Background
H. pylori is a Gram-negative bacterium that colonizes the human gastric epithelium.
H. pylori infection is characterized by mucosal inflammation (gastritis) and may result in peptic ulcer disease or gastric adenocarcinoma [
1].
H. pylori is transmitted between humans by a variety of routes including gastro-oral and fecal-oral mechanisms that include contaminated water and food. It has also been postulated that oral cavity may play a role in
H. pylori transmission and possibly act as a reservoir [
2]. For example,
H. pylori has been detected in the oral cavity using the polymerase chain reaction (PCR).
H. pylori has also been successfully cultured from saliva from individuals with positive results of both saliva
H. pylori antigen test and
H. pylori flagella test [
3].
The oral cavity is one of the most complex and largest microbial habitats that harbors hundreds of different bacteria which play important roles in maintaining oral homeostasis and influencing the development of both oral and systematic diseases [
4]. Many factors in the oral environment including intraoral pH and salivary iron concentration as well as expression of serum malondialdehyde and lipid profile have been reported to have significant relationships with oral microbial communities [
5,
6]. Several recent studies detected the efficacy of antibiotics or drug composed of herbal on oral microbiol communities [
7,
8]. However, there was few reports about
H. pylori and its relationship with the microbial community structure in human saliva.
Currently, most oral bacteria species cannot be easily cultivated in vitro using traditional cultivation methods requiring the use of molecular biological techniques, such as checkerboard hybridization, microarray chips, and the quantitative real-time PCR to identify and classify the currently uncultivable bacteria [
9]. However, many low-abundance bacteria species still cannot be detected using these approaches which impede the comprehensive and in-depth understanding of oral bacteria diversity. In this study, we used amplicon sequencing of 16S rDNA V3-V4 hypervariable regions to define the bacterial composition, abundance, and structure of salivary microbiome in people with and without active
H. pylori infections. In addition, we also characterized the salivary biodiversity of a subgroup of subjects before and after the
H. pylori eradication. As
H. pylori passes through saliva, it is important to detect its expression in oral cavity as well as the efficacy of its infection and eradication on the oral microbiol communities.
Discussion
In this study, using the technology of high-throughput sequencing, we found H. pylori exit in the oral cavity of infected (12/34), uninfected (11/24) and successful eradicated (15/22) subjects, composing 0.0139% of the total sequences.
A few studies have shown that
H. pylori can be spread by oral-oral (or fecal-oral) way [
2]. Feeding, kissing, and tableware shared play the important role in the transmission. Previous studies have detected the presence of
H. pylori in saliva [
3]. Bacteria in plaque adhere to the gums, which are relatively fixed. It is more reasonable to study the saliva instead of dental plaque in this situation. A comprehensive and thorough investigation of the bacterial diversity of salivary microbiota is essential for understanding the how or whether
H. pylori infection alters the salivary microbiota. The technology of high-throughput sequencing has provided new cognizance of the structures and compositions of microbiota communities.
By comparing the alpha diversity indexes we found that the bacterial diversity in saliva was similar among the
H. pylori uninfected and
H. pylori infected people. Our study is consistent with the notion that
H. pylori in the stomach has little or no effect on the bacterial diversity of the oral cavity [
20]. The Shannon diversity index, ACE richness index, and Chao 1 richness estimator all declined after eradication of
H. pylori compared to the baseline samples (
p < 0.05), which was consistent with the prior studies that use of PPI and antibiotics may affect the oral microbiome [
21,
22].
According to the beta diversity analysis based on the unweighted UniFrac distances, the community structures of saliva microbiota were different in
H. pylori uninfected and infected individuals, which was contrary to the results of Schulz’s study [
20]. Samples from the
H. pylori infected subjects tended to cluster together, while the microbiota in the uninfected subjects appeared to be more variable suggesting that gastric
H. pylori infection may affect oral bacterial components. Clear segregations by the PCoA and NMDS analysis among individuals before and after
H. pylori eradication therapy demonstrated that successful eradication or eradication therapy changed the oral bacterial components to some extent.
