Effects Of IbeRAT Operon On Biofilm Formation Of Meningitis Escherichia Coli

RESEARCH BACKGROUD AND PURPOSE Bacterial meningitis is a serious infectious disease of the central nervous system,with high mortality rate and neurological sequelae.Antibiotics have achieved a certain successe in treating infections,but when antibiotics are used more often,the drug resistance of bacteria increases.The formation of bacterial biofilm can lower the susceptibility of bacteria to antibiotics and induce drug resistance.In meningitis pathogenic Escherichia coli RS218,ibeA gene located on GimA gene island is an invasive factor;ibeR gene is a regulatory factor;ibeT gene is related to protein transport;and the entire ibeRAT operon is intimately tied to biofilm formation.The study on the biofilm formation mechanism of meningitis pathogenic Escherichia coli in ibeRAT operon is crucial for the prevention and treatment of bacterial infection.RESEARCH METHODS AND RESULTS Methods:(1)The effects of ibeA,ibeR and ibeT genes on biofilm formation were studied using mutant ZD1,mutant BR2,and mutant BTD2-3.(2)The model of cerebral microvascular endothelial cells was cultured to further study the adhesion and invasion of bacteria to cells.(3)The effect of indole on biofilm inhibitor was analyzed,as well as the mechanism by which the ibeRAT operon inhibits biofilm formation.Results:(1)The results of bacterial growth curve and MTT cell viability test showed that the growth and proliferation of bacteria were unaffected after gene knockout.P>0.05;(2)The biofilm formation of each group was observed by crystal violet staining.After 96 h,the obvious biofilm were observed in the wild strain,but the biofilm structure of the mutants was not obvious;(3)In the biofilm formation experiment,the biofilm forming ability of the three mutants ZD1,BR2,and BTD2-3 was decreased as compared to the wild strain.The mutant ZD1,BR2,and BTD2-3 levels were 0.40,0.79 and 0.53 times higher than E44,respectively.Gene mutation decreased the biofilm formation ability of bacteria,P<0.05;(4)The results of cell invasion assay showed that the invasion rate of bacterial mutant ZD1 decreased significantly.The invasion rates of wild strain E44 and mutant ZD1 were(0.88±0.09)%and(0.30±0.06)%,respectively.The invasion ability of wild strain E44 to brain microvascular endothelial cells was significantly higher than that of bacterial mutants.P<0.01;(5)The results of indole drug experiment in wild strain E44 showed that indole,7-hydroxyindole and isatin could effectively reduce the biofilm formation of bacteria,P<0.05.0.5 mM dose of indole could significantly inhibit the biofilm formation of Escherichia coli in meningitis,with a biofilm formation index of 0.27,P<0.01;(6)Indole decreased the expression of IbeA protein in wild strain E44,but there was no significant difference in IbeA protein expression in mutant strain.P<0.01.CONCLUSION (1)The wild strain E44 can form a bacterial biofilm with obvious structure,but the mutant does not;(2)After gene knockout,the biofilm formation of the mutant was inhibited,the biofilm formation index was decreased,and the ibeRAT operon played an important role in biofilm formation when compared to the wild strain;(3)Knockout of ibeA gene reduces bacterial invasion of brain micro vascular endothelial cells,which confirmed that ibeA gene is the invasion factor of meningococcal coli;(4)Indole drugs inhibit biofilm formation,regulate ibeA expression via the ibeR gene,and clarify the mechanism of drug action.Therefore,indole might be employed to develop a therapy for Escherichia coli-caused meningitis.