Analysis of by high-throughput sequencing: Helicobacter pylori infection and salivary microbiome

Yingjie Ji, Xiao Liang, Hong Lu, Yingjie Ji, Xiao Liang, Hong Lu

Abstract

Background: There have been reports of Helicobacter pylori (H. pylori) in the oral cavity and it has been suggested that the oral cavity may be a reservoir for H. pylori reflux from the stomach. High-throughput sequencing was used to assess the structure and composition of oral microbiota communities in individuals with or without confirmed H. pylori infection.

Methods: Saliva samples were obtained from 34 H. pylori infected and 24 H. pylori uninfected subjects. Bacterial genomic DNA was extracted and examined by sequencing by amplification of the 16S rDNA V3-V4 hypervariable regions followed by bioinformatics analysis. Saliva sampling was repeated from 22 of the 34 H. pylori infected subjects 2 months after H. pylori eradication.

Results: High-quality sequences (2,812,659) clustered into 95,812 operational taxonomic units (OTUs; 97% identity). H. pylori was detected in the oral cavity in infected (12/34), uninfected (11/24) and eradicated (15/22) subjects by technique of high-throughput sequencing, occupying 0.0139% of the total sequences. Alpha diversity of H. pylori infected subjects was similar to that of uninfected subjects (Shannon: 1417.58 vs. 1393.60, p > 0.05, ACE: 1491.22 vs. 1465.97, p > 0.05, Chao 1: 1417.58 vs. 1393.60, p > 0.05, t-test). Eradication treatment decreased salivary bacterial diversity (Shannon, p = 0.015, ACE, p = 0.003, Chao 1, p = 0.002, t-test). Beta diversity analysis based on unweighted UniFrac distances showed that the salivary microbial community structure differed between H. pylori infected and uninfected subjects (PERMANOVAR, pseudo-F: 1.49, p = 0.033), as well as before and after H. pylori eradication (PERMANOVAR, pseudo-F: 3.34, p = 0.001). Using LEfSe analysis, 16 differentially abundant genera were defined between infected and uninfected subjects, 12 of which had a further alteration after successful eradication.

Conclusions: Our study using high-throughput sequencing showed that H. pylori was present commonly in the oral cavity with no clear relation to H. pylori infection of the stomach. Both H. pylori infection and eradication therapy caused alterations in community and structure of the oral microbiota.

Trial registration: clinicaltrials.gov, NCT03730766. Registered 2 Nov 2018 - Retrospectively registered, https://ichgcp.net/clinical-trials-registry/NCT03730766" title="See in ClinicalTrials.gov">NCT03730766.

Keywords: 16S rDNA; Helicobacter pylori; High-throughput sequencing; Salivary microbiota.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Distribution of the predominant bacteria at different taxonomic levels (phylum, class, order, family, and genus). The predominant taxa (> 1% relative abundance) in each level are shown. Red represent P group, green represent N group, and blue represent E group. N = uninfected group, P = infected group, E = eradicated group
Fig. 2
Fig. 2
Principal coordinate analysis (PCoA) of unweighted UniFrac analysis. a. PCoA analysis demonstrated that subjects of P group were significantly different from N group (PERMANOVAR, pseudo-F: 1.49, p = 0.033). N = uninfected group, P = infected group. b. PCoA analysis showed that the overall microbial composition showed significant difference between PE and E group (PERMANOVAR, pseudo-F: 3.34, p = 0.001). E = eradicated group, N = uninfected group, PE = pre-eradicated group
Fig. 3
Fig. 3
Comparison of microbial variations at the genus level, using the LEfSe online tool. a. Histogram of the LDA scores for differentially abundant features among groups. The threshold on the logarithmic LDA score for discriminative features was set to 2.0. N = uninfected group, P = infected group. b. Cladogram for taxonomic representation of significantly differences among groups. Differences are represented in the color of the most abundant taxa (red indicating P group, green indicating N group, and white indicating non-significant). N = uninfected group, P = infected group
Fig. 4
Fig. 4
H. pylori in oral cavity of three groups. Red represent P group, yellow represent N group, and blue represent E group. N = uninfected group, P = infected group, E = eradicated group

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