The Effect of Lactobacillus salivarius SGL03 on Clinical and Microbiological Parameters in Periodontal Patients

Gingivitis and periodontitis comprise a large group of diseases of a complex etiology (Hasan and Palmer 2014; Dahlen et al. 2019). Among them, plaque-induced gingivitis and periodontitis constitute diseases in which the primary etiological factors are bacteria in dental plaque and other types of biofilms present in the oral cavity (Bartold and Van Dyke 2019; Geisinger et al. 2019). Epidemiological studies show that these diseases pose a serious problem to public health, they may lead to systemic diseases such as diabetes and cardiovascular diseases (Caton et al. 2018; Dahlen et al. 2019). Therefore, prevention and treatment of periodontitis are crucial not only for the maintenance of teeth and oral health but also for the whole human.

Periodontal tissue destruction in the course of inflammation occurs due to both direct bacterial action and activation of indirect immune-inflammatory mechanisms in host tissues (Dahlen et al. 2019). The constant presence of pathogenic bacteria in the oral cavity, through a number of factors such as pro-inflammatory cytokines or proteolytic enzymes, supports mechanisms of chronic destruction of connective and bone tissue, which causes disease progression and hinders its treatment. For years research studies were mainly concentrated on the composition of dental biofilm microbiota and attempt to determine a specific bacterium eliminating of which would allow effective treatment of the disease (Hasan and Palmer 2014; Dahlen et al. 2019; Proctor 2000). Initially, subgingival biofilm with a range of anaerobic bacteria was considered pathogenic, then species such as Aggregatibacter (previously Actinobacillus) actinomycetemcomitans or Porphyromonas gingivalis were identified as particularly pathogenic. In turn, studies by Socransky et al. (1998) proved the pathogenic role of not individual bacterial species, but rather bacterial complexes such as the red complex: P. gingivalis, Tannerella forsythia, and Treponema denticola. So far, however, no specific species have been identified that would be responsible for developing of the disease (Bartold and Van Dyke 2019). At present, nonspecific bacterial plaque is considered pathogenic in gingivitis, while in periodontitis, the role of additional risk factors, such as genetic and epigenetic factors, nicotinism, diabetes, as well as lifestyle and other environmental factors is emphasized (Hasan and Palmer 2014; Meyle and Chapple 2015; Bartold and Van Dyke 2019; Dahlen et al. 2019). The contemporary model of periodontal disease pathogenesis emphasizes the importance of dysbiosis and inflammation as the factors that directly lead to the destruction of periodontal tissues (Van Dyke et al. 2020). Simultaneously, concepts of so-called health-promoting biofilm appear, which are based on the theory of symbiosis between bacterial species and the specific response of the host to a given type of biofilm. According to these hypotheses, the development of periodontitis results from pathogenic bacteria, lack of beneficial bacteria, and host risk factors (e.g., genetic susceptibility (Haukioja 2010; Devine et al. 2015; Meyle and Chapple 2015; Dahlen et al. 2019)).

Bacterial biofilm reduction is still the basis of treating gingivitis and periodontitis. For many years, new therapeutic methods have been sought to increase biofilm reduction effectiveness, such as scaling and root planning (Geisinger et al. 2019). Supportive treatment includes local and systemic antibiotic therapy, local use of antiseptics, laser therapy, or photodynamic therapy. However, these methods are expensive, may cause undesirable general and local effects, and there is a lack of certain studies confirming their beneficial effects. Therefore, the use of probiotics (or pro- and prebiotics) seems to be particularly beneficial in this respect, which – by influencing the composition of the bacterial biofilm – may simultaneously contribute to the regulation of the immune-inflammatory response in the periodontium (Haukioja 2010; Gruner et al. 2016).

