Aims: To study the growth, thermotolerance and biofilm formation of the emergent pathogen Enterobacter sakazakii in infant formula milk (IFM). Methods and Results: The temperature range, death kinetics and biofilm formation of E. sakazakii were determined using impedance microbiology and conventional methods. In IFM the organism grew as low as 6°C and optimally at 37–43°C. In faecal coliform tests, 23% of strains (n = 70) produced gas from lauryl sulphate broth (LSB) at 44°C after 48 h incubation. Three strains failed to grow in LSB at any of the temperatures. The D-value of cells suspended in IFM was determined between 54 and 62°C. The resultant z-value was 5·7°C. The organism was able to adhere and grow on latex, polycarbonate, silicon and to a lesser extent stainless steel. Conclusions: Enterobacter sakazakii was able to grow at refrigeration temperatures and on infant-feeding equipment. The thermotolerance of the organism was similar to other Enterobacteriaceae and should be killed during standard pasteurization treatment. Significance and Impact of the Study: Enterobacter sakazakii has been associated with infant meningitis through consumption of contaminated IFM. Enterobacter sakazakii is able to grow in IFM during storage at refrigeration temperatures and attach to infant-feeding equipment, which may become reservoirs of infection.

A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii (strain 1330^T, isolated from spiced meat purchased in Slovakia), two previously assigned to Cronobacter genomospecies 1 (strains NCTC 9529^T and 731, isolated from water and a leg infection, respectively) and two previously assigned to Cronobacter turicensis (strains 96 and 1435, isolated from onion powder and rye flour, respectively) was carried out. The analysis included phenotypic characterization, 16S rRNA gene sequencing and multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). 16S rRNA gene sequence analysis and MLSA showed that strain 1330^T formed an independent phylogenetic lineage in the MLSA, with Cronobacter dublinensis LMG 23823^T as the closest neighbour. DNA–DNA reassociation and phenotypic analysis revealed that strain 1330^T represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed (type strain 1330^T = CECT 7863^T = LMG 26250^T). Strains NCTC 9529^T, 731, 96 and 1435 clustered together within an independent phylogenetic lineage, with C. turicensis LMG 23827^T as the closest neighbour in the MLSA. DNA–DNA reassociation and phenotypic analysis confirmed that these strains represent a novel species, for which the name Cronobacter universalis sp. nov. is proposed (type strain NCTC 9529^T = CECT 7864^T = LMG 26249^T).

The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10–17% absence of genes. CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.