Isolation of Cronobacter spp. from Dry Powdered Products

The procedure adopted for the isolation of Cronobacter spp. was based on Iversen (2007) [24 (link)] and the original Enterobacter sakazakii technical specifications (ISO/TS 22964, 2006). Briefly, a 100 g DRP sample was homogenized with 900 mL buffered peptone water (BPW; OXOID, Hampshire, UK) and incubated at 37°C for 18 hr as a pre-enrichment step. Subsequently, 1 mL of the BPW suspension was transferred to 10 mL modified lauryl sulfate tryptose broth (OXOID, Hampshire, UK), and after further incubation at 42°C for 24 hr, the broth was streaked on Druggan Forsythe Iversen (DFI, Oxoid, Hampshire, UK) agar and incubated at 37°C for 24 hr. Greenish/blue colonies on DFI agar were considered presumptive Cronobacter spp. The suspected isolates were subjected to the biochemical galleries by VITEK 2 compact GN (bioMèrieix, France) for identification of Cronobacter, according to the manufacturer's instructions. Two reference strains Cronobacter muytjensii ATCC51329 and Cronobacter sakazakii ATCC 25944 purchased from American Type Culture Collection. Supplemental biochemical differentiation tests were performed according to Farmer (1980) and Iversen (2006) [25 –26 (link)].