| Background&PurposeNonalcoholic fatty liver disease(NAFLD)usually refers to a variety of causes of abnormal fat accumulation in the liver,which has been beyond the viral hepatitis to become the world's largest liver disease.At present,the pathophysiology of nonalcoholic fatty liver disease is not clear,widely recognized as the "two strike doctrine",which includes the role of fat and systemic immunity.In recent years,with the development of intestinal flora,the researchers found that the two strike theory can not fully explain the pathogenesis of the disease.The role of intestinal microflora closely related to nonalcoholic fatty liver disease.Oral flora plays an important role in the composition of the digestive tract flora.Oral flora with saliva were swallowed into the digestive tract,some of the bacteria were killed by stomach acid and the remaining enter into intestinal tract.Li Lanjuan et al found that the about 54%intestinal microbiome of patients with cirrhosis were similar to oral microbiome,indicating that they were invading the gut from the mouth.Therefore,we speculate that there is a certain correlation between oral flora and NAFLD.Therefore,noninvasive method was used to collect saliva,and we detect oral flora diversity.The purpose of this study was to explore the relationship between NAFLD group and oral bacteria,find the bacterial markers of early diagnosis and treatment.MethodsSubject section:Saliva samples were collected from patients with 24 nonalcoholic fatty liver disease and 22 healthy control in Guang dong General Hospital(Guang dong Academy of Medical Sciences).Patients with nonalcoholic fatty liver disease are diagnosed according the guidelines for the diagnosis and treatment of nonalcoholic fatty liver disease published in 2010 by the Chinese Academy of Medical Sciences.Standardize the collection of sample collection.Bacterial DNA was extracted strictly in accordance with the guideline of the kit instructions.PCR amplification of the 16S rRNA gene V4-V5 region:The extracted DNA was detected by agar gel electrophoresis.All samples had obvious main bands,no pollution and no degradation.The amplified PCR was purified,and database was operated according to the NEBNext Ultra DNA Library Prep Kit for it Illumina standard procedures,finally then sequenced using Illumina Hiseq2500 PE250 sequencing platform.Data processing:According to the sequence of Barcode and PCR primer sequence,we split sample data from primary data.In the end,we can get Effective Tags.OTU clustering and analysis:We clustered all the samples of all valid data(Effective Tags)by Qiime software.The total reads were then sorted according to average quality value and grouped into operational taxonomic units(OTUs)using UCLUST with a sequence identity threshold of 97%.By selecting the representative sequences of OTUs,according to the principle of calculation,the sequences with the highest frequency in OTUs are selected as the representative sequences of OTUs.We analyzed all the data including operational taxonomic units,diversity indexes and Species annotation,etc.Statistical analysis:all data were processed by SPSS20.0 software,P<0.05 was statistically significant.Age and the relative abundance of bacteria difference between groups was tested by Mann Whitney U test.Gender and ethnic differences was tested by chi square test,and OTUs number and alpha diversity index was tested two independent sample t test.LEfSe analysis was performed using Kruskal-Wallis and Wilcoxon rank sum test.ResultThis study included 24 cases of patients with nonalcoholic fatty liver disease,22 cases of healthy control group.nonalcoholic fatty liver disease group the average age is 42.5±9.5,the healthy control group 39.9±12.9.groups in age,gender and ethnicity had no difference(P>0.05).Saliva samples were sequenced on Illumina Hiseq2500 PE250 sequencing platform and 1799438 sequences were obtained.After processing and quality control,we get 39118 optimized sequences in the saliva of each group,including NAFLD group was 38152,and the healthy control group was 40172.Nearly 21797 tages were used to clustering OTUs,in the end,we obtained clustering OTUs about 308±48 innonalcoholic fatty liver group and clustering OTUs about 305±53 in healthy control group.Alpha diversity analysis:the results showed that there was no significant difference between the Chao1 and Ace values of the NAFLD group and the HC group,indicating that the overall content of salivary bacteria in NAFLD patients did not change significantly.Shannon index,Simpson index and PD_whole_tree value were not statistically significant,indicating that the diversity of the two groups were significantly different.The saliva flora of saliva samples of two groups were analyzed in the phylum,class,order,family and genera.The composition of the microbiome was little difference.We obtained 375 genera,247 families,161 orders and 87 classes,which belong to 37 phylum.Located in the top 10 species in phylum:Bacteroidetes?Proteobacteria?Firmicutes?Fusobacteria?SR1?Spirochaete?Actinobacteria?Tenericutes?GN02?Acidobacteria.In the top 8 in class were Bacteroidia?Clostridia?Betaproteobacteria?Gammaproteobacteria?Fusobacteriia?Flavobacteriia?Bacilli?Spirochaetes.The main eight kinds of bacteria in order were Bacteroidales?Clostridiales?Neisseriales?Pasteurellales?Fusobacteriales?Flavobacteriales?Lactobacillales?Spirochaetales.The dominant families were Prevotellaceae?Veillonellaceae?Neisseriaceae?Pasteurellaceae?Pasteurellaceae?Porphyromonaclaceae?[Paraprevotellaceae]?Flavobacteriaceae?Strptococcaceae?Spirochaetaceae.In genus most of the 10 species bacteria were Prevotella?Neisseria?Veillonella?Haemophilus?Fusobacterium?Porphyromonas?[Prevotella]?Capnocytophaga?Aggregatibacter?Streptococcus.Beta diversity analysis:Nonalcoholic fatty group and healthy control group of saliva flora in bacteria community PCoA analysis showed the close clustering(PC113.05%and PC27.62%),indicated that they were difference.Saliva samples of NAFLD group and normal control group were analyzed by LEfSe,there were significant differences between the 21 species:Prevotella?Provotellaceae?melaninogenica?Gallionella?Gallionellales?Gallionellaceae?solibacteraceae?Candidatus Solibacter?Bacilli?Lactobacillales?Streptococcaceae?Streptococcus?Gemellales?Carnobacteriaceae?Granalicatella?ochracea?planctomycetia?Corynebacterium?Corynebacteriaceae?BSV43.The first 8 species were enriched in HC,and the latter 13 were enriched in NAFLD group.ConclusionThe composition of saliva samples in patients with nonalcoholic fatty liver disease and normal control group was basically the same as that in the phylum,the class,the order,the family and the genus.But the relative abundance of bacteria showed differences,which Bacteroidetes(Prevotella spp.,Poulet Was melaninogenica)was a relatively high abundance in healthy control group and Firmicutes of nonalcoholic fatty liver group(bacilli,lactobacillales,Streptococcus,Streptococcus)is the high relative abundance.The salivary microbiome composition of nonalcoholic fatty liver disease is different from healthy control.In the NAFLD group,the abundance of Firmicutes was increased,and the abundance of the bacteria was decreased.It was speculated that the imbalance of the salivary bacteria is associated with nonalcoholic fatty liver disease. |
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