The Intestinal Microbiome And Its Relationship To Bronchopulmonary Dysplasia In Preterm Infants

Objective:Bronchopulmonary dysplasia(BPD)is a major respiratory disease of premies leading to long term impact till adulthood.So far its etiology and pathogenesis have not been fully understood.Intestinal microbiome is thought to play an important role in chronic lung diseases.This study aimed to explore the effect of the intestinal microbiome on BPD development by high-throughput 16SrDNA sequencing from fecal samples of premature infants,and then provide some reliable evidence for the prevention and treatment of BPD.Methods:Preterm infants within 24 hours after birth(gestational age≤32weeks)were enrolled in the neonatal intensive care unit(NICU).Nested Case Control was employed for statistical analysis and all patient’s clinical data were colleted.Fecal samples were respectively collected on 1st,4ht,7th,14th,21st,and 28th day after birth.Patients were allocated into BPD group and non-BPD group:the non-BPD group was randomly selected in a 1:1 matching ratio from preterm infants who did not develop BPD.BPD can be categorized into two subgroups:mild and morderate/severe interms of National Institute of Child Health and Human Development(NICHD)and other disease committees in 2000.The results of the clinical data of aforementioned two group,the severity of BPD and 16SrDNA high-throughput sequencing method of intestinal microbiome were analyzed.Results:A total of 151 preterm infants were involved in the cohort,58 infants were designated in BPD group by diagnostic criteria including 26 mild patients and 32 moderate/severe patients,while BPD free infants were randomly matched with the BPD group at a ratio of 1:1,there were 116 patients in both groups.Between two groups:(1)there were no significant difference regarding gender distribution,birth weight,gestational age,mode of delivery,IVF incidence,multiple births,apgar scores at 1 and 5 minutes after birth,and general maternal conditions,Neonatal respiratory distress syndrome(NRDS),pulmonary hypertension,periventricular intraventricular hemorrhage(PVH-IVH),periventricular leukomalacia(PVL),neonatal pneumonia,respiratory failure,anemia,sepsis,age when breastfeeding onset,PICC placement,initial antibiotics regime(restricted level or unrestricted level)and duration(P>0.05).(2)In the BPD group,patent ductus arteriosus(PDA),frequent apnea,retinopathy of prematurity(ROP),parenteral nutrition associated cholestasis(PNAC)and extrauterine growth restriction(EUGR)were significantly higher than non-BPD groups.Blood transfusion≥3 times,invasive ventilation,non-invasive ventilation,and the use of single-special antibiotics were significantly higher than those of non-BPD groups.The period of PICC catheterization,invasive ventilation and non-invasive ventilation days,duration of oxygen supplementation,duration of antibiotic use,days of parenteral nutrition,and hospital stay were significantly longer than those of the non-BPD group(P<0.05).Logistic multivariate regression analysis showed that frequent apnea(OR=0.134,95%CI:0.030～0.603),blood transfusions≥3 times(OR=0.081,95%CI:0.015～0.429),and non-invasive ventilation days(OR=1.251(95%CI:1.111～1.409)were independent high-risk factors for BPD.Analysis of relative abundance of species showed that at the level of phylum classification,proteobacteria were transiently increased in both the BPD group and the non-BPD group.Compared with non BPD group,the relative abundance of Proteobacteria in BPD group peaked at the 14th day after birth,7 days later than no BPD group,furthermore,the relative abundance of Proteobacteria in BPD group was pesistant lower than non BPD group from 4 to 28 days of age.(2)Alpha diversity analysis results:①Alpha diversity analysis results between BPD group and non-BPD group:The cho1 and observed species index of BPD group temporarily increased at day 7 then guradually dwindled while in non BPD group they declined transiently at day 7 and gradually stabilized at a certain level since 14th day of life.The chaol index and observed species index of the BPD group were always higher than those of the non-BPD group on the 4th to 28th day of life,and the chaol index and the observed species index of the BPD group were significantly higher than those of the non-BPD group on the 4th and 7th day after birth(P<0.05).The shannon and simpson index of the BPD group decreased with age,Whilist in non BPD group they decreased transiently on day 7 then increased gradually.The BPD group’s shannon index and simpson index were higher than those of the non-BPD group at 4 to 14 days.Since 21 days,the two groups’indexes were basically equal.At the 4th and 7th,the BPD group’s shannon index and simpson index were significantly higher than those of the non-BPD group.