Impact Factors Of Necrotizing Enterocolitis In Premature Infants And Multi-Omics Of Intestinal Flora

Research BackgroundNecrotizing enterocolitis(NEC)is a common and serious intestinal inflammation in neonates.The basic pathological manifestations are ischemic necrosis of the gastrointestinal tract and gas accumulation in the intestinal wall and portal vein.In severe cases,intestinal perforation,peritonitis or even death may be involved.Seriously threatening people’s lives and health.The leading cause of death from gastrointestinal diseases in premature infants.Current studies show that the incidence of NEC is about 8.8%,and premature infants account for about 90%.The incidence of NEC was mainly in very low birth weight infants and preterm infants<32 weeks,with a mortality rate of 21%to 39%.Therefore,it is urgent to study the influencing factors and pathogenesis of NEC in premature infants.The pathogenesis of NEC is influenced by many factors,including intestinal immaturity,intestinal microbiome,and ischemia and/or reperfusion injury.However,the pathogenesis of NEC is complex and remains controversial.Some studies suggest that NEC in premature infants is due to intestinal damage caused by inflammatory storm caused by the imbalance of immune regulation between pro-inflammatory mediators and anti-inflammatory mediators.However,some studies do not support this view,suggesting that multiple factors,such as individual genetic variation,environmental factors,and intestinal microbiome imbalance,all contribute to the development of NEC.Obviously,our understanding of the factors and mechanisms of NEC pathogenesis is still in its infancy,and further research is needed to clarify it.As an indispensable part of human body,intestinal microbes are mutually beneficial and symbiotic with the host,and can affect the life process of the host through their metabolites acting on the target organs.The early shaping and evolution of gut microbes,as well as the production of metabolites,are critical to the development and maturation of the newborn’s immune system,which,when maladjusted,can cause disease.More and more studies have shown that intestinal microbes are closely related to NEC,and the imbalance of intestinal flora is considered to be the main cause of NEC.Multiple lines of evidence suggest that the gut microbiome plays an important role in the development of NEC,and that risk factors that increase the likelihood of NEC(such as antibiotics,formula feeding,and acid inhibitors)influence the gut bacterial community.Multiple studies in preterm cohorts have found that NEC is associated with enrichment of Proteobacteria(Klebsiella,Escherichia coli,and Enterobacter)and corresponding reductions in Firmicutes and Bacteroidetes.Although enrichment of enterobacteriaceae is the most common microbiome feature of NEC,it has limited predictive value for infant gut microbiome.Current studies focus on cross-sectional studies to study the relationship between intestinal microbes and NEC at specific time points.A common feature in these studies is the common use of 16sRNA detection methods with very small sample size.At present,less is known about the dynamic changes of intestinal flora and metabolites in premature infants with NEC before,during and after treatment.Up to now,metagenomics combined with metabonomics longitudinal cohort has not been reported in NEC intestinal microbiota studies in premature infants,which is worthy of further study.The above research background drives us to conduct the following research on the premature infants admitted to the neonatal care unit of our Center.It is expected to understand the factors affecting the occurrence of NEC in premature infants in our center,so as to facilitate early clinical identification and reduce the risk of NEC.At the same time,we tried to understand the impact on premature infants with NEC from the dimension of intestinal microbes,to understand the dynamic changes of intestinal flora and metabolites in premature infants before the occurrence of NEC,during the course of disease and after treatment and rehabilitation,and to explore the pathogenesis of NEC,explore potential therapeutic targets and effective intervention strategies.Part 1:Influencing factors of necrotizing enterocolitis in premature infantsObjectiveTo explore the influencing factors of necrotizing enterocolitis,so as to help clinicians identify early and reduce the risk of NEC.MethodsThis study used a nested case-control study of premature infants admitted in the NICU of our center from January 1,2019 to December 30,2021.The subjects were divided into two groups,namely,case group(NEC group)and control group(non-NEC group).The case group included 105 premature infants with NEC,and 781 non-NEC premature infants in