Study On The Protective Effect And Mechanism Of Lactobacillus Rhamnosus Supernatant Against Neonatal Rat E.Coli Infection And Analysis Of Effective Components

BACKGROUND AND AIMSSepsis and meningitis remain a major cause of high mortality and severe neurological morbidity in neonates,especially in the preterm.Escherichia coli?E.coli?is the most predominant gram-negative bacteria that cause neonatal sepsis and meningitis.The incidence of E.coli infections may further increase because of the recent emergence of antibiotic resistant E.coli strains.Therefore,it is necessary to develop alternate treatment strategies for preventing neonatal E.coli sepsis and meningitis.Immaturity of intestinal barrier function is the major reason responsible for high sensitivity of neonates with E.coli infection.Thus,the blockage of bacterial adherence to enterocyte and translocation across the intestinal barrier into the bloodstream is a potential approach to prevent neonatal E.coli sepsis and meningitis.Accumulating evidence shows that Lactobacillus rhamnosus GG Culture Supernatant?LCS?has a preventive effect against several intestinal disorders through competition with pathogens,maintenance of intestinal barrier integrity and promoting immune function.However,it is not very clear whether LCS could exert preventive effect against neonatal E.coli infection.In the present study,we have explored the protective effect of LCS on neonatal E.coli infection and identified the specific active ingredients in LCS.METHODS AND RESULTSMethods:Firstly,we have explored the enhancement effect of LCS on intestinal barrier and the protective role against E.coli-induced dysfunction of the intestinal barrier using human colonic carcinoma cell line Caco-2.Then,the neonatal rat model of gut-derived E.coli infection was used to determine the prophylactic effect and mechanisms of LCS on neonatal systemic E.coli infection.At last,the liquid chromatography-tandem mass spectrometry?LC-MS/MS?was performed to identify the specific active ingredients in LCS.Results:Our in vitro results showed that LCS could block adhesion,invasion and translocation of E.coli to Caco-2 monolayer via up-regulating mucin production and maintaining intestinal integrity.Then,we found pre-treatment with LCS significantly decrease susceptibility of neonatal rats to oral E.coli infection as reflected by reduced bacterial intestinal colonization,translocation,dissemination and systemic infections.Further,we found that LCS treated neonatal rats have higher intestinal expressions of Ki67,MUC2,ZO-1,IgA,mucin and lower barrier permeability than those in untreated rats.These results indicated that LCS could enhance neonatal resistance to systemic E.coli infection via promoting maturation of neonatal intestinal defense.Furthermore,a protein termed hypothetical protein HMPREF0539₂242 was identified from LCS and seemly exert the protective role on neonatal rats against neonatal E.coli infection.CONCLUSIONVertical transmission from the mother's birth canal during delivery is the primary source of E.coli.Thus,successfully colonize and across the gut barrier have been considered as the critical steps for meningeal invasion of E.coli.This implication indicate that the blockage of bacterial adherence to enterocyte and translocation across the intestinal barrier into the bloodstream would be a potential approach to prevent neonatal E.coli sepsis and meningitis.The immature intestinal defense in the neonatal period may provide opportunities for pathogenic translocation across the gut barrier into the blood stream,leading to systemic inflammation and infection.In the present study,we reported for the first time that administration with LCS could enhance the resistance of neonatal rats to E.coli infection.Further study found that LCS could promote intestinal proliferation and differentiation,accelerate intestinal barrier formation and increase mucin and IgA production in the gut of neonatal rat.These data indicated that neonatal administration with LCS accelerates the maturation of intestinal defense and confers a high resistance to intestinal infection.At last,we identified a protein from LCS using LC-MS/MS,and found this protein seemly exert a preventive effect on neonatal rats against E.coli infection.Our data indicated that LCS has a great potential to become an effective prophylaxis for neonatal E.coli sepsis and meningitis.