(P<0.05).② Alpha diversity analysis results between different subgroups of BPD:The chaol and observed species index of the mild BPD subgroup gradually decreased from the 4th day,and reached to the nadir on the 21st day,and then rebounded gradually;the moderate/severe BPD subgroup of chaol and observed The species index surged transiently on day 7,then reduced on day 14.The index of chaol and observed species of the mild BPD subgroup was always higher than that of the moderate/severe BPD subgroup,and there was a significant difference between the two groups on the 4th day after birth(P<0.05).The shannon and simpson indexes of the mild BPD subgroup decreased to the lowest at 21 days after birth,and then increased.The shannon and simpson indexes of the medium/severe BPD group decreased transiently on the 14th day,and then gradually increased.On the 4th day after birth,the Shannon index of the mild BPD subgroup was significantly higher than that of the moderate/severe BPD subgroup(P<0.05),while the simpson index between the two subgroups was similar at each time point(P>0.05).(3)Beta diversity analysis results:At 4 and 7 days after birth,the differences in flora between the BPD group and the non-BPD group were prominent,and samples from the same group were gathered.(4)Significant analysis of the differences in flora community structure:① Compared with the non-BPD group,the BPD group had a differential flora from the 4th day after birth.From the 4th day after birth,the Proteobacteria(P=0.028)in the BPD group was significantly reduced,and the thick-walled Mycoplasma(P=0.008),Bacteroides(P=0.003),Actinomycetes(P=0.002),Rhodobacter spp.(P=0.000)increased significantly;Bacteroides(P=0.000)in the BPD group increased significantly on day 7 after birth,Klebsiella(P=0.006),Enterobacteriaceae(P=0.004)was significantly reduced;on the 14th day after birth,Enterobacter(P=0.003),Klebsiella(P=0.016),Lactobacillus(P=0.016)in the BPD group were significantly reduced,and Staphylococcus(P=0.018)significantly increased;on the 21st day after birth,Enterobacter(P=0.008)decreased significantly,Corynebacterium(P=0.000),Precepsia(P=0.000)increased significantly;on the 28th day after birth In the BPD group,Enterobacter(P=0.003)and Streptococcus(P=0.042)were significantly reduced,and Corynebacterium(P=0.004)was significantly increased,and the difference was statistically significant.②Comparison of mild BPD and moderate/severe BPD subgroups:Compared with mild BPD subgroups,moderate/severe BPD subgroups had significantly reduced streptococci(P=0.043)and bifidobacteria(P=0.022).Conclusions:(1)Frequent apnea,blood transfusions≥3,and days of non-invasive ventilation are high risk factors of BPD.(2)The colonization pattern of intestinal flora of preterm infants in the BPD group and non-BPD group showed a transient increase in Proteobacteria,and the relative abundance was lower than that in the non-BPD group from 4 to 28 days after birth.The peak time of proteobacteria abundance in the BPD group(day 14 after birth)was later than that in the non-BPD group(day 7 after birth).(3)Compared with non-BPD,the intestinal flora diversity of preterm infants with BPD increased significantly on the 4th day after birth,and then increased significantly on the 4th and 7th days.Compared with mild BPD,the diversity of intestinal flora was significantly reduced on the fourth day in preterm infants with moderate/severe BPD(4)Preterm infants who develop into BPD had different bacterial flora from the 4th.The BPD group has fewer Enterobacter and Lactobacillus,and then more Bacteroides,Staphylococcus,and Corynebacterium.(5)There was different flora among premature infants in different degrees of BPD.The reduction of bifidobacteria and streptococci was associated with the occurrence of moderate/severe BPD.