Changes In The Gastrointestinal Microbiota Of Children With Acute Lymphoblastic Leukemia And Its Association With Antibiotics In Short Terms

BackgroundAcute lymphoblastic leukemia(ALL)is the most common cancer and leukemia in children.But the etiology and pathogenesis of ALL are still unclear,which may be related to the disorder of immune microenvironment.As an important component of immune system,digestive tract is inhabited by plenty of bacteria.The development of gastrointestinal(GI)immune relies on the interaction with its bacteria to some extent.The alteration of GI bacteria may cause the variation of the healthy status of host;meanwhile,the disorder of immune or metabolism caused by disease can alter GI bacteria.So it can be speculated that ALL may have an intimate relationship with GI microbiota.Objective1.To detect the alteration of gut bacteria of children with ALL and search biomarker to diagnose ALL.Provide a laboratory basis for further research on microbes and mucosal immunity at cellular and molecular levels.2.Analysis the impact of short-term-use of antibiotics to the effect caused by ALL.3.Find out the relationship between GI microbiota and effect of chemotherapy,as well as infectious complications.Methods1.Subjects’ recruitment and sample collection.Collect 30 fecal samples from new-diagnosed children with ALL and 33 samples from healthy children.According to the medication history of antibiotics(ATBx)for three months prior to this study,we divided them into four groups entitled ALL+ATBx,ALL,CON+ATBx and CON.2.DNA extraction and sequencing.After extracting genomic DNA,PCR and product purification,high throughput 16S rRNA sequencing was performed based on Illumina platform.3.Statistical analysis based on bioinformatics analysis.①Alpha diversity was utilized to describe richness and species diversity of the samples.②Beta diversity was applied to evaluate sample differences in species complexity between samples.③ Compositional changes were described by the difference in abundance of difference bacteria at different taxonomy hierarchy.④The microbial features differentiating the fecal microbiota were characterized by the linear discriminant analysis effect size method(LEfSe)to detect bacteria with significant difference according to LDA score and find microbiological markers.4.Follow-up and retrospective analysis.Explore the relationship between GI microbiota and effect of chemotherapy,as well as infectious complications,by follow-up visit and retrospective study.Results1.In a diversity:The comparison of indices indicating species diversity showed:Shannon index(ALL)<Shannon index(CON),P<0.05;Shannon index(CON+ATBx)<Shannon index(CON),P<0.05;Shannon index(ALL+ATBx)<Shannon index(CON),P<0.01;Simpson index(ALL)>Simpson index(CON),P<0.05;Simpson index(CON+ATBx)>Simpson index(CON),P<0.05;Simpson index(ALL+ATBx)>Simpson index(CON),P<0.01;There were no significant difference between each two groups in Chao 1 index and Ace index which indicate richness.2.In β diversity:ALL and CON were separated clearly into different clusters in the graphs gotten by PCoa and NMDS,so as ALL+ATBx and CON+ATBx.3.In the composition of bacteria:There were differences among the 4 groups at different taxonomy hierarchy.The Firmicutes/Bacteroidetes ratio(F/B)were different as F/B(CON+ATBx)<F/B(CON),(P<0.05).4.The number of bacteria with obvious difference between groups was higher in the paired groups ALL and CON than ALL+ATBx and CON+ATBx.The comparison between ALL group and CON group showed that the area under the ROC(AUC)of Bacteroidales and Enterococcaceae were 0.735 and 0.724 respectively.5.There were no significant differences of Shannon indices of GI microbiota between patients with minimal residual disease(MRD)≥ 1%in the 19th day of chemotherapy and patients with MRD<1%,either patients with infectious complications and without;and the F/B manifested in same manner.Conclusion1.ALL can induce changes in the gut microbiota structure.2.The administration of antibiotics in short terms disturbs the changes induced by ALL in alpha diversity,and leads to changes in GI bacterial composition of children with ALL.3.With high LDA sore and AUC,Bacteroidales and Enterococcaceae are rich in children with ALL,which could be used as a microbial marker for diagnosing ALL on the condition that no medication history of antibiotics recently.4.There were no clear link between GI microbiota and effect of chemotherapy,nor infectious complications.