Effects Of Empirical Antibiotic Use Duration On Gut Microbiota And Immune Function Of Premature Infants And The Screening For Biomarkers In Neonates With Early-Onset Sepsis

PART ⅠEFFECTS OF EMPIRICAL ANTIBIOTIC USE DURATION ON GUT MICROBIOTA AND IMMUNE FUNCTION OF PREMATURE INFANTSObjective: The neonatal period is a critical period for the formation of gut microbiota,which plays an irreplaceable role in regulating of the developing of intestinal immune system.Premature infants at high risk of infection are often given antibiotics empirically immediately after birth,and antibiotics have a significant impact on gut microbiota composition.The purpose of this study is to clarify the effects of antibiotic use duration on gut microbiota and intestinal immune function of premature infants,so as to provide theoretical basis for guiding the rational use of antibiotics in clinical practice.Methods: The subjects of the study were premature infants hospitalized in Children’s Hospital of Chongqing Medical University immediately after birth.According to the duration of antibiotic use duration,the infants were divided into two groups,namely discontinuation after 3-days of continuous antibiotic use(A3 group)and discontinuation after 5-days of continuous antibiotic use(A5 group),and the control group was the non-antibiotic use group(AF group).Fecal samples were collected on day 3,day 5,and day 7in the 3 groups.Intestinal flora composition of the infants in each group was analyzed based on 16 S r DNA high-throughput sequencing,and an experimental antibiotic preterm mouse model was established.Feces and intestinal tissues of mice were collected for HE staining,immunohistochemistry for MUC1 and MUC13 proteins,flow cytometry for ILC3 cells,and QPCR/ELISA for cytokines.To detect the difference of gut microbiota and immune function between each group.Results: At the portal level,Firmicutes and Proteobacteria were dominant in each group on day 3,day 5 and day 7,followed by a small proportion of Bacteroidetes and Actinomycetes,and there was no significant difference in the proportions of the four bacteria groups over time(P > 0.05).On day 3,the Firmicutes in Group A5 and the Proteobacteria in group A3 had the highest proportion.On day 5,the proportion of each bacterial colony in the three groups was similar to that on day 3,but Bacteroidetes increased in the AF group.By day 7,Firmicutes and Proteobacteria were dominant in each group.The changes of microbiota in each group on day 3,day 5 and day 7 were compared and analyzed.Firmicutes increased gradually in Group A3,while Proteobacteria,Bacteroidetes and Actinomycetes decreased.In group A5,Firmicutes increased,Proteobacteria decreased,and Bacteroidetes and Actinomycetes contained in the microbiota were very few.However,the Firmicutes to Proteobacteria ratio in AF group did not change significantly with time,and the Bacteroidetes increased on the 5th and 7th days.In the level of three groups of microbiota proportion is 3 days,found that Shigella A3 group share to the highest in three groups,Enterococcus bacteria genera of A5 group proportion is highest in three groups,Staphylococcus aureus accounted for the A5 group for the highest in three groups,Klebsiella species proportion is highest in the three groups of A3 group,narrow Fusobacterium of AF group microbiota proportion is highest in the three groups.On day 5,The proportion of Escherichia coli in Group A3 was the highest,and the proportion of Enterococcus in group A5 was the highest,Staphylococci accounted for the highest proportion of microbiota in group A5,Klebsiella accounted for the highest proportion in group A5,and Clostridium narrow accounted for the highest proportion of microbiota in group AF.Comparing the level of microbiota of the three groups on day 7,the proportion of Escherichia was the highest in Group A3,the proportion of Enterococci was the highest in group A5,and the proportion of Staphylococcus was the highest in group A3.Klebsiella occupies the highest proportion in Group A5,and Clostridium narrow occupies the highest proportion in group AF.The proportions of Enterococcus and Shigella in each group on day 3,day 5 and day 7 were compared.The proportions of Enterococcus and Shigella in the A3 group on day 5 were increased compared with that on day 3,while the proportions of Enterococcus and Shigella in the group on day 7 were decreased compared with that on day 5,and were lower than that on day 3.The proportion of Shigella in group A5 on day 5 and day 7 was higher than that on day 3.The proportion of Enterococcus and Shigella in AF group was lower than that on day 3 on day5 and lower than that on day 5 on day 7,and the difference was statistically significant(P<0.05).With the extension of antibiotic intervention preterm mouse model showed that significantly reduced expression of ILC3 in the intestinal tissue led to the lower secretion of IL-22 the increased expression of inflammatory factors,and anti-inflammatory factors decreased(P<0.05).Conclusion: Antibiotics use duration had a significant effect on the colonization of gut microbiota,the proportion of pathogenic microbiota in the antibiotic group increased significantly,and the diversity of microbiota in the antibiotic group for 5 days was decreased compared with that for 3days.The prolonged of antibiotics can cause disturbance of gut microbiota and destruction of intestinal immune homeostasis.The mechanism may be related to the ILC3-IL-22 axis.PART Ⅱ THE SCREENING FOR BIOMARKERS IN NEONATES WITH EARLY-ONSET SEPSISObjectives: Neonatal early-onset sepsis(EOS)is associated with high morbidity and mortality.Accurate early diagnosis is crucial for prompt treatment and a better clinical outcome.We aimed to identify new biomarkers for the diagnosis of EOS.Methods: A total of 152 neonates with a risk of EOS were divided into EOS group and non-EOS group according to the conventional diagnostic criteria.Blood samples were collected within 0-24,24-48,and 48-72 hours after birth.Serum levels of PGRN,IL-33,IL-17 a,IL-23,IL-6,TNF-α,IFN-γ,GM-CSF,PCT,and CRP were determined.Results: PGRN levels were significantly elevated in the EOS neonates compared with the levels in the non-EOS neonates(1.53 vs.0.77 ng/ml(median),P < 0.001),with an area under the receiver-operating characteristic(ROC)curve(AUC)of 0.76(P< 0.001).Compared with PGRN,IL-33,IL-17 a,IL-23,IL-6,PCT,and CRP showed significant(AUC>0.70)but slightly less predictive power for EOS within the same time range.Stepwise multivariate regression analysis identified PGRN,IL-33,and PCT as independent predictors of EOS.In addition,the combined measurements of PGRN,IL-33,and PCT showed significantly higher predictive power for EOS than any of the three markers alone.Conclusion: PGRN showed greater efficacy for predicting EOS than the traditional markers PCT and CRP as well as other potential markers tested in this study.PGRN may serve as an effective biomarker for the early diagnosis of EOS.