Food Safety, Great Plains Institute of

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Research from the Great Plains Institute of Food Safety. The institute website may be found at https://www.ag.ndsu.edu/foodsafety

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Browsing Food Safety, Great Plains Institute of by Subject "Enterobacter sakazakii."

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    An In-vitro Co-Culture Model to Study the Disruption of the Blood Brain Barrier by Cronobacter sakazakii. (formerly Enterobacter sakazakii)
    (North Dakota State University, 2010) Koval, Erin Louise
    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.
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    Relatedness ofisolates of a Novel Genus, Cronobacter, Formerly Known as Enterobacter sakazakii
    (North Dakota State University, 2010) Solseng, Tracy Anne
    Members of the genus Cronobacter were once classified as Enterobacter sakazakii. These bacteria are opportunistic pathogens that are associated with necrotizing enteritis, sepsis and meningitis in neonatal or low-birth-weight infants and can result in death, slowed development, or extensive neurological disorders. In adults, they have been documented as a cause of bacteremia, osteomyelitis, and vaginitis. Previously, E. sakazakii was found in the midgut of stable flies. Research by Nangoh et al. determined that Cronobacter spp. (previously identified as E. sakazakii) are present in bison and bovine feces. In addition to the bison and bovine fecal isolates of Cronobacter spp. found by Nangoh et al., other isolates of Cronobacter spp. were analyzed phenotypically and genetically for biochemical typing and genotyping. The additional isolates include several American Type Culture Collection isolates, an isolate from a neonatal meningitis case, and multiple isolates of various origins received from Cornell University. These isolates were further categorized using four different biochemical tests. The results of these tests placed the isolates into one of the six different species or subspecies within the genus Cronobacter. For genotyping, the isolates were tested for the gene specifically responsible for the a-glucosidase activity. In addition, Pulsed-Field Gel Electrophoresis using two different enzymes, Xbal and Spel, was performed to determine possible genetic similarity of isolates from the bison and bovine feces to other isolates found in food, clinical and environmental settings. The X'baI enzyme showed two Cornell isolates, F6-049 and F6-051, had a high degree of similarity; both of these isolates were from the same clinical source. Isolates from bison and bovine feces, 52 and N72, respectively, have a high degree of dissimilarity to each other, ~ 75%. Isolate 52 showed ~ 35% dissimilarity to an isolate from a food source, and N72 showed ~45% dissimilarity to an isolate from a clinical source. The results using the Spel enzyme showed a wide diversity among the isolates. This study shows that very few of the Cronobacter spp. isolates are closely related and that there is a high level of diversity based on pulse-field gel electrophoresis and biochemical analysis.