates associated with the presence of this organism in powdered infant milk formula. A new chromogenic medium (Druggan-Forsythe-Iversen agar, DFI) is described for the selective detection of this emergent pathogen. The medium is based on the alpha-glucosidase reaction which is detected using 5-bromo-4-chloro-3-indolyl-alpha,D-glucopyranoside (XalphaGlc). Ent. sakazakii hydrolyses this substrate to an indigo pigment, producing blue-green colonies on this medium. DFI was compared with the current method of detection on violet red bile glucose agar (VRBGA) followed by pigment production on tryptone soy agar (TSA) after 48-72 h at 25 degrees C and subsequent biochemical profile determination using Biomerieux API20E. Ninety-five clinical and food strains of Ent. sakazakii were detected on the DFI chromogenic medium 2 days sooner than the alternative method. The characteristics of 148 strains representing 17 genera of non-Ent. sakazakii Enterobacteriaceae were compared using the two methods. Only 16/18 Escherichia vulneris strains, 2/3 strains of Pantoea spp. and 1/8 Citrobacter koseri strains gave false positive results on DFI agar. Eight alpha-glucosidase positive strains were identified as Pantoea using their API20E biochemical profile, but had higher percentage identification as Ent. sakazakii using ID32E. Therefore the DFI medium enables the detection of Ent. sakazakii within mixed cultures of Enterobacteriaceae, whereas the organism could be missed when using VRBGA since the latter is a general Enterobacteriaceae selective medium. In addition, the common use of API20E to check yellow pigmented colonies on TSA may lead to false negative results and consequently the acceptance of a batch of infant formula milk (IFM) that contains Ent. sakazakii.