Cronobacter spp. (formerly Enterobacter sakazakii) is an opportunistic pathogen associated with contaminated powdered infant formula. It causes necrotizing enterocolitis (NEC) and sepsis, which can develop into severe meningitis and brain abscess formation in infants. Very little is known about the specific pathogenic mechanisms of this organism. In this study, we propose a model to investigate the ability of Cronobacter spp. isolates to disrupt the blood-brain barrier. Biofilm formation of the Cronobacter spp. isolates used in the model was also assayed. Secondary mouse endothelial and astrocyte cells were grown to con.fluency on polyethylene terephtalate (PET) membranes (1.0 μm pore size) in 24-well plate hanging culture inserts (Millipore® Billerica, MA) to achieve a culture system similar to the physiological structure of the blood brain barrier in vivo. Initially, a monoculture system was tested containing only endothelial cells and then a co-culture system was developed with both cell types. Selected Cronobacter spp. isolates were added to the cell culture systems. Escherichia coli KI and Kl2 strains were used as positive and negative controls, respectively. Some of the treatments did not have bacteria added to them to serve as cell controls, and membranes without cells were included as media control blanks. The transendothelial electrical resistance (TEER) was measured across the cells to determine if the cell barrier was disrupted. Initially, measurements were taken at 0, 6, 24, and 48 hours after adding bacteria. Due to overall loss of cell integrity at 48 hours, a second experiment was performed where measurements were taken at 0, 6, and 24 hours after adding the bacteria. Biofilm formation was analyzed using a method described by O'Toole (72) and Skyberg (86). The monoculture and co-culture systems worked based on TEER measurements. The positive control (E.coli Kl) and Cronobacter sakazakii isolates disrupted the tight junctions between cells evidenced by significant decrease in TEER over time. The negative control (E.coli K12) did not have any significant effect on the cells. The cell control and negative control (E.coli Kl2) maintained the highest resistance values in both the monoculture and co-culture experiments. The positive control (E. coli Kl) and the Cronobacter sakazakii isolates caused the resistance across the cells to significantly decrease over time in both experiments. According to the results of our biofilm assay, none of Cronobacter spp. isolates used in the culture models formed biofilms. Further testing would need to be done using other biofilm detection procedures to confirm this conclusion Overall, we successfully constructed a co-culture model to depict the BBB. The selected Cronobacter sakazakii decreased resistance in this model of the BBB similar to the positive control Kl E.coli isolate. This assay can be used in future studies to test the potential pathogenicity of Cronobacter spp. bacteria as well as other bacteria involved with central nervous system diseases.