Probiotics are defined by World Health Organization (WHO) and Food and Agriculture Organization (FAO) as non-pathogenic live microorganisms, which – when administered inappropriately, e.g., as dietary supplements – improve the host’s health (Teughels et al. 2008; Zarco et al. 2012; Laleman and Teughels 2015). The term a biotherapeutic agent is used in the literature to describe microorganisms that accelerate the treatment or prevent complications of the disease, and their effectiveness has been scientifically proven, involving large groups of patients, randomized trials, placebo tests, and double-blind studies (Elmer et al. 1996; Mcfarland 2000). Attempts to use probiotics in dentistry date back to the 1990s and concern primarily prevention and treatment of caries and periodontal disease. The most popular probiotics belong to the genera Lactobacillus and Bifidobacterium (Gruner et al. 2016). However, the majority of studies in this area are based on in vitro experiments or small groups of subjects, while clinical trials involving large groups of patients are mainly retrospective. With the development of microbiological research, the effect of probiotics on oral microbiota as well as on clinical and immunological parameters in periodontal tissues is being studied increasingly.

The aim of the study was to assess the clinical (plaque index – PI; bleeding on probing – BOP; mean pocket depth – PD; maximal pocket depth – PD max) and microbiological parameters (colony-forming unit – FU, from supragingival plaque) in patients before and after 30 days of use of the dietary supplement containing Lactobacillus salivarius SGL03 in the form of an oral suspension, compared to the subjects receiving placebo.

All patients finished the study. The authors excluded one of them because of their doubts about whether he understood the instruction for use well. Finally, in group A there were 26 and in group B 25 patients. Demographic parameters (sex and age) of patients enrolled in study (A), and control (B) groups are shown in Tables I and II, respectively. In the study group, there were 19 females (73.1%) and seven males (26.9%), while in the placebo group – 16 females (64.0%) and nine males (36.0%). The study group’s mean age was 55.35 years, and in the placebo group – 53.28 years (p = 0.585).

The study (A) and control (B) groups did not differ at baseline in terms of plaque index, bleeding index, mean pocket depth, and maximum pocket depth (Table IV). The mean plaque index in the L. salivarius SGL03 group decreased from 55.38% to 51.61%, while in the placebo group – from 56.81% to 52.92%. The differences were not statistically significant (Table V). Similarly, the mean bleeding index decreased in both the L. salivarius SGL03 group (from 20.39% to 18.11%), and the placebo group (from 20.3% to 17.57%), but the differences were not statistically significant. However, the reduction of bleeding index in the whole group participating in the study (A + B) turned out to be statistically significant (from 20.34% to 17.84%, p = 0.011) (Table V). The average maximum pocket depth in the L. salivarius SGL03 group decreased from 4.88 to 4.58, while in the placebo group from 4.96 to 4.84. The differences were not statistically significant (Table V). In turn, the mean pocket depth in the L. salivarius SGL03 group underwent a statistically significant reduction from 2.50 to 2.42 (p = 0.027) (Table V). This parameter also decreased in the placebo group from 4.96 to 4.84, but the difference was not statistically significant. There was also no significant difference in this parameter between the L. salivarius SGL03 and placebo groups (Table V).

The average number of bacterial colonies cultured from samples of supragingival plaque, collected from patients in the L. salivarius SGL03 group, was 5.32× 10⁷ before the study and 8.77×10⁷ after the study (Table VI). The difference was not statistically significant. In the placebo group, the average number of colonies was 1.18×10⁸ before the study, and 1.09 ×10⁸ after the study – the difference was also not statistically significant.

Correlations of clinical indices with microbiological parameters are shown in Table VII. In the study (A) group, a negative correlation was found between the maximum depth of periodontal pockets before and after treatment (PD max T0, PD max T1) and the number of bacteria before treatment (CFU T0). A positive correlation was recorded between plaque index before treatment (PI T0) and the number of bacteria after treatment (CFU T1) in the study group (A). In the placebo (B) group, in turn, a positive correlation was observed between the following parameters: the pre- and post-treatment bleeding on probing (BOP T0, BOP T1) indices and the number of bacteria after treatment (CFU T1); the mean pocket depth both before and after treatment (Mean PD T0, Mean PD T1) and the number of bacteria after treatment (CFU T1); the mean pocket depth before treatment (Mean PD T0) and the change in the number of bacteria (ΔCFU); the maximum pocket depth before and after treatment (PD max T0, PD max T1) and the change in the number of bacteria (ΔCFU); the plaque index before treatment (PI T0) and the number of bacteria before treatment (CFU T0) as well as between the plaque index after treatment (PI T1) and the number of bacteria after treatment (CFU T1). Similarly, a positive correlation was found between the change in the plaque index (ΔPI) and the number of bacteria after treatment (CFU T1) and the change of the number of bacteria in the samples (ΔCFU).

