Identification and characterization of beneficial oral bacteria

Periodontitis is an oral infectious pathology which affects the surrounding and supporting tissues of the teeth, leading to permanent loss of the teeth. In terms of prevalence, about 50 percent of the population suffers from periodontitis and around 15 percent of them from periodontitis in its more severe form. In addition, periodontitis is also associated with a large number of systemic disorders.

In healthy persons, the oral microbiota and host are in equilibrium (homeostasis). However, genetic, systemic and environmental changes can disrupt the equilibrium and cause the overgrowth of periodontal pathogens. In this pathological condition, the periodontal pathogens can directly inflict damage to the host tissues and subsequently provoke a strong activation of the inflammatory host response. The combination of the bacterial infection and the subsequent immune response causes the progressive destruction of host tissues. The current standard treatments are primarily based on the eradication of pathogenic micro-organisms by means of mechanical subgingival debridement, often supplemented with the use of antiseptics or antibiotics. The outcome of these therapies strongly depends on the establishment of a non-pathogenic/host-compatible microbiota in the subgingival area. However, they often only result in a temporary microbial shift. The pathogenic bacteria that either survived the therapies or that translocated from other intra-oral habitats, are soon recolonizing the pockets. Therefore, these traditional periodontal therapies are often insufficient to result in the formation of stable, non-pathogenic/host-compatible biofilms. Additionally, increasing levels of antibiotic-resistant bacteria requires the development of treatments that do not rely on antibiotics. Therefore, alternative and/or complementary therapies are needed to replace and/or complement the inefficient conventional treatments.

As part of the indigenous oral microbiota, the beneficial bacteria play an important role in the limitation of the periodontal pathogens. They can produce a wide spectrum of antimicrobial substances to antagonize the growth of the oral pathogens. The application of beneficial oral species, that are able to produce specific antimicrobial substances effective against the periodontal pathogens, could be an alternative or complementary treatment in periodontitis therapy. Based on this, the objective of the first study was to identify commensal bacteria with inhibitory activity against the main periodontopathogens, to determine the antimicrobial substances responsible for these inhibitory activities and to study the influence of the experimental and environmental conditions on the magnitudes of inhibition. The Streptococcus species S. sanguinis, S. cristatus, S.gordonii, S. parasanguinis, S.mitis and S. oralis were classified as beneficial bacteria because of their inhibitory properties against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia. Hydrogen peroxide (H2O2) production by the commensals was found to be the main mechanism of inhibition and the bacterial antagonism was shown to be species-specific and dependent on as well experimental as environmental conditions. These results indicate that these bacterial species could possibly be applied as new probiotics in a potential future treatment. Furthermore, they also provide new insights into the important role of H2O2 in the formation and composition of oral biofilms and into the major influence that several environmental conditions can have on the magnitudes of inhibition.

Concerning the influence of these environmental factors, the interfering effect of blood compounds with the inhibitory mechanisms was investigated. The addition of blood in the agar plates completely blocked the inhibitory effect of the beneficial streptococcal species identified in the first experiment. Based on these results, we hypothesized that some blood components could interfere with H2O2 and neutralize its antimicrobial effect. Serum, hemin and hemoglobin from the blood were selected based on their capacity to react with H2O2. The application of these blood compounds on oral biofilms subsequently caused dysbiosis, blocking the antimicrobial effect of H2O2 and stimulating the growth of A. actinomycetemcomitans, P. gingivalis and P. intermedia. This study brings new knowledge on the process of dysbiosis and leads to a new understanding of the compounds involved in the microbial shift that accompanies the transition from health to disease.

The next studies addressed which kind of compounds present in the gingival crevicular fluid (GCF) are also able to interfere with H2O2 and to neutralize the inhibitory effect of H2O2 produced by the beneficial bacteria. It is known that GCF emerges from the host tissues to control the microbial challenge posed by the periodontal pathogens. Several studies analyzed the composition of GCF both in healthy individuals and in periodontitis patients, and showed that myeloperoxidase (MPO) concentrations are significantly higher in periodontitis patients. The concentration of MPO found in the GCF of healthy individuals and periodontitis patients was subsequently used in the in vitro experiments to test the capacity of MPO to produce dysbiosis in complex oral biofilms. In the same way, the neutralization capacity of other enzymes (lactoperoxidase and catalase) was also tested using their salivary concentrations. Both the peroxidases originating from the GCF and from saliva showed a significant neutralization of the inhibitory effect of H2O2. The results obtained during this study allow us to make a correlation between the concentrations of peroxidases found in GCF and saliva on the one hand and the dysbiosis process that occurs in oral biofilms on the other hand.

The objective of the next study was to demonstrate that the microbial shift of the oral biofilms caused by exposition to serum, hemoglobin, hemin and peroxidase could produce an increase in the host inflammatory response due to the outgrowth of periodontal pathogens. The possible overexpression of several inflammatory mediators (IL-8, IL-6, TNF-α...) was evaluated. Furthermore, the expression of virulence genes from the periodontal pathogens was also analyzed. This research showed that the presence of dysbiosis-inducing compounds can cause the outgrowth of the periodontal pathogens and increase their virulence. These dysbiotic biofilms also increased the inflammatory response from the host tissues.

The last study focused on an important side effect of the traditional but unspecific and ineffective treatments in periodontitis. It is already known that oral bacteria developed different strategies to overcome the effects of these standard treatments. Necrotrophic growth could be one of these strategies, since it was already demonstrated that Legionella pneumophila bacteria were able to survive in refrigeration pipes by using dead bacteria as nutritional source. The experiments conducted during this study showed that the main periodontal pathogens indeed could feed from parts of dead oral bacteria, thereby increasing their own growth and virulence. The presence of dead bacteria plus living bacteria also caused an increase in mortality of cell cultures. This study demonstrated that necrotrophy is an important side effect of the current periodontitis treatments and that new, more specific therapies are highly necessary for an efficient treatment of periodontitis.

All together, the obtained results offer a new insight into the development of periodontitis. These insights were obtained by means of characterization of the important role beneficial oral bacteria play in the maintenance of homeostasis. The most important function of H2O2 was demonstrated, namely limiting the growth of periodontal pathogens in oral biofilms. The initiation of dysbiosis is related to the neutralization of the inhibitory effect of H2O2 produced by beneficial bacteria during inflammation, and more specifically to myeloperoxidase in gingival crevicular fluid that is responsible for this neutralization. Furthermore, the dysbiotic biofilms could also induce an increased inflammatory response. Standard treatments supplemented with the use of antimicrobials can thus enhance the pathogens growth and increase their virulence. A next generation of oral treatments of periodontitis should be based on the stimulation of the advantageous properties of the beneficial bacteria by using prebiotics and leave behind the unspecific killing procedures.