Enterobacter sakazakii is a bacterial contaminant of powdered infant formula milk that has been associated with necrotising enterocolitis, bacteraemia and a rare form of infant meningitis. The presence, persistence and growth of the organism in infant formula milk needs to be better understood to limit the occurrence of infection, and improved isolation methods need to be developed in order for companies to implement appropriate food safety management systems. A collection of E. sakazakii isolates from diverse clinical, food and environmental sources was compiled. Isolates identified biochemically as E. sakazakii formed four genomic clusters when housekeeping gene sequences (165 rONA and hsp60 loci) were compared. The reliability of presumptive isolate identification using commercial biochemical galleries was investigated in comparison to identification by 165 sequencing. The Biolog GN2 system appeared to be the most reliable identification gallery. A novel chromogenic medium, based on the a-glucosidase reaction, was developed to improve the efficiency of E. sakazakii isolation methods and is commercially available as Chromogenic Enterobacter sakazakii medium, Oruggan-Forsythe-Iversen formulation (OFI), CM1055, Oxoid ltd. The sensitivity and specificity of the OFI medium was compared with other proposed media. Also 486 food samples were tested for the presence of E. sakazakii. The organism was isolated from 67 samples using the OFI medium compared with only 19 using the conventional method. A novel enrichment medium was also investigated to improve recovery of E. sakazakii. Preliminary investigation of factors that may be associated with increased risk of acquiring E. sakazakii infection from contaminated infant formula indicated that E. sakazakii strains are able to survive in a desiccated state for over 6 months. They are also able to form biofilms on infant feeding equipment, can attach and invade human epithelial (CaCO-2) cells in vitro and can survive in human serum. Some strains may persist in macrophages, and many produce exopolysaccharide capsules which enhance biofilm formation and may contribute to evasion of host immune defences.