In addition to the presence of different bacterial members, the abundance of some bacteria also differed significantly among groups. We clearly observed that some bacteria in the saliva of
H. pylori infected individuals showed a significantly reduced abundance, among which
Aggregatibacter,
Klebsiella,
Fusobacterium, and
Parvimonas species were pathogenic bacteria, causing infective endocarditis, liver abscess, pneumonia, meningitis, systemic infections, et al. [
23‐
25]. However, the abundance of other bacteria significantly increased in saliva of
H. pylori infected individuals, most of which were oral microbiota composition, including
Sphingomonas,
Ochrobactrum,
Afipia,
Leptotrichia,
Oribacterium, and
Moryell, except
Acinetobacter species causing infectious diseases like pneumonia and urinary tract infections [
26], and
Leptotrichia species, a potential cariogenic bacteria [
27]. While in Schulz’s study, no significant difference in oral communities between
H. pylori infected and uninfected individuals was detected at genus level [
21], this may be due to the different target sequencing region of 16 s rDNA, sample size, or geographic location. Interestingly, most
H. pylori-enriched genera increased after the eradication, including
Ralstonia,
Leptotrichia,
Sphingomonas,
Leptothrix,
Oribacterium, and
Acinetobacter. The exception was
Ochrobactrum. However, genera low expressed in
H. pylori infected saliva experienced a further decline after
H. pylori eradication therapy, including
Alloprevotella,
Aggregatibacter,
Leptotrichlaceae__G_1_,
Parvimonas, and
Fusobacterium. Our study suggests that
H. pylori infection may change the salivary microbiota, eradication therapy would further change the bacteria composition of saliva microbiota. Although the clinical significance of these alterations was not known,
H. pylori unexpectedly and clearly altered the oral microbiota composition.
In our study, we found that
H. pylori infection didn’t change the abundance and diversity, but changed the composition structure of salivary microbiome.
H. pylori is colonized in the stomach, and there is a certain distance from stomach to the oral cavity. Even though
H. pylori can cause a large change in the abundance of the microbiota in the stomach, it is difficult to affect the abundance of the salivary microbial flora. Previous studies have reported acid inhibition in upper gastric tract may have an effect on the oral microbiome leading to alterations in the microbiota [
28]. In addition, changes in gastric pH could also lead to an alteration in the pH of oral cavity [
29]. We proposed that
H. pylori likely changed the community and structure of oral microbiota through changes in the acidic environment in stomach by generating large amount of urease, an enzyme which decomposes urea into ammonia and carbon dioxide and transiently reduce the acidic environment in the stomach [
30], leading to the further changes in pH of oral cavity. The use of PPIs during the eradication therapy would further inhibit the pH in stomach, leading to further alteration in saliva microbiota, which can partially explain why genera enriched in
H. pylori infected individuals would further increase and genera low expressed in
H. pylori infected individuals would decline after successful eradication. Although the precise mechanism has yet to be clarified, to our knowledge this is the first study to clearly show oral microbiota alterations as a result of
H. pylori infection in a cohort of subjects. Additional studies like metagenomics sequencing or metabolomics to investigate these possible causal relationships would likely provide interesting findings.
Using amplicon sequencing of 16S rDNA V3-V4 hypervariable regions we detected
H. pylori in the oral cavity of almost half of the subjects regardless of whether they had gastric infection with
H. pylori. Subjects who did not have
H. pylori in the oral cavity before eradication surprisingly had
H. pylori detected in saliva samples after
H. pylori eradication therapy. Clearly, using these techniques the prevalence of
H. pylori in oral cavity is not clearly associated with colonization status in the stomach which is not consistent with the notion that the oral cavity represents a secondary site for
H. pylori colonization [
31]
. The gastric and oral mucosa differ markedly. For example, of the two only the gastric mucosa expresses Lewis
b antigen, an ABO blood group antigen, which enables adherence of
H. pylori to the epithelial surfaces. It has been proposed that
H. pylori is a passerby in oral cavity, rather than a colonizer and it may be also be included in the material in gastroesophageal reflux. The natural history of
H. pylori infection has been that after
H. pylori eradication from the stomach, gastric reinfection is rare and when it occurs early it can often be shown to be recrudescence (the same genotype) whereas later reinfections are most often reinfection with a different genotype [
32]. The hypothesis that
H. pylori was a common passerby rather than a colonizer would partly explain why recurrences are most common in areas with poor sanitation and a high prevalence of
H. pylori and rare in developed countries whose frequency of
H. pylori infection had become much lower than that of poor regions.
Strengths and limitations
Our study showed that
H. pylori infection and the eradication treatment resulted in alterations of oral microbiota. However, there were limitations to our study. The technique of high-throughput sequencing we used in our study could not detect
H. pylori-specific virulence factors such as VacA, CagA, OipA, etc. or full sequence [
33]. Although the samples of the first set of cross-sectional analysis were collected at different times in August 2018, the collection interval between the first and the last collection were equal. All the samples underwent DNA extraction and sequencing analysis at the same time to make our result were comparable. One issue with the interpretation is that there was no control sample of
H. pylori uninfected individuals receiving the same antimicrobial therapy which precluded determination about whether the presence of
H. pylori, the antimicrobial therapy, or both were dominant factors in changing the within-individual diversity of the oral cavity.
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