the control group.The general conditions and characteristics,perinatal factors,diagnosis and treatment information were collected,and the differences between the two groups were compared in general conditions and perinatal factors by t-test,rank sum test,chi-square test,and the relevant influencing factors of NEC were evaluated by logistic regression analysis.Results1.Case collectionAccording to the inclusion and exclusion criteria,a total of 886 premature infants were included in this study,including 105 NEC children;There were 547 males and 339 females;There were 51 cases with gestational age<28 weeks,199 cases with gestational age≥28 weeks and<32 weeks,and 636 cases with gestational age ≥32 weeks.113 cases of maternal amniotic fluid contamination;143 preterm infants were treated with immunoglobulin before NEC;Invasive respiratory support 327 cases;PICC catheterization in 167 cases;Erythropoietin was used in 225 cases;122 cases of anemia;There were 187 cases of hypoproteinemia.2.General situationGestational age and birth weight were statistically significant compared with preterm infants in the two groups.Gestational age<28 weeks accounted for 10.48%of NEC children,gestational age≥28 weeks and<32 weeks accounted for 35.24%of NEC children,accounting for a higher proportion than non-NEC group,and the difference was statistically significant.There were no significant differences in Apgar score,asphyxia history at birth,intrauterine distress and delivery mode between the two groups.3.Perinatal factorsThere were 23 cases of amniotic fluid contamination in the NEC group and 90 cases in the non-NEC group.There was a statistical difference between the two groups,indicating that amniotic fluid pollution may increase the risk of developing NEC.There were no significant differences in maternal age,gestational hypertension,prenatal glucocorticoid use,prenatal magnesium sulfate use,umbilical cord abnormalities between the two groups.4.Clinical diagnosis and treatmentThe NEC group was treated with non-invasive breathing and erythropoietin before the occurrence of NEC.Compared with the non-NEC group,the difference between the two groups was statistically significant.There was no significant difference between the two groups in the application of immunoglobulin and PICC catheterization before the onset of NEC.5.ComplicationsPneumonia and asphyxia were not statistically significant between the two groups.Preterm infants with anemia and hypoproteinemia before developing NEC in NEC group were statistically different from those in non-NEC group.6.Univariate analysisConsidering many variables and the possibility of multicollinearity between variables,two-way stepwise logistic regression was carried out to screen variables.Four variables were selected by multivariate stepwise logistic regression analysis,including birth weight,amniotic fluid pollution,application of erythropoietin and hypoproteinemia,indicating that birth weight,amniotic fluid pollution,erythropoietin and hypoproteinemia were independent influencing factors of NEC.The results showed that for every 100g increase in birth weight,the risk of developing NEC decreased by about 13%.Exposure to erythropoietin in preterm infants during the course of the disease reduces the risk of NEC.The risk of NEC in patients with amniotic fluid contamination was 2.08 times higher than that in patients with clear amniotic fluid.Preterm infants with hypoproteinemia are at increased risk of developing NEC.Summary1.Birth weight,sheep contamination,propin,and hypoproteinemia were independent factors affecting NEC in preterm infants.2.Preterm infants with amniotic fluid contamination and hypoproteinemia may have increased risk of NEC;preterm infants with relatively large birth weight and eryproins may have reduced risk of NEC.Part 2:Analysis of gut microbiota and metabolites before NECObjectiveTo further study the characteristic intestinal microbiota and related metabolites related to the pathogenesis of NEC,identify the driving microorganisms of NEC,and analyze the association between characteristic intestinal microbiota,related metabolites and clinical phenotypes,in order to explore potential therapeutic targets.MethodsAll premature infants admitted to the neonatal care unit of our Center from January 1,2019 to December 30,2021(same as the subjects in the first part of the study)were recruited.Stool was collected from the neonatal care unit,with a sample collection interval of about 1 week.30 NEC premature infants were included in the case group,namely the NEC group.A total of 30 non-NEC premature infants matched which according to gestational age,feeding pattern and perinatal factors similar to NEC premature infants were included in the control group(non-NEC group)in a ratio of 