A method for the detection of Cronobacter strains in powdered milk-based foods using enrichment and real-time PCR

Cronobacter spp. are emerging food-borne pathogens implicated in fatal infections in neonates and infants. As the contaminated powdered infant formula (PIF) has been identified as the major source of infection, a zero tolerance of Cronobacter spp. is required for these products. The aim of our study was to evaluate real-time PCR-based method for Cronobacter detection in milk powder-based products. TaqMan real-time PCR targeting the dnaG gene of MMS-operon was used. The system was evaluated as 100% specific for Cronobacter spp., which was determined using 97 Cronobacter strains and 85 non-Cronobacter strains with a PCR detection limit of 1×10E2 CFU per ml. Shortened two-step enrichment was used. Out of 50 milk powder-based samples analysed, six were positive by the PCR-based method and five by the standard method. Out of 15 artificially contaminated PIF with Cronobacter at 10 CFU per 10 g, 15 and 13 were positive by respective methods. Low detection limit of the complete method was not compromised either by Enterobacteriaceae background up to 10E7 CFU per 10 g, or by dryness and cold stress of Cronobacter cells used for artificial contamination. This method facilitated a reliable detection of Cronobacter spp. alternative to the currently available method, providing a considerable time reduction in comparison to the microbiological standard detection method.