Table VIII presents the survey results regarding the subjective evaluation of the taste of the preparation, convenience of use, effect on the state of gingiva and mucosa, and the adverse effects of the dietary supplement in the L. salivarius SGL03 and placebo groups. Among the respondents, 61.5% rated the taste of L. salivarius SGL03 as good, 26.9% as neutral, and for 11.5%, it was unpalatable. Similarly, 61.5% of patients thought that the preparation was convenient to use, 26.9% – neutral, and 11.5% – uncomfortable. Among patients using L. salivarius SGL03 46.2% said that their gums had improved (vs. 60.0% placebo) and 53.8% that they had remained unchanged (vs. 40.0% placebo). No patient reported any deterioration of gingiva. Regarding oral mucosa condition assessment, 57.7% of patients reported that the condition had improved (vs. 52.0% placebo) and 42.3% (vs. 48.0% placebo) that it had not changed. No patient reported deterioration of mucosa. Three patients (11.5%) from the study group and one patient (4.0%) from the control group reported adverse reactions, and they were related to gastrointestinal disorders.

The mechanism of probiotics in the oral cavity is not fully understood (Haukioja 2010; Umar et al. 2015; Laleman and Teughels 2015; Gruner et al. 2016; Seminario-Amez et al. 2017). In caries, probiotics are associated with reducing the number of colony-forming units (CFUs) of cariogenic bacteria (primarily Streptococcus mutans). Simultaneously, in periodontal disease, inhibition of periopathogens and so-called biofilm modification is observed (Iniesta et al. 2012; Montero et al. 2017; Barboza et al. 2020).

An interesting issue is the impact of probiotics on oral microbiota. Lactic acid probiotic bacteria produce antibacterial substances such as hydrogen peroxide, bacteriocins, and lactic acid, which provide a probiotic effect (Laleman et al. 2015; Takahashi 2015; Morales et al. 2016, Barzegari et al. 2020). In addition, by reducing levels of proinflammatory cytokines, elastase and prostaglandin E2 (PG E2), they inhibit an inflammatory response – humoral and cellular – in periodontal tissues (Haukioja 2010; Devine et al. 2015). It is also believed that probiotic bacteria compete with pathogens for adhesion surfaces and nutrients. L. salivarius, like other lactobacilli, is a species detected much more often in individuals with healthy periodontium compared to patients with periodontitis (Kõll-Klais et al. 2005). This species has strong antibacterial properties against pathogenic bacteria in the periodontium. The mechanism of its action is not entirely clear. Nissen et al. (2014) showed that L. salivarius inhibits the expression of toxins secreted by A. actinomycetemcomitans, including leukotoxin A, thus inhibiting its virulence.

Research on the use of probiotics in periodontology can be divided into several groups. Laboratory tests involve assessing probiotic bacteria’s effect on growth or functions (such as adhesion, coaggregation, secretion of antibacterial substances) of other bacterial strains in culture. Clinical studies concern assessing the effect of probiotics on clinical, microbiological, and immunological parameters in experimentally induced gingivitis or patients with disease – gingivitis or periodontitis. In these studies, probiotics are used as the only treatment or as an addition to conventional treatment (scaling and root planning). It is worth emphasizing that there is a lack of recommendations in the literature in which disease entities and phases of treatment probiotics should be used in periodontology. In this study, it was decided to use a probiotic in the maintenance phase of periodontal disease treatment to strengthen or maintain the effects achieved in the causal phase by modifying the microbiota’s composition and its impact on inflammatory, immunological and microbiological parameters in the periodontium.

