| dc.description.abstract | 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. | en_US |
