Study On Characterization Of Gut Microbiota In Children With Cyanotic Congenital Heart Disease

Aims and content:Patients with cyanotic congenital heart disease(CCHD)are in a chronic persistent hypoxia(CCH)state for a long time.The main complications include poor growth and necrotizing enterocolitis(NEC),which have severe effects on the growth and development of the body.At present,there is a lack of relevant research on the changes of gut microbiota in the human body under the condition of long-term hypoxia.Therefore,we took patients with CCHD as the research object,the changes of gut microbiota in children with CCHD in different age groups were systematically analyzed in order to find out the adaptive changes of gut microorganisms when the body is in a long-term hypoxic state and screen bacteria related to hypoxia adaptation.NEC is a common gastrointestinal emergency in premature infants and full-term infants with CCHD,the pathological manifestations are separation and destruction of the mucin layer and lamina propria,which results in translocation of intestinal microbial products(such as lipopolysaccharide),triggering local and systemic inflammation.Previous reviews have detailed the risk factors of NEC and gut microbiota dysbiosis in premature infants,and there are few studies characterize the gut microbiota of the term infants with CCHD.In this study,healthy newborns and CCHD newborns were studied,and the differences of the gut microbiota composition between the two groups were analyzed and compared,hoping to find suitable strains for clinical intervention and treatment.In addition,gut microbiota dysbiosis induced by antibiotic treatment has been a clinical concern.Therefore,we observed the dynamic changes of gut microbiota in a pair of newborn twins with CCHD during the surgery and antibiotic treatment,and explored how the drug resistance of Enterobacter cloacae strains isolated at different treatment time points changed during treatment,so as to provide reference for the clinical application of antibiotics and the dynamic changes of gut microbiota during neonatal hospitalization.Methods:15 children with CCHD of different ages were included and divided into newborns,1month ~ 6 months old,6 months ~3 years old and over 3 years old groups according to their age,numbered N1~N4.Meanwhile,19 healthy newborns were included.Fresh fecal samples of these patients and healthy newborns were cultured and identified by culturomics.Three mediums were used to increase the detection rate of bacteria.The composition and changes of gut microbiota were analyzed by 16 S r DNA sequencing.The drug resistance of Enterobacter cloacae isolated at different treatment timepoints was detected by in vitro drug sensitivity test.Results:The culturomics results showed that Enterococcus was common in all CCHD patients,and the isolation rates of Clostridium,Escherichia,Peptoniphilus and Klebsiella were more than 50%.There were more common parts of N3 and N4 groups at species level and genus level.The sequencing results showed that there was significant difference in Shannon index between group N1:N3,and no significant difference in β diversity among children with CCHD in different age groups.Besides,there was significant difference in Enterococcus between group N1: N4 and group N2: N4,and in Clostridium_Xl Va between group N2: N4.As patients get older,the average relative abundance of Megasphaera gradually increased,Enterococcus and Citrobacter gradually decreased.There were individual differences of the gut microbiota in healthy neonates,the bacterial species could be isolated were limited,and the isolation rates of Enterococcus,Enterobacter,Staphylococcus,Streptococcus,Klebsiella and Raoultella were not less than 50%.There was no significant difference in α diversity and β diversity between healthy neonates and CCHD neonates,but the relative abundance could be more than 100 times different between the two groups in Veillonella,Gemmiger,Dialister and other genera.The average relative abundance of Enterococcus in CCHD neonates was higher,and that of Bifidobacterium in healthy neonates was higher.Both culturomics and sequencing results showed that the composition and abundance of bacteria in fecal samples of the twins changed significantly at different treatment timepoints.Culturomics results suggested that the richness of fecal samples has decreased before the surgery due to the use of antibiotics.After long-term hospital environmental exposure,multiple opportunistic pathogenic bacteria were detected.After 4 months of breastfeeding,the bacterial composition of fecal samples changed significantly,and probiotics such as Lactobacillus and Bifidobacterium were isolated.16 S r DNA sequencing results showed that the Shannon indexes and the genus with highest relative abundance were different at different treatment timepoints under the influence of various factors.The drug sensitivity test showed that Enterobacter cloacae gradually resistant to piperacillin during treatment.Conclusions:1.Under the survival stress caused by long-term chronic hypoxia stimulation,some gut microbiota gradually adapted and abundance increased,while some bacteria were opposite.The relative abundance of Megasphaera in CCHD children gradually increased with age,while Enterococcus and Citrobacter gradually decreased.It is speculated that Megasphaera is more dominant under long-term hypoxia stimulation,while Enterococcus and Citrobacter are gradually at a disadvantage.2.The richness of gut microbiota in healthy newborns was higher than that in CCHD newborns at genus level,and the relative abundance of some genera was different more than100 times between two groups,suggesting that the colonization of some genera in CCHD newborns may be delayed.In addition,additional supplementation of Bifidobacterium to CCHD neonates may alleviate gut microbiota dysbiosis.3.Antibiotics,feeding patterns,external environment exposure and many other factors could affect the composition of the gut microbiota in newborns.Adhere to breastfeeding can restore the disturbance caused by antibiotics treatment to a certain extent,and make the composition of gut microbiota healthier.4.The long-term use of antibiotics will significantly affect the composition and the diversity of the gut microbiota in hospitalized patients,and lead to gradual resistance of opportunistic pathogenic bacteria during treatment.