According to literature, the most frequently evaluated clinical parameters comprise the plaque index (PI), bleeding on probing index (BOP), gingival index (GI), and pocket depth (PD), less frequently also the effect of probiotics on gingival crevicular fluid (GCF) volume. Many researchers – using various probiotic bacteria (e.g., Lactobacillus reuteri, L. salivarius, Streptococcus oralis, Streptococcus uberis, Streptococcus ratti) – reported an improvement in these clinical parameters used in periodontology; however, these differences often were not statistically significant in comparison to the control groups (Krasse et al. 2006; Shimauchi et al. 2008; Laleman et al. 2015).

In our study, no effect of probiotic on the plaque index (PI) was observed. In both groups, it oscillated around 50% before the test, and it slightly decreased after using the probiotic but remained within 50%. The differences between the initial and final visits and between the groups were not statistically significant. As the study group consisted of patients in the maintenance phase of treatment, it is not surprising, i.e., individuals with established hygiene habits. It was assumed that the probiotic administration is only intended to help maintain microbial balance within the bacterial biofilm, partially achieved after the causal phase of treatment, and to sustain this treatment’s effects. The relatively high average plaque index values (about 50%) probably resulted only from the lack of hygiene procedures on the day of the examination because they did not correspond to average bleeding indices. The bleeding index decreased in the study group (20.39% vs. 18.11%) and the control group (20.3% vs. 17.57%). Still, the differences were not significant in either of the groups. However, BOP reduction was significant in the whole group (study + control) of patients participating in the study (20.34 vs. 17.84). The lack of plaque index changes and the reduction of the bleeding index in the study and placebo groups indicated that it was not only the result of dental plaque.

It is worth mentioning that in literature, reductions in plaque index and bleeding index were found mainly in those studies where a probiotic was used as an adjunct to conventional therapy, i.e., during active treatment of gingivitis (natural or experimentally induced) and periodontitis (Vivekananda et al. 2010; Teughels et al. 2013; Morales et al. 2016). A statistically significant reduction in the mean pocket depth (PD) was observed in the study group in our study. However, there was no significant difference between the study and control groups. Penala et al. (2016) obtained similar results.

As mentioned in this paper, the study group consisted of patients in the maintenance phase of treatment. From the clinical point of view, reduction of pocket depth is an expected and beneficial therapeutic effect. For patients in the maintenance phase of periodontitis treatment, the most crucial goal is to maintain a low bleeding index, a symptom of active inflammation in the periodontium, and maintenance or progress of reduction of pocket depths obtained during the active treatment phase. In the causal phase of treatment, reduction of pocket depth mainly results from a reduction in the number of bacteria, thus reducing active inflammation in the periodontium. As a result, tissue hyperemia and swelling are reduced. In turn, further reduction of pocket depths in the maintenance phase of treatment may result from the regulation of additional destructive mechanisms in periodontal tissues. Maintaining favorable composition of bacterial biofilm, obtained from the elimination of bacteria in the causal phase, means that the inflammation does not recur, bleeding does not intensify, and inflammatory-immunological mechanisms are gradually modulated. In periodontium, healing processes begin to prevail over destruction processes.

As mentioned earlier, in our study, both plaque and bleeding indices were not significantly reduced, which could mean that the pocket reduction process included additional mechanisms. It may be indirectly confirmed by the results of a clinical parameter correlation analysis (Table VII). In the placebo group, positive correlations were observed between plaque and bleeding indices as well as the mean and maximum pocket depths vs. reduction in the number of bacteria; therefore, changes in the number of bacteria affected clinical parameters. Such correlations were not found in the study group, which may mean that the significant reduction in pocket depth observed in this group was not due to a change in clinical parameters and the number of bacteria but rather a change in biofilm composition and its effect on inflammatory and immunological parameters. In the study group, only a negative correlation between the maximum depth of periodontal pockets and the number of bacteria before treatment was noted. It means that the greater the maximum depth of periodontal pockets before treatment, the smaller the number of bacteria were detected in tested samples. It may indicate that patients with the most advanced disease were very well motivated to maintain proper oral hygiene. This correlation disappeared after treatment, which may be associated with biofilm composition changes after using L. alivarius SGL03.

