Colonization Of Lactobacillus Plantarum RS-09 In The Intestine Of Mice And Its Inhibitory Effect On Salmonella Enteritidis Infection

Background:Salmonella Enteritidis(S.Enteritidis,SE)is a relatively common foodborne pathogen.In recent years,S.Enteritidis has become an important pathogen causing gastroenteritis and food poisoning.S.Enteritidis is mainly colonized in the cecum and colon,and can cause up-regulation of proinflammatory cytokines such as IFN-?and IL-6 in intestinal tissues.The long-term use and abuse of antibiotics have led to the emergence of multi-drug resistance of S.Enteritidis,posing a new challenge to the prevention of S.Enteritidis.How to prevent S.Enteritidis has become a major problem that plagues human health.In recent years,studies have shown that L.plantarum,as the main probiotic group in the intestine,can increase the diversity of intestinal flora and enhance the body's immunity.The antibacterial peptides,bacteriocins and organic acids produced by the plant can inhibit the growth of pathogenic microorganisms.Breeding,so the use of probiotics to prevent or inhibit pathogen infection has caused widespread concern.Objective:In recent years,studies on L.plantarum have focused on in vitro adhesion experiments and their effects.There are relatively few studies on the colonization of L.plantarum in the intestine and the mechanism of inhibition of S.Enteritidis.In this study,a well-adhered L.plantarum was isolated from fermented apple juice,and its colonization in the intestine of mice was studied.At the same time,L.plantarum inhibited the colonization of S.Enteritidis in the intestine of mice.And infections were studied.Methods:(1)Screening isolates were screened by Ca CO₃-MRS medium and Gram stain for positive acid-producing positive bacilli,16s r DNA sequencing and biochemical reaction.The phylogenetic analysis of the isolated strains was carried out to determine the species of the isolated strains.(2)To evaluate the acid-producing ability and growth performance of the strain by measuring the acid-producing curve and growth curve of the strain,to determine its acid-tolerant and bile-salt-capacity and adhesion performance,and to evaluate the colonization ability of the strain;(3)using c FDA-SE the marker model was constructed as a marker dye;(4)After 5?10⁹CFU-labeled L.plantarum RS-09 was administered to mice,flow cytometry and frozen sections were used to monitor the colonization of the marker strain in the intestine of mice;The Oxford Cup method and the cell experiment to investigate the in vitro inhibition of RS-09 against Salmonella enteritidis;(6)In vivo experiments:pretreatment of mice,continuous administration for 9 days 5?10⁹CFU of L.plantarum RS-09,The mice were challenged with S.Enteritidis on the 10th day,and the mice were sacrificed on the 15th day.The isolates were evaluated for inhibition in vivo by organ index,intestinal bacterial count,immunological indicators and high-throughput techniques.The role of S.Enteritidis infection.Results:(1)A L.plantarum was isolated from the fermented apple liquid and named as RS-09,and deposited by the Culture Collection,CMCCC No.17118.The strain has good acid-producing ability,acid-tolerant and bile-salt-resistant ability and antibacterial effect in vitro,and exhibits excellent adhesion to intestinal epithelial cells HT-29.(2)Successful use of c FDA-SE as a marker dye Fluorescently labeled L.plantarum RS-09 strain model;(3)The strain was administered to mice at a dose of5?10⁹ CFU,and the test was carried out at different time points.It was found that the labeled strain was basically colonized to the intestinal surface epithelium at 48h.The cells were dynamically balanced with the intestinal flora after 96 hours,indicating that the labeled strain has good colonization ability,and its colonization rules are:colon>cecum>ileum>jejunum>duodenum.(4)In vitro bacteriostatic experiments demonstrated that the fermentation broth of L.plantarum RS-09 can inhibit the proliferation of S.Enteritidis,Staphylococcus aureus and Escherichia coli;(5)RS-09inhibits S.Enteritidis mainly by rejection and competition.Adhesion of HT-29 cells;(6)In vivo experiments showed that L.plantarum RS-09 pretreatment can slow down the weight of mice,increase spleen index and liver index,reduce the number of colonization of S.Enteritidis in the intestine of mice;serum cytokines The results showed that the expression levels of pro-inflammatory factors IFN-?and IL-12,anti-inflammatory factors IL-10 and IL-4 were increased after challenge with S.Enteritidis;and the pretreatment of L.plantarum RS-09 was reduced.The expression levels of pro-inflammatory factors IFN-?and IL-12 promoted the up-regulation of the levels of the anti-inflammatory factors IL-4 and IL-10;the results of flow cytometry showed that Th1 in the mouse's Peyer's knot after challenge with S.Enteritidis The level of activation was significantly increased,and pretreatment of L.plantarum RS-09 reduced the level of activation of Th1 cells in the Peyer's patch.High-throughput sequencing results showed that some pathogenic bacteria of Serratia marcescens and Bacteroides in the S.Enteritidis group had significant biological differences compared with the control group;L.plantarum RS-09 pretreatment could reduce microorganisms caused by Salmonella The degree of community change maintains the dynamic balance of the intestinal flora.Conclusion:This study successfully obtained a strain of L.plantarum RS-09 with good growth performance,acid-producing ability,acid-tolerant bile-salt resistance and strong adhesion ability.It was firstly analyzed by c FDA-SE labeling technique in vivo long-term follow-up study.The colonization of probiotics and the establishment of L.plantarum RS-09 in the intestinal tract of mice:colon>cecum>ileum>jejunum>duodenum;preliminary investigation of L.plantarum RS-09 to prevent S.Enteritidis infection The mechanism of mice proves that L.plantarum RS-09 pretreatment can interfere with the colonization of S.Enteritidis in the intestine of mice,which can reduce the inflammatory reaction caused by S.Enteritidis infection and improve the intestinal microbial composition.Provides a preliminary basis for subsequent follow-up on L.plantarum RS-09 and S.Enteritidis interaction mechanisms.