1:1.Pre-onset fecal samples of NEC children(group S1)and control group(group S6)were collected,and metagenomic sequencing and non-targeted metabolomics analysis were performed.Results1.Metagenomic and untargeted metabolome studies showed that the α-diversity was significantly decreased and β-diversity was significantly different before the onset of NEC.2.Comparative analysis of microbiota composition showed that before the onset of NEC,the relative abundance of Proteobacteria increased,while the relative abundance of Firmicutes,actinobacteria and Bacteroidetes decreased.At the genus level,the relative abundance of Klebsiella increased,and the relative abundance of Escherichia coli decreased.3.LEfSe analysis showed that Klebsiella,Bacteroides,Enterococcus faecium and other bacteria significantly increased before the onset of NEC,while the abundance of bifidobacterium,Lactococcus,Streptococcus and other beneficial bacteria decreased.4.NetShift analysis showed that microorganisms in various genera including Enterococcus,Bacillus,Enterobacter,Lactobacillus,and Lactococcus were related to the pathogenesis of NEC.5.Through functional analysis of intestinal flora,this study found that the abundance of genes related to amino acid biosynthesis,tyrosine and tryptophan biosynthesis,pantothenic acid and coenzyme A biosynthesis pathways were significantly higher than those in the control group before the occurrence of NEC.However,the abundance of T3SS and effector genes was lower than that of the control group.6.Untargeted metabolome analysis showed that 564 metabolites were significantly up-regulated and 748 metabolites were significantly down-regulated before the onset of NEC.In group S1,the biosynthesis of amino acids,the biosynthesis of tyrosine and tryptophan,the biosynthesis of arginine,the metabolism of alanine,aspartate and glutamic acid,and the metabolic pathways of histidine were significantly increased in the metabolic pathways enriched with differential metabolites.7.Untargeted metabolome analysis showed that the area under the ROC curve AUC was 0.88,95%,CI,respectively,when the Top10 differential metabolites were used to construct the NEC incidence assessment model.0.772-0.97,indicating that the model can effectively distinguish NEC children before onset and control children,which can be effectively used as a molecular marker for early warning of NEC,and make up for the shortcomings of traditional diagnostic methods.8.Through "microorganisms-functional genes-metabolites" correlation analysis found that:the pathogenic bacteria klebsiella bacteria and a variety of metabolites,enrichment of pathways found tryptophan metabolism and biosynthesis of amino acids,tyrosine and tryptophan biosynthesis,pantothenic acid,coenzyme a biosynthesis pathways and so on.However,the correlation between bifidobacterium,clostridium,lactobacillus and differential metabolites was relatively low,with the absolute value of correlation between 0.57 and 0.76,which was lower than the threshold range above 0.77 of the top20.Therefore,it was speculated that it was not the main factor affecting the difference of metabolites.Summary1.In this study,we found that intestinal microbiota disorder occurred before the onset of NEC,and it was preferred to clinical symptoms.The results showed that the relative abundance of Klebsiella increased before the onset of NEC,while the abundance of beneficial flora such as Bifidobacterium and Lactococcus decreased.2.Before the occurrence of NEC,T3SS and effector genes were significantly decreased,while amino acid biosynthesis,tyrosine and tryptophan biosynthesis,pantothenic acid and coenzyme A biosynthesis pathways,tryptophan metabolism and other related genes were significantly increased,which could be used as an early warning target in the future.3.Klebsiella may be involved in the development of NEC with a variety of substances,such as rimenidine and amphetamine.Klebsiella and such metabolites can be further explored as a target to provide help for prevention and treatment.Part 3:Dynamic changes of gut microbiota and metabolome after the occurrence of NECObjectiveTo analyze the dynamic changes of intestinal microbiota and metabolites in children with NEC after treatment,so as to provide a scientific basis for the formulation of effective intervention strategies.MethodsThe subjects of this part are the same as the case group included in the second part,namely the NEC group.Fecal samples of premature infants in NEC group were collected before onset(S1 group),fasting and anti-infection treatment group after NEC treatment(S2),resuming feeding group(S3),discharge day group(S4),and 1 month after discharge group(S5)for metagenomic sequencing comparative analysis and untargeted metabolome analysis.Results1.Analysis