Study On Characteristics Of Invasiveness On The Main Cells Of BBB With Cronobacter Sakazakii

Cronobacter sakazakii is a significant food-borne pathogenic bacterium that can cause serious neonatal bacteremia, enterocolitis and meningitis. Meningitis is a kind of serious disease that infects the central nervous system(CNS), and even causes death. Previous studies have shown that Cronobacter sakazakii can enter into blood and be transmitted by blood and invade to CNS through blood brain barrier(BBB) to make human to be infected. However,the mechanism on how Cronobacter sakazakii passes from BBB to CNS is still unclear. In this study, 40 representative Cronobacter sakazakii strains, which have strong correlation with clinical pathogenicity, were firstly selected according to the results acquired in the previous studies. Secondly, the pathogenic mechanism on Cronobacter sakazakii causing human meningitis was explored by using human brain microvessel endothelial cell(HBMEC) and U251 cells, which were the two main components of BBB, for their ability of cellular invasion, transcytosis of Cronobacter across transwell-cultured, and cytokines detection. The main results are as follows:(1) After the 40 Cronobacter sakazakii strains were subtyped by MLST and ERIC-PCR,23 different ST subtypes were appeared. The most common ST subtypes were ST1, ST4 and ST93. Thirteen new ST subtypes, which were ST441、ST442、ST443、ST444、ST445、ST446、ST447、ST448、ST449、ST450、ST451、ST452 and ST453, were detected. Combined with results of ERIC-PCR, isolates 35-15-1(ST4), 36-8-1(ST4), 6-16(2)(ST4), 2-9(1)(ST4) and12-12(2)(ST93) were selected for further studies.(2) Abilities of cellular invasion and transcytosis of Cronobacter sakazakii were tested across transwell-cultured experiments by using HBMEC and U251 cells. The results indicated that strain 36-8-1(ST4) had the strongest ability to invade both of the two cells with invasion efficiency of 0.11% and 0.0076%, respectively, which were significantly higher than the control group(Cronobacter sakazakii ATCC BAA-894). Strain 35-15-1(ST4) had the strongest transcytosis ability, the transcytosis efficiency of this isolate to pass through HBMEC was 0.23% after six hours of infection. Meantime, the bacteria were observed in the outside of the cells, inside of the cells and on the cell membranes after their location were observed by using transmission electron microscopy(TEM). Our results exhibitedCronobacter sakazakii recovered from powdered infant formula(PIF) and rice cereal could pass through the main component cells of BBB to result in infections.(3) TNF-α and IL-1 in HBMEC and U251 cells were tested using Enzyme-linked immunosorbent assay(ELISA) kits after the two kinds of cells were invaded by Cronobacter sakazakii. The results indicated that TNF-α and IL-1 were detected in the highest secretion level in HBMEC and U251 after they were infected by strain 36-8-1(ST4), the secretions of TNF-α and IL-1 in the two cells were significantly higher than those in control group(ATCC BAA-894). Our results illustrated that the Cronobacter sakazakii could induce TNF-α and IL-1 secretion in cerebrospinal fluid(CSF) after they passed through the main cells of BBB.(4) In LDH release test, we found that the level of LDH releasing activity in HBMEC and U251 cells was increased with the extension of Cronobacter sakazakii invasion time. The results suggested that the increaseing invasion time aggravated the damage of the membrane of these two types of cells, and BBB can be seriously damaged.