Influence of Helicobacter pylori culture supernatant on the ecological balance of a dual-species oral biofilm

Abstract Dental caries is a chronic progressive disease occurring in the tooth hard tissue due to multiple factors, in which bacteria are the initial cause. Both Streptococcus mutans and Streptococcus sanguinis are main members of oral biofilm. Helicobacter pylori may also be detected in dental plaque, playing an important role in the development of dental caries. Objective The aim of this study was to investigate the effect of H. pylori culture supernatant on S. mutans and S. sanguinis dual-species biofilm and to evaluate its potential ability on affecting dental health. Material and methods The effect of H. pylori supernatant on single-species and dual-species biofilm was measured by colony forming units counting and fluorescence in situ hybridization (FISH) assay, respectively. The effect of H. pylori supernatant on S. mutans and S. sanguinis extracellular polysaccharides (EPS) production was measured by both confocal laser scanning microscopy observation and anthrone-sulfuric acid method. The effect of H. pylori supernatant on S. mutans gene expression was measured by quantitative real-time PCR (qRT-PCR) assays. Results H. pylori supernatant could inhibit both S. mutans and S. sanguinis biofilm formation and EPS production. S. sanguinis inhibition rate was significantly higher than that of S. mutans. Finally, S. mutans bacteriocin and acidogenicity related genes expression were affected by H. pylori culture supernatant. Conclusion Our results showed that H. pylori could destroy the balance between S. mutans and S. sanguinis in oral biofilm, creating an advantageous environment for S. mutans, which became the dominant bacteria, promoting the formation and development of dental caries.


Introduction
Helicobacter pylori is implicated in several diseases such as gastritis, gastric ulcers and gastric carcinoma 20,26 . Approximately 10% of individuals suffer from gastritis or gastric ulcer due to H. pylori infection 28 .
H. pylori can also be detected in saliva, on the dorsum of the tongue, on the surface of oral ulceration and in dental plaque 8,15,22 , the latter representing a crucial location, playing an important role in the development of dental caries. The prevalence of H. pylori infection in the oral cavity of gastric H. pylori-positive people is significantly higher than that of gastric H. pylorinegative people 30 . Researchers increasingly consider H. pylori as a conditional pathogen that exists in the oral cavity of both healthy people and patients with gastritis 14 . H. pylori infection in the oral cavity is associated with dental caries and poor oral hygiene.
The caries rate in H. pylori-positive people is higher than that in H. pylori-negative people 14 .
According to the World Health Organization (WHO), dental caries has been one of the most important global oral health issues, accounting for 60-90% school-aged children of most of the industrialized countries 27 .
Streptococcus mutans is considered a crucial agent in caries pathogenesis because of its cariogenic traits 9,23 . Glucans are essential to the adhesion of S. mutans to the tooth surface and to other oral bacteria, as well as to the formation of dental biofilms matrix 11,29 . Furthermore, S. mutans possesses aciduric properties, allowing it to perform glycolysis at low pH values within the matrix of the biofilm, which result in dental enamel demineralization 24 . Streptococcus sanguinis is usually colonizing oral biofilm 12 , having been considered a "good" member in the oral biofilm, since its presence is associated with the absence of caries 13 . S. mutans and S. sanguinis inversely affect each other in the formation of dental plaque 13,21 .
Previous studies have shown that the interspecies interaction between S. mutans and S. sanguinis is mediated by S. mutans acidogenicity (production of lactic acid by L-lactate dehydrogenase, encoded by ldh) and production of bacteriocin (two major mutacins, mutacin IV and mutacin V, encoded by nlmAB and nlmC, respectively) 13 . These two streptococci compete for teeth colonization, since elevated levels of S. sanguinis in the early colonization results in a delayed colonization by S. mutans. Conversely, S. mutans teeth colonization is associated with low levels of S. sanguinis 13 . Indeed, caries-free children have high levels of S. sanguinis in their saliva and dental plaque compared to children with carious lesions who, instead, showed an elevated concentration of S. mutans 7 . Therefore, the imbalanced microecology of dental plaque was considered a key factor leading to caries. Currently, several studies 10,14,15,25   After the 24-h incubation period, biofilms were fixed in 4% paraformaldehyde, labeled with oligonucleotide probes (Probe 5′-ACTCCAGACTTTCCTGAC-3′ specific for S. mutans was labeled with FITC and probe 5′-GCATACTATGGTTAAGCCACAGCC-3′ specific for S. sanguinis was labeled with ROX) and analyzed by species-specific FISH as previously described 3 .
Micrographs from at least five randomly selected fields of each sample were captured. S. mutans to S. Anthrone-sulfuric acid method to determine biofilm insoluble EPS production

Effect of H. pylori culture supernatant on dualspecies biofilm
Since H. pylori could be detected in dental plaque and is related to the presence of dental caries, we    pylori infection, dental caries and recurrent aphthous mouth ulcerations has been investigated in other researches 6,25 . A previous study showed that in H. pylori positive participants, caries prevalence rate was two times higher than in the participants without H. pylori 14 . Previous studies already have epidemiologic surveys regarding the relationship between dental caries and oral H. pylori, but the interaction between oral H. pylori and dental caries-related bacteria such as S. mutans and S. sanguinis has not been explored.
Oral biofilm can be defined as a diverse community of microorganisms, working as a system allowing bacterial adhesion and antibiotic resistance 16 . Oral biofilm is the key factor that causes dental caries, not bacterioplankton 2,17 . Thus, in this study we focused on oral biofilm to understand its role in caries formation. mutans, which became the dominant bacteria. We also found that the production of EPS of S. mutans and S. sanguinis was inhibited by H. pylori supernatant.
Thus, we hypothesized that H. pylori supernatant contained some specific substances that may be secreted effectors, small molecules or metabolites, and that these substances could inhibit streptococcal EPS synthesis, affecting, therefore, the biofilm formation.
However, this hypothesis needs further studies to be confirmed.
We also found that H. pylori supernatant had no significant effect on planktonic growth although having clear effects on biofilm and EPS formation. The biofilm formation and EPS production of S. mutans were known to be regulated by several signal transduction systems, like two-component system and second messenger signaling 3 . It is possible that the function of these signal systems was affected by substances in H. pylori supernatant. Our further research will focus on the specific mechanisms of the anti-biofilm effects of H. pylori supernatant.
In conclusion, our results showed the ability of H. pylori to destroy the balance between S. mutans and S. sanguinis in oral biofilm, creating an environment in which S. mutans is the dominant bacteria, promoting the formation and development of dental caries.