Study On The Relationship Between Oral And Esophageal Microbial Characteristics Of The Occurrence And Development Of Esophageal Squamous Cell Carcinoma

ObjectivesBased on the people in the high incidence area of esophageal squamous cell carcinoma in China,this study explored the effects of smoking and drinking on the microbiota in the saliva and three segments of the esophagus（upper,middle,and lower）in healthy individuals.At the same time,the paired saliva and esophageal samples of normal people,patients with low-grade dysplasia,high-grade dysplasia and esophageal squamous cell carcinoma were collected to compare the differences in the composition of oral and esophageal microbiota among groups,screened out the biomarkers in each group,and explored the relationship of microecological changes among groups.This study provided new microbial evidence for the etiological exploration,screening and treatment of esophageal cancer.Materials and methods1.The effects of smoking and drinking on the oral and esophageal microbiota of healthy people:Saliva and cell brush samples of upper,middle and lower esophagus were taken from 76 healthy people who participated in the upper gastrointestinal cancer screening project at the high incidence site of esophageal cancer in Feicheng,Shandong Province.The samples were sequenced based on Ion S5TM XL platform,and biologically analyzed by 16S ribosomal RNA（16S rRNA）sequencing technology and QIIME2（Quantitative insights into microbial ecology）software,and the differences of microbiological composition of saliva,upper,middle and lower esophagus between healthy non-smoking and non-drinking people and healthy smoking and drinking people were compared.Linear discriminant analysis effect size（LEfSe）was used to find the specific bacteria in every group.2.Characterization of the oral and esophageal microbiota in esophageal precancerous lesions and squamous cell carcinoma:Saliva and esophageal cell brush paired samples were collected from 276 patients with normal,low-grade dysplasia,high-grade dysplasia and esophageal squamous cell carcinoma in Cancer Hospital of Chinese Academy of Medical Sciences,Linzhou Cancer Hospital of Henan Province,Cixian Cancer Hospital of Hebei Province and Feicheng People’s Hospital of Shandong Province.Based on Ion S5TMXL platform,16S rRNA sequencing technology and QIIME2 software were used to compare the microbial composition of saliva and esophageal cell brush samples in different pathological stages of esophageal cancer.LEfSe and random forest were used to screen out specific microorganisms（genus level）in every group,and random forest modeling was used to evaluate the diagnostic efficacy of specific bacteria in disease groups.PICRUSt2 was used for functional prediction.The differences of species with the same pathological degree in saliva and cell brush samples at the genus level were analyzed by DESeq2.Results1.For the non-smoking and non-drinking group,there were 13 specific bacteria in the saliva,such as Prevotella,Neisseria and Porphyromonas.In the upper esophageal specimens,there were four specific bacteria:Streptococcus,Haemophilus,Gemella and Butyrivibrio.In the middle esophageal specimens,there were 6 specific bacteria,such as Bacteroides,Johnsonella and Acidovorax.In the lower esophageal specimens,there were 18 species of specific bacteria,such as Lactobacillus,Arthrospira and Bosea.2.For the smoking and drinking group,there were 15 specific microorganisms in saliva,such as Prevotella,Neisseria and Porphyromonas.In the upper esophageal specimens,there was one specific microorganism（Streptococcus）.In the middle esophageal specimens,there were 35 specific microorganisms such as Bosea,Caulobacter and Cupriavidus.Two specific microorganisms,Haemophilus and Gemella,were found in the lower esophagus.3.Compared with the non-smoking and non-drinking group,the saliva samples of the smoking and drinking group had 15 characteristic microorganisms such as Prevotella,Porphyromonas and Fusobacterium.There were 14 characteristic microorganisms in the upper esophagus,including Streptococcus,Alloprevotella and Gemella.There were 32 characteristic microorganisms in the middle part of esophagus,such