of the whole dynamic process of the occurrence,development,treatment and rehabilitation of NEC found that:NEC children with intestinal flora gradually recover from a state of disorder to close to healthy controls state of flora,klebsiella,e.coli,freund’s citric acid bacilli and wei rong bacteria fungi and other pathogens,abundance presents a significant declining trend,and thick wall door,bacteria-the bacteroidetes and other probiotic group of abundance,in the steady growth and maintain the stability of engraftment after discharge characteristics,However,compared with the control group,there are still some differences in the balance of bacteria,mainly reflected in the abundance of Streptococcus,Enterobacteria and actinobacteria,while some bacteria of Enterococcus and Bacteroidetes are relatively less,which means that the children still need a longer follow-up recovery process.2.Opportunistic pathogens such as Escherichia coli and Klebsiella pneumoniae may be highly related to the occurrence and development of NEC.However,the role of Proteobacteria in the occurrence,development,attenuation and recovery of NEC still needs to be further explored.3.Metagenomic function analysis of intestinal microbiota showed that the microbiota changed dynamically with the improvement of NEC in children.KEGG modules such as type III secretion system and type III effector genes,succinate dehydrogenase genes,NADH quinone oxidoxidases genes were identified to have significant changes during the recovery period after NEC treatment.4.Metabolomic analysis results show that the process of healing in the treatment to the NEC,enrichment of metabolites significant difference to the bile secretion pathway and amino acid metabolism pathway,NEC children in development condition,the bile secretion,histidine metabolism,arginine biosynthesis and physiological functions such as amino acid biosynthesis and metabolism of butyric acid was serious interference,They gradually recovered after treatment and recovery.Summary1.The current treatment regimen helps the children’s intestinal microbiota gradually recover to be close to the control group,and the dynamic change of the microbiota can be used as a scale to evaluate the treatment effect.2.Opportunistic pathogens such as Klebsiella,Escherichia coli,Castrobacter freundii and Veronella are highly positively correlated with the occurrence and development of NEC.They may affect the occurrence and development of NEC through pathogenic systems such as T3SS and type 3 effectors.3.NEC children may be affected by the bile secretion,histidine metabolism,arginine biosynthesis and physiological functions such as amino acid biosynthesis and metabolism of butyric acid can lead to the development of illness occurs,maintain and restore the physiological functions,such as by improving and optimizing the intestinal flora,rebuild normal bile acid enterohepatic circulation,assisted with necessary metabolite,May contribute to the improvement and rehabilitation of NEC.4.This is the first to NEC children from prior to your illness recovery explore the dynamic changes of intestinal flora and metabolites,using multiple omics tools and multi-level joint analysis strategy,complete depicts the NEC after intestinal flora and metabolic reconstruction process,effective strategy choice for late NEC medical intervention to provide important theoretical basis.Conclusion1.According to the analysis of clinical factors of necrotizing enterocolitis,the risk of NEC may increase in premature infants with sheep contamination and hypoproteinemia,and NEC with relatively large birth weight around gestational age may decrease the risk.Therefore,help clinicians to identify and warn early warning.2.Preterm infants had intestinal flora disturbances before the onset of NEC,which occurred before clinical symptoms.The species and abundance of microflora are decreased,which is manifested as the decrease of beneficial bacteria and the increase of opportunistic and pathogenic bacteria.Therefore,timely probiotic intervention should be given after the establishment of gastrointestinal feeding in premature infants to achieve a balance of symbiotic microflora.3.Before the onset of disease in infants,this study established a corresponding prediction model by using the differential microorganisms and metabolites found,which can better distinguish whether premature infants suffer from NEC and play a role in clinical early warning.4.After the children suffered from NEC,this study found that with the recovery of the disease,the symbiotic flora gradually reached a stable state,showing a decline in pathogenic bacteria;Beneficial bacteria are gradually established and changes in intestinal flora can be used as promising prognostic indicators of disease.