Microbiological testing of the oral cavity microbiota comprises several approaches, including qualitative or quantitative culture methods for detecting particular species (with the use of selective culture media) or groups of bacteria. PCR techniques are particularly useful as they enable detecting specific species of bacteria, including non-viable or non-cultivable microorganisms. Both probiotic and pathogenic periodontal species (e.g., P. gingivalis or A. actinomycetemcomitans) may be detected with this method. Metagenomic methods are increasingly used in dentistry, and they make it possible to detect the composition of microorganisms that make up the biofilm, as well as the percentage of individual types of bacteria (Xu and Gunsolley 2014; Dabdoub et al. 2016). These techniques also allow the discovery of new periopathogens, including bacteria that cannot be isolated using classical culture methods (Hiranmayi et al. 2017; Torres et al. 2019). A better understanding of oral microbiota composition and mutual interactions of microorganisms present in the course of the disease will allow for the use of more effective therapeutic procedures, including patients with periodontitis (Proctor et al. 2020).

Several types of samples are used in microbiological studies of the oral cavity; however, in periodontology, they mainly comprise specimens of supragingival and/or subgingival plaque.

According to the authors of the analysis concerning periodontal disease, the use of probiotics improves clinical parameters such as BOP, PD, GI, but not the number of colony-forming units (CFU) of bacterial periopathogens (Seminario-Amez et al. 2017). However, it should be remembered that the microbiology of periodontal pockets is very complex and comprises both periopathogens as well as aerobic, pioneering, and seemingly nonpathogenic bacteria.

A meta-analysis by Gruner et al. (2016), in which three papers on the effectiveness of therapy with probiotics containing Lactobacillus bacteria were evaluated, did not show their effect on the examined periopathogens: A. actinomycetemcomitans, P. gingivalis, and Prevotella intermedia. However, despite a reduction in gingival inflammation indices (GI and BOP), no impact of probiotics on the plaque index was observed in the analyzed studies. Therefore, the authors of the analysis conclude that its effect results from the influence on host response, not on bacteria themselves.

Several bacteriological studies revealed the effect of probiotic strains of Lactobacillus spp. on the reduction of A. actinomycetemcomitans, P. gingivalis, P. intermedia, and T. forsythia in the subgingival plaque of patients with periodontitis, with no significant effect on clinical indices in these patients (Mayanagi et al. 2009; Vivekananda et al. 2010; Iniesta et al. 2012; Montero et al. 2017). On the other hand, in the paper mentioned above by Hallström et al. (2013), the use of L. reuteri lozenges did not affect biofilm composition in experimental gingivitis. Similarly, in our research, no influence of the probiotic on the number of bacteria in supragingival plaque samples was observed, with a slight statistically not significant reduction in the number of bacteria in the placebo group and an increase (not statistically significant) in the number of bacteria in the study group. This rise may be related to different bacterial species composition in samples before and after applying the probiotic. Given the statistically significant reduction of pocket depths in the study group, it can be assumed that probiotic use could have a beneficial effect on changing the composition of dental plaque microbiota to microbiota with lower pathogenic potential. However, further studies are needed to evaluate the biofilm composition concerning specific bacterial species.

Long-term use of a probiotic may positively affect the composition of oral microbiota and interactions between individual types of bacteria and inhibit proinflammatory effects of periopathogens; however, it cannot replace daily hygiene procedures (Laleman et al. 2015). The authors of a systematic review of the literature regarding the effect of probiotics on experimentally induced gingivitis in humans concluded that probiotics may be an alternative to rinses containing chlorhexidine (CHX), which could have undesirable adverse effects (Barboza et al. 2020).