as Bosea,Ca ulobacter and Mycoplasma.There are 15 characteristic microorganisms in the lower esophagus,such as Rothia,Streptococcus and Gemella.4.For saliva samples,at the genus level,the specific microorganisms of esophageal squamous cell carcinoma were Peptophilus,Peptostreptococcus,Bosea,Lachnospiraceae_[G-9],Gemella,Solobaterium and Streptococcus.The specific microorganisms of high-grade dysplasia of esophagus were Bacillus,Peptiphileae_[G-3],Bordetella,Peptophilus,Parvimonas,Agrobacterium,Gemella,Lactobacillus,Granulicatella,Lachnospiraceae_[G-9]and Streptococcus.The specific microorganisms of low-grade dysplasia of esophagus were Enterococcus,Lachnoanerobaculum,Atobium,Veillonella,Parvimonas,Gemella,Solobaterium,Actinomyces,Leptotrichia,and Saccharibacteria_TM7[G-7],Schaalia,Granulicatella,Rothia and Streptococcus.Streptococcus was screened by LEfSe and random forest.It belongs to the common specific bacteria of low-grade dysplasia,high-grade dysplasia and ESCC patients.5.For cell brush specimens,at the genus level,the specific microorganisms of esophageal squamous cell carcinoma were Parvimonas,Centipede,Helicobacter,Peptostreptococcus,Veillonella and Saccharibacteria_TM7[G-1],Schaalia,Solobacterium,Gemella,Klebsiella,Leptotrichia,Acinetobacter,Absconditabacteria_SR1[G-1],Rothia,Fusobacterium and Bosea.The specific microorganisms of high-grade dysplasia were Lachnospiraceae_[G-2],Lactobacillus and Bosea.The specific microorganisms of lowgrade dysplasia were Schaalia,Leptotrichia,Helicobacte,Fusobacterium,Granulicatella,Gemella,Rothia and Streptococcus.Among them,Bosea was screened by LEfSe and random forest.It belongs to the common specific bacteria of low-grade dysplasia,highgrade dysplasia and ESCC patients.6.The area under the curve（AUC）of the top respectively bacteria in saliva and cell brush samples used to distinguish low-grade dysplasia,high-grade dysplasia,ESCC patients and normal people were 91.16%,80.28%and 87.16%;91.2%,73.97%and 89.13%respectively.7.PICRUSt2 results showed that,in the cell brush sample,the gene abundance encoding nitrite reductase decreased significantly in patients with high-grade dysplasia and ESCC.In saliva samples,the gene abundance of nitrite reductase in high-grade dysplasia and ESCC group decreased significantly,and the gene abundance of nitrate reductase in ESCC group increased significantly（P<0.05）.Conclusions1.Smoking and drinking will change the composition of microbiota in the oral cavity and upper,middle and lower parts of esophagus,resulting in an increase in the abundance of bacteria related to periodontal disease,dental caries and oral cancer（Porphyromonas gingivalis,Streptococcus,Prevotella,etc.）.However,the characteristic microorganisms in the upper,middle and lower esophagus of smokers and drinkers are not the specific bacteria of Barrett’s esophagus,esophageal adenocarcinoma and ESCC found in previous studies.2.There were significant differences in the composition of microbiota in saliva and lesion surface among normal people,low-grade dysplasia of esophagus,high-grade dysplasia and ESCC patients.Streptococcus was screened by LEfSe and random forest.It belongs to the common specific bacteria in the saliva of patients with low-grade dysplasia,high-grade dysplasia and ESCC.Bosea is the common specific bacterium on the esophageal surface of the above patients.3.The microbial characteristic bacteria in the saliva and on the lesion surface of esophageal low-grade dysplasia,high-grade dysplasia and ESCC patients have good diagnostic efficacy to distinguish between normal people and patients,and can be used as clinical diagnostic markers for ESCC screening,early diagnosis and early treatment.Functional prediction analysis showed that the ability of producing nitrite in the oral cavity of ESCC patients increased,and the function of metabolizing nitrite in the oral cavity and esophagus decreased.The future research on the interaction between specific bacteria and host in different esophageal disease states found in this study will contribute to the etiology research,disease diagnosis and treatment of ESCC in the future,as well as the development of noninvasive early cancer screening products.