Metagenomic Studies Of The Oral And Gastric Microbiomes In Gastric Adenocarcinoma Patients

Gastric cancer is the third leading cause of cancer-related death worldwide,with an estimated 1 million new cases a year.Helicobacter pylori?H.pylori?infection is the most important risk factor for gastric cancer.Microbiome studies using high-throughput sequencing and bioinformatics tools have established that gastric microbiota other than H.pylori may constitute a complex ecosystem in human stomach and be closely associated with gastric diseases,including gastric cancer.Some studies have demonstrated that a dysbiotic gastric microbiota were closely associated with gastric cancer,and some specific microbes were promising to be potential biomarkers for gastric cancer.Interestingly,several studies found that bacteria usually colonizing in oral cavity were more abundant in the stomach of gastric cancer patients than patients with superficial gastritis,but few researches have studied the microbiota of oral cavity in gastric cancer directly.However,these studies were based on 16 S rRNA marker gene analysis,which,although contributing remarkably to our understanding about microbiota in gastric carcinoma,have also limited our ability to characterize other key microbial genes of the community in this specific niche,and the function of microbial community can only be predicted through this method.Therefore,we performed shotgun metagenomics survey on the gastric and oral microbial communities of advanced gastric adenocarcinoma?GC?cases and chronic superficial gastritis?SG?controls to characterize the compositional and functional changes of microbiota associated with gastric malignancy.Six individuals with GC and 5 with SG were recruited with strict exclusion and inclusion criteria.In this study,gastric wash samples and mouth wash samples collected from all the subjects were processed to prepare sequencing libraries and conduct high-throughput sequencing.The raw sequencing data were then trimmed and filtered with strict quality control methods,and finally we obtained microbial DNA sequences with high quality.The processed reads were further used to characterize the taxonomic profiles and functional capacity by MetaPhlan2 and HUMAnN2 respectively,generating the datasets of taxonomic composition and metabolic pathways of each sample.We then performed the Alpha and Beta diversity analyses of the microbial communities.LEfSe was further used to conduct comparative analyses on the composition and function of microbiota to discover microbial biomarkers of GC.Major research results of this study were as follows:1.We obtained high-quality microbial DNA sequences data after quality control.Taxonomic profiles through MetaPhlAn2 showed that the majority of mapped reads corresponded to bacteria?99.46±0.66%,mean±S.D.?and a minimal proportion corresponded to viruses.Totally,204 and 190 microbial taxa were identified in the oral cavity and stomach of subjects respectively.Functional profiles through HUMAnN2 showed that more than 99% identified pathways corresponded to bacteria.301 and 349 microbial pathways were identified in the oral cavity and stomach of subjects respectively.2.The microbial Alpha diversity analyses showed that: Species richness?Observed species?was highly dependent on clinical diagnoses with higher richness values for SG compared to GC in both oral and gastric samples.While Shannon Index,was significantly different between groups only in oral samples.Suggesting that the diversity of bacteria in SG group was higher than that of GC group.3.Beta diversity analyses showed that: The overall differences of microbial communities between GC and SG evaluated by Jaccard index were significant in both oral and gastric samples.While the differences evaluated by Bray-Curtis dissimilarity were only significant in oral samples.These statistics showed that the microbial structures were significantly different between GC and SG groups.4.Comparative analyses of microbial communities and biomarker discovery: LEfSe analysis on the relative abundances of all bacterial taxa generated by MetaPhlan2 showed that totally 44 bacterial taxa were identified to be differentially abundant between groups in gastric samples,including 8 families,12 genera,18 species and 6 strains.We further screened out the most representative taxa which were differentially abundant between clinical diagnoses based on their relative abundance and prevalence.We finally obtained 5 GC-enriched taxa?Neisseria,Alloprevotella,Aggregatibacter,Porphyromonas_endodontalis.t_GCF₀00174815,Streptococcus_mitis_oralis_pneumoniae?and one SG-enriched taxon?Sphingobium_yanoikuyae?.13 bacterial taxa were identified to be differentially abundant between GC and SG in oral samples.Notably,three most representative taxa,including Neisseria,Alloprevotella and Porphyromonas_endodontalis.t_GCF₀00174815 were enriched both in oral and gastric samples of GC patients.5.Comparative analyses of microbial functional capabilities: PCoA analysis fund that the microbial metabolic pathways were significantly different between groups both in oral and gastric samples.LEfSe analysis identified 34 microbial metabolic pathways which were differently abundant between groups in gastric samples,and 55 metabolic pathways were identified to be differentially abundant in oral samples.Generally,these pathways corresponded to the metabolic of sugar,amino acid,lipid and nucleotide.Specifically,pathways corresponded to lipopolysaccharide biosynthesis were more abundant in GC,while pathways corresponded to the yielding of short chain fatty acids were more abundant in SG.In conclusion,we conducted shotgun metagenomics survey on the gastric and oral mi crobiome of patients with GC and SG for the first time,and newly identified some GC-related bacteria and functional gene pathways that are closely associated with GC,which extended the current knowledge about the role of digestive tract microbiota in GC,providing new perspective and information in discovering microbial targets for GC.