Although some studies do not show an improvement in clinical parameters (e.g., plaque and bleeding indices) after the use of probiotic, they indicate the possibility of its action by modulating inflammatory response, e.g., by reducing the level or activity of PGE2, proinflammatory cytokines or proteolytic enzymes (elastase, MMP-3 metalloproteinase) in gingival crevicular fluid (GCF) or saliva (Staab et al. 2009; Lee et al. 2015; Kuru et al. 2017).

Attention should be paid to the form of the supplement used in the study – Salistat SGL03 is a rinse solution, while the majority of supplements on the market are oral tablets or lozenges. This probiotic supplement (and the placebo) in the form of a solution enables the accurate distribution of the suspension on tooth and gum surfaces as well as the mucosa of the entire oral cavity. In addition, as recommended by the manufacturer, bacteria constituting the contents of the package are kept in the mouth for about 30 seconds before being swallowed. It seems to be a much better and more effective form of probiotic application than tablets or lozenges. It also requires slightly increased patient involvement in the evening application procedure after thorough toothbrushing, which could have contributed to the improvement of clinical parameters in the placebo group.

The effects of probiotics reported in the literature on clinical parameters and inflammatory markers are variable, ranging from no effect to statistically significant decrease in PI, GI, and BOP indices and in GCF volume (Slawik et al. 2011; Iniesta et al. 2012; Hallström et al. 2013; Kuru et al. 2017). Interestingly, Kuru et al. (2017) reported that beneficial effects were also observed after cessation of toothbrushing for five days, which may indicate a beneficial effect of the probiotic in patients with temporarily reduced performance of hygiene procedures, e.g., for health reasons. Further studies are needed to clarify this issue.

It should be noted that in this study, only one probiotic preparation has been evaluated (containing L. salivarius SGL03), so our findings cannot rule out other effects of the use of other probiotics.

In this study, the authors also analyzed a questionnaire on subjective assessment of the probiotic preparation containing L. salivarius SGL03 (its taste perception, the convenience of use, the effect on the state of gums and mucosa as well as potential adverse effects of dietary supplement), in comparison to the preparation administered to the patients in the placebo group. Over 61% of patients in the study group were satisfied with both the taste of this dietary supplement as well as convenience of its use (in comparison to 80.0% and 76% in the placebo group, respectively). It is important to emphasize that the addition of probiotic strains of microorganisms may alter the taste and aroma of the final food product or dietary supplement due to the production of different metabolites (e.g., organic acids) during fermentation, and extended storage (Terpou et al. 2019). It may determine the patient’s adherence to therapy, particularly during long-term treatment for several weeks or months. In this study, 46.2% and 57.7% of patients in the study group reported the improved effect on the gums and oral mucosa compared to 60.0% and 52.0% in the placebo group. As reported in the literature, probiotic dietary supplements increasingly used in dentistry – apart from their direct effects (e.g., inhibition of oral pathogenic microbiota) – may also contribute indirectly to the regulation of mucosal permeability and local immunity in the oral cavity as well as decreased gum bleeding and reduced gingivitis (Krasse et al. 2006; Anusha et al. 2015). There were adverse reactions reported in the questionnaire by three patients in the study group and one individual in the placebo group; however, it should be noted that no patient had reported these adverse effects during therapy or ceased the use of the preparation (probiotic or placebo) because of them.

The use of the probiotic-containing L. salivarius SGL03 in the form of an oral suspension in patients in the maintenance phase of periodontitis treatment could have contributed to a reduction in the periodontal depths pockets, with no change in other clinical parameters and the number of bacteria in supragingival plaque. The use of probiotics seems to be justified in the maintenance phase of treatment to sustain the microbiological balance obtained during its causal phase. They can then be an alternative to additional therapies with antiseptics. Further research is needed on individual bacterial species’ clinical and microbiological parameters to confirm the long-term effect of the preparation.