The Intestinal Microbiome And Its Relationship To Necrotizing Enterocolitis In Preterm Very Low Birth Weight Infants

Objective:Neonatal necrotizing enterocolitis(NEC)is an inflammatory intestinal disorder that seriously threatens the life of preterm infants.The etiology and pathogenesis of NEC remains uncertain.The intestinal microecological imbalance is considered to play an important role in the occurrence and development of NEC.The aim of this study was to explore the effect of intestinal microbiome on the occurrence and development of premature VLBWI NEC by high-throughput 16SrDNA sequencing from fecal samples of premature infants and provide a reliable basis for the prevention and treatment of NEC.Methods:Preterm infants within 24 hours after the birth(birth weight<1500 g)were included in the neonatal intensive care unit(NICU)of Children’s Hospital of Soochow University and/or Suzhou Municipal Hospital Parent and Child Center from April 20,2018 to November 20,2018.A prospective nested case-control(NCC)study design was used and the basic clinical data of all premature infants were collected.The fecal samples were collected on the first,fourth and seventh days after birth and then once a week after the first week.The end point of the fecal specimens had been reached when NEC occurred,at the time of discharge or at the 8th week after birth(whichever happened first).The objects were divided into NEC group and VLBW control group.According to the NEC diagnostic criteria,the enrolled objects developed NEC were taken as the NEC group and the VLBW control group was randomly selected in a 1:1 matching ratio from preterm infants who did not develop NEC.NEC group was divided into NEC Ⅰ,NEC Ⅱand NEC Ⅲ subgroups according to Bell stage criteria.The clinical information and fecal samples of the NEC group and VLBW control group were prepared.Based on the 16SrDNA high-throughput sequencing method,the clinical data and intestinal microbiome of NEC group,VLBW control group and different NEC subgroups were analyzed.Results:First,clinical information:A total of 184 preterm infants were enrolled in the cohort among which 39 developed NEC,including 16 NECI phase,16 NEC Ⅱ phase,and 7 NECⅢ phase.The age of onset of NEC was 4 to 53 days with an average of 16(10,23)days.There were 145 premature infants with no NEC in the cohort,the VLBW control group(39 cases)randomly matched the NEC group in a ratio of 1:1.There were no significant differences in gender distribution,birth weight,gestational age,age at admission,mode of delivery,natural pregnancy,multiple births,small gestational age,history of asphyxia,and basic information of mothers(P>0.05).The incidence of sepsis,infectious pneumonia,respiratory failure,and parenteral nutrition associated cholestasis(PNAC)in preterm infants in the NEC group was significantly higher than the VLBW control group.The NEC group had a fast milking rate and a long period of antibiotic use.The application rates of treatments such as use of two or more special grade antibiotics,blood transfusion>3 times,invasive ventilator and continuous positive airway pressure(CPAP)were significantly higher than that of VLBW control group with significant difference(P<0.05).Second,results of high-throughput sequencing of fecal 16SrDNA:(1)Analysis of the relative abundance of species showed that Firmicutes had a transient decline in the NEC and VLBW control groups.It hit the lowest on the 14th day after birth in the NEC group and 7 days after birth in the VLBW control group.The relative abundance of Firmicutes in the NEC group was lower than that of the VLBW control group from the 14th day after birth.Proteobacteria was relatively abundant in the NEC group.(2)Results of Alpha diversity:① Alpha diversity between NEC cases and VLBW controls:Both chao1 index and the observed species index showed a transient on the 7th day and increased with age.After 7 days of birth,the chao1 index and observed species index of NEC group were lower than those of VLBW control group with no statistical significance(P>0.05).The shannon index and simpson index of the VLBW control group had a transient decline on the 7th day,and then increased with age,while the 2 indexes of the NEC group did not reflect the same changes accordingly.The shannon index and simpson index of NEC group was lower than that of the control group after 7 days of birth and the difference was statistically significant on the 21st and 28th day(P<0.05).②Alpha diversity of different subgroups of NEC:The observed species index was statistically different among NECI,NECⅡ,and NECⅢ(P=0.013).There was a significant difference in observed species index between NECI and NECII(P=0.002),however no significant difference between either NECI and NECⅢ or NECⅡ and NECⅢ.There was a significant difference(P=0.049)for the higher shannon index of NEC Ⅱ stage than NECⅠ stage,but no significant difference between NECI stage and NECⅢ stage,NECⅡ stage and NECⅢstage(P>0.05).(3)Beta diversity analysis:The difference between the NEC group and the VLBW control group,of which the same group was gathered together,was prominent for the 21 days and 28 days after birth.(4)The differences in the community structure of the flora:① NEC group versus VLBW control group:Analysis of the difference in community structure showed that the NEC group had a different flora from the 4th day after birth compared with the VLBW control group.At the level of the class,the reduction of Erysipelotrichi in the NEC group was statistically significant(P=0.006).From the 14th day after birth,Bacteroidetes and Betaproteobacteria increased significantly(P=0.005;P=0.01).On the 21st day after birth,Firmicutes,Clostridia and Bifidobacteriaceae was significantly reduced(P=0.003;P=0.03;P=0.04).The 28th day after birth,the decrease in Firmicutes,Clostridium and Clostridiaceae were observed decreased obviously(P=0.006;P=0.001;P=0.001).②The differences in the community structure of the flora of NEC subgroups:Coriobacteriia and Coriobacteriaceae in NEC Ⅱ and NEC Ⅲ stage significantly increased compared with NEC Ⅰ(P=0.0001;P=0.002).Compared with NECⅡ phase,Bifidobacterium longum and veillonella dispar significantly decreased in NEC stage Ⅲand NECⅡ stage(P=0.003;P=0.032).Conclusion:(1)VLBWI who developed NEC,intended to have high incidence of sepsis,infectious pneumonia,respiratory failure,PNAC,with long antibiotic use time,use of two or more special grade antibiotics,blood transfusion>3 times,invasive ventilator,CPAP and other treatments.The overall enteral nutrition time and hospitalization time were extended resulting in more hospital expense.(2)The colonization pattern of the microbiome show that it had a transient decline in Firmicutes of the NEC and VLBW control groups.It hit the lowest on the 14th day after birth in the NEC group and 7 days after birth in the VLBW control group.(3)The diversity of intestinal microbiome in NEC group was significantly lower than that in VLBW control group on the 21st day after birth.There was no significant decrease in the diversity of intestinal flora between different Bell stages.(4)From the fourth day after birth,premature infants of NEC cases had different flora compared with the control group in an age related flora changes pattern.(5)Floras were different between different Bell stages.The increase of Coriobacteriia and Coriobacteriaceae is related to severe NEC.