Analysis Of Tongue Coating Microecological Flora Of Children With Anorexia Spleen Failure Syndrome

Purpose:This paper discusses the microflora of tongue coating specific to children with Syndrome of Spleen Dysfunctional type Infantile Anorexia（IA）/Avoidant/Restrictive Food Intake Disorder（ARFID）by analyzing the microbiome of tongue coating in children with IA/ARFID and healthy children,providing an important basis for syndrome differentiation and treatment of this diseaseMaterial and method:From January 2019 to January 2020,22 children with“Spleen dysfunction type pediatric anorexia”（儿童厌食症脾运失健证 as TCM diagnosis）were enrolled from the pediatric outpatient department of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine（Test group=FED group;According to the thickness of tongue coating,subgroup analysis was also carried out:15 children in FED1 thin white coating subgroup and 7 children in FED2 thick white coating subgroup）.Western medicine diagnosis was Infantile Anorexia（IA）and/or Avoidance/Restrictive Food Intake Disorder（ARFID）.17 healthy children of the same age group were selected as the control group（HC group）.Tongue scraping samples were collected from the two groups according to the Standard Tongue Coating Sample Collection Method,and then the microbial composition in each group of samples were analyzed with 16S rDNA high-throughput sequencing technology（①Community composition analysis②α,β diversity analysis③Species difference and significance analysis）Results:1.Comparison of general data:There were 22 patients in the FED case group（15 children in FED1 thin white coating subgroup and 7 children in FED2 thick white coating subgroup）,including 14 boys and 8 girls,with an average age of 6.3±2.0 years and an average BMI of 14.1±1.3;17 children were in the HC control group（Thin white tongue coating）,including 12 boys and 5 girls,with an average age of 7.3±2.2 years and an average BMI of 15.7±1.9 None of the selected children had used antibiotics or active bacterial preparations within one month before the study.There was no significant difference between the two groups in gender and age（Pgender=0.65>0.05,Page=0.25>0.05）,while the difference in BMI between the two groups was significant（PBMI=0.005<0.01）2.Community composition analysis:Taxonomic analysis:8 phyla,13 classes,23 orders,43 families,67 genera,122 species and 203 OTUs were found in 39 samples of the two groups The bacterial types at family level making up most in proportion（in descending order）were In FED group,Streptococceae（22.18%）,Prevotellaceae（21.04%）,Veionellaceae（15.63%）,Neisseriaceae（8.0%）and Actinomycetes（7.67%）.In HC group,Streptococceae（27.54%）,Veionellaceae（12.20%）,Prevotellaceae（9.32%）,Neisseriaceae（9.24%）and Actinomycetes（7.95%）accounted for the most.At OTU level,there was only one OTU in FED group not existing in HC group:OTU230（an unclassified species in the genus Corynebacterium）.The bacterial composition in FED1 and FED2 subgroups were basically consistent with that of the main FED group.Compared with FED1 subgroup at the OTU level,FED2 lacked OTU22（f.Nocardiaceae g.Rhodococcus s.Rhodococcus erythropolis）and OTU117（f.Veillonellaceae g.Selenomonas 3 unclassified species）Summary:① Prevotellaceae had the biggest change in proportion ratio between FED and HC groups（9.32%vs.21.04%）;②OTU230 was present in FED group,while not in HC group;③The microbial composition of the two subgroups FED1 and FED2 was basically the same as that of the main FED group;④OTU22 and OTU117 were present in FED2 subgroup,while not in FED13.Diversity analysis:Analysis was carried out at OTU level.Rank abundance curve:most of the curves in the two main groups decreased smoothly,indicating that the species diversity of all the samples was relatively high.α diversity analysis:Among the 7 indices of Sob,Shannon,Simpson,Ace,Chao,Shannoneven and Simpsoneven that were analyzed,only Sob and Chao indices were statistically significant（P<0.05）,showing that there is significant difference in species richness but similar diversity among the two main groups.There was no statistical significance between FED1 and FED2 subgroups（P>0.05）.β diversity analysis:PCoA analysis（Bray-Curtis weighted distance method）results showed that the difference in microbial communities between the FED,HC two main groups was statistically significant（P=0.027,r=0.1022）,but the since the r value was not high（indicating that the separation between the two groups was low and significance of P value may not be very large）,NDMS analysis was carried out,the result of which was stress=0.163,showing that the analytic graph had interpretative significance.Again using Bray-Curtis weighted distance calculation method,Adonis significance analysis regarding the total variance and statistical significance of ANOSIM analysis showed that R2=0.082,PR（P=0.006）,indicating that the reliability of the test was high.Summary:At the OTU level,① Species diversity of FED and HC groups was relatively high,but there was no significance difference（P>0.05）②The species richness of tongue coating microflora in FED group was significantly higher than that in HC group（P<0.05）③ There was significant difference between the two groups in the composition of microbial community（P=0.027）④ The test reliability was high（R2=0.082,P=0.006）⑤There was no statistical significance between FED1 and FED2 subgroups in diversity analysis.4.Species difference between the two groups and its significance:At the family level,Welch’s t-test showed that Prevotellaceae showed the most significant difference between the two main groups（P=0.003）,and there were 8 other species with statistical significance.The most significant difference between the two subgroups was Streptococcus（P<0.05）,and there were three other species with statistical significance.LEfSe analysis:Compared to HC group,Bacteroidetes had significant dominance（log10>3,P<0.01）in FED group at the phylum level,and among its families,Prevotella had the most significant effect on the difference（log10=4.75,P=0.001）,while Microsporaceae,Fusobacteridae,and Phyromonaceae showed significant increase in HC group（log10>3,P<0.01）.There was no significant difference in abundance between the two subgroups（log10<3）.Metagenomic analysis:Results supported the results of Welch’s t-test,showing that the family Prevotellaceae and 8 others were of significance.At the genus level,Prevotella₇,Prevotella₆,Alloprevotella and others were of significance（P<0.05）Summary:① Microbial family showing the most significant difference in abundance ratio between FED and HC groups:Prevotellaceae（P=0.003）;②FED group:dominance was observed from phylum level（Bacteroides,log10>3,P<0.01）,among which Prevotellaceae（log10=4.75,P=0.001）was the most influential family.HC group:dominance was observed from family level（log10>3,P<0.01）③There was no significant difference between FED1 and FED2 subgroups in abundance（log10<3,P>0.05）④The results of macrogenomic analysis supported Welch’s t-test results.Prevotella₇,Prevotella 6 and Alloprevotella from the Prevotellaceae family were statistically significant（P<0.01）Conclusion:1.The mean BMI of children with ARFID or IA of spleen dysfunction type was significantly lower than that of healthy children and not much difference was found in bacterial diversity or abundance between the thin white tongue coating and thick white tongue coating subgroups2.Compared with the tongue coating of healthy children,the microflora in thin/thick white tongue coating samples of children with ARFID or IA showed higher species abundance Bacteroidetes dominated significantly at the phylum level and among several others,Prevotellaceae showed the greatest influence on the significance.No significant difference was found in bacterial diversity between the two groups.