The Tongue Coating Microbiome In Lung Cancer Patients As Revealed By Next-generation Sequencing

Objective:Tongue examination is a unique diagnostic method used in traditional Chinese medicine(TCM).The tongue diagnosis analysis of the obeservation of the tongue color and the thickness of tongue coating to determine human physiological fuction and pathological conditions.At present study,the generative mechanism of tongue coating is yet unclarity,but the tongue microbial is an important reason for the formation of tongue coating.Microbial researches of traditional molecular biology methods such as Real-Time PCR,Denaturing Gradient Gel Electrophoresis(DGGE)not only heavy and time-consuming,and the detected tongue coating microbial abundance is limited.Next-generation sequencing(NGS)technology is the most advanced detection method currently used for the analysis of complex microbial community structures.Illumina Miseq sequencing technology,remedied the defects of traditional molecular biological technology,through comprehensive analysis of the tongue of microbial abundance and diversity to meet the comprehensive understanding of tongue coating bacteria.Lung cancer is the most commonly diagnosed cancer.Illumina Miseq sequencing technology was used to explore the characteristics of the tongue coating microbial in patients with lung cancer,further reveal the formation of tongue coating principle,promote the objectification and standardization of TCM tongue diagnosis.Methods:A total of 41 patients with lung cancer based on pathological diagnosis were recruited from the First Affiliated Hospital of Wannan Medical College.Additionally,43 healthy controls without respiratory disease who were matched to the lung cancer cohort according to gender and age were recruited.This study was approved by the Medical Ethical Committee of the First Affiliated Hospital of Wannan Medical College,China.All patients and healthy volunteers signed an informed consent form.The DS01-B tongue manifestation acquisition instrument was used to photograph the tongue coatings of patients and healthy volunteers and to objectively determine the thickness of the tongue coating.All samples were collected from the participants after gargling before breakfast.Using a sterile swab,the tongue was repeatedly scraped 30 times to collect the tongue microbes.Bacterial cells in the swabs were dislodged via vigorous stirring in 2 ml of PBS.Finally,the samples of 14 patients with lung cancer and 7 healthy controls were used;the remaining samples were withdrawn from this study due to non-standard operating procedures.We performed NGS on the V2-V4 hypervariable regions of 16 S r DNA to analyze the overall structure of the microbiome in lung cancer patients with different tongue thickness and in healthy controls.Results:1.The common tongue coating types in 41 patients with lung cancer,21 cases were thin and whitish fur,10 cases were white and greasy fur,8cases were yellowish greasy fur,2cases were no coating.42 of the 43 case healthy people were thin and whitish fur,the other is thin and yellowish fur.The thickness of tongue coating in patients with lung cancer were significantly higher than those of healthy people(314.86 + 222.47 vs.80 + 75.84,P < 0.01).2.To obtain accurate results from NGS analysis,27 of the 41 lung cancer patients and 35 of the 43 healthy controls were withdrawn from the study due to non-standard operating procedures.Of the 14 included patients,8 patients were classified in the thick group,and the others were classified in the thin group.We characterized the diversity of the tongue-coating microbiome via targeted 16 S r RNA gene sequencing.In this study,we obtained 24,191 high-quality V2-V4 sequences per sample from508,003 sequences,which 99.99% of the sequencing length for 401-500 bp.3.The sequence coverage in each group was greater than 97%,indicating that the 16 S r DNA sequences identified in these groups represent the majority of bacteria present in the samples examined in this study.Diversity analysis showed that the thick group on microbial community richness(Ace 、 Chao)and diversity(Shannon)were lower than the other two groups.4.The relative abundance of microbial sequences according to specific phyla revealed that the microbiome of the lung cancer group(both the thick and thin groups)exhibited close similarity to that of the healthy control group.The microbiome of the lung cancer group was predominated by Firmicutes(43.6±17.8%),Proteobacteria(16.5±13.3%)and Bacteroidetes(19.9±12%).Another represented phylum was Actinobacteria(9.8±6%),and 0.34% of the OTUs were unclassified at the phylum level.Compared to the lung cancer group,the healthy control group microbiome exhibited a significantly higher abundance of Proteobacteria(30.5±9.8% vs.16.5±13.3%,P=0.030,Mann-Whitney U test),but the abundance of other phyla was lower in the microbiome of the healthy control group than in that of the lung cancer group.The relative abundance of the tongue coating microbiome was notably different at the genus level as a percentage of reads assigned between the groups.There were132,129 and 123 genera in the microbiome of the thick,thin and healthy control groups,respectively.As shown in Figure 4,the dominant genera in all groups were Prevotella,Streptococcus,Veillonella,Neisseria,and Lactococcus.Prevotella,one of the most abundant genera in all three groups,was more abundant in the microbiome of the thin group than in that of the thick and healthy control groups.The abundance of Streptococcus,Veillonella,Lactococcus,Leptotrichia,Actinomyces,Pseudomonas and Lactocacillus were significantly higher in the thick group than in the thin and healthy control group.In contrast,Neisseria,Haemophilus,Granulicatella and Fusobaterium were more abundant in the healthy control group than in the other groups.Conclusion:Screening the tongue coating microbiome is a simple and noninvasive diagnostic method that may be useful in the early diagnosis of lung cancer.