Presence of Streptococcus dentisani in the dental plaque of children from different Colombian cities

Abstract Streptococcus dentisani has been identified as an oral cavity probiotic due to its beneficial characteristics. One of its beneficial features is the production of bacteriocins, which inhibit the growth of cariogenic bacteria, and another is its buffering capacity through the production of ammonium from arginine. The purpose of this study was to determine the presence of S. dentisani in the dental plaque of Colombian children and whether the presence of this bacterium is related to oral health and other conditions. Dental plaque and information on diet and oral hygiene habits were collected from children between 6 and 12 years of age from four Colombian cities, divided into caries‐free children (International Caries Detection and Assessment System [ICDAS] 0, Decayed Missing Filled Teeth index [DMFT] 0), children with ICDAS 1 and 2, and children with ICDAS >3. Plaque DNA was extracted and quantified, and real‐time polymerase chain reaction was performed using specific primers. This bacterium was identified in all samples, with a median of 0.46 cells/ng DNA (interquartile range [IQR] 0.13–1.02), without finding significant differences between the groups (P > 0.05). In caries‐free children, a median of 0.45 cells/ng DNA (IQR 0.14–1.23) was found. In children with ICDAS 1 and 2, the median was 0.49 cells/ng DNA (IQR 0.11–0.97), and in children with ICDAS >3, the median was 0.35 cells/ng DNA (IQR 0.12–1.07). However, statistically significant differences were found in the origin of children (P < 0.01), the use of fluoride‐containing products (P < 0.01), and the frequency of food intake (P < 0.05). In conclusion, the presence of S. dentisani was quantified in children from four Colombian cities, without finding significant differences in oral health status. Nevertheless, three conditions showed a possible relationship with S. dentisani.


| INTRODUCTION
The oral cavity includes several niches, such as the surfaces of teeth and cheeks, periodontal pockets, tongue, saliva, gingival sulcus, and soft and hard palates, among others. Each region of the mouth has its own characteristics, with unique microenvironments that allow the establishment of the oral microbiome, where bacteria predominate with over 700 different species (Kilian et al., 2016). The literature describes the composition of the oral microbiome, analyzes its roles in healthy and unhealthy mouths, and infers the interactions between the oral microbiome and its host (Jiang, Gao, Jin, & Lo, 2016;Kilian et al., 2016;Simón-Soro, & Mira, 2015;Zaura, Nicu, Krom, & Keijser, 2014).
After birth, a newborn acquires a wide variety of microorganisms.
Only part of them are able to colonize the individual, which can influence the subsequent colonization by other microorganisms (Sampaio-Maia & Monteiro-Silva, 2014). The initial colonization process is dominated by Streptococcus, which make up to 80% of microorganisms of the biofilm (Kreth, Merritt, & Qi, 2009), followed by Actinomyces and other bacteria.
Advances in molecular biology have allowed the development of methods favoring the knowledge of the diversity and composition of the oral microbiome, the understanding of the dynamics and establishment of microorganisms, and their roles in health and disease (Benn, Heng, Broadbent, & Thomson, 2018). Real-time quantitative polymerase chain reaction (qPCR) allows simultaneous amplification and quantification of the amplicons using a fluorescent dye. This dye or fluorophore binds only to double-stranded DNA after each amplification cycle; therefore, the fluorescence intensity reflects the number of DNA amplicons generated. The point at which fluorescence intensity increases above the detection threshold corresponds proportionally to the initial number of molecules of the sample DNA template. This point is called the quantification cycle (Cq) and allows the absolute amount of target DNA to be determined according to the constructed calibration curve. This technique is highly accurate and sensitive for the quantification of individual bacterial species as long as appropriate and specific primers are used (Kralik P, & Ricchi, 2017). Different bacterial species are associated with mouth diseases, such as Streptococcus mutans, Lactobacillus (Becker, Paster, & Leys, 2002) and Scardovia wiggsiae (Kressirer et al., 2017)

in caries;
Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia (Mineoka et al., 2008), Prevotella intermedia, and Prevotella nigrescens (Zhang et al., 2017) in periodontal disease; or Fusobacterium nucleatum in halitosis (Lee, Mak, & Newsome, 2004). In contrast, healthy mouth conditions are associated with other species with interesting characteristics to control the dysbiosis. Streptococcus salivarius K12 (Burton et al., 2013), the Streptococcus strain designated A12 (Huang et al., 2016), and Streptococcus dentisani (López-López, Camelo-Castillo, Ferrer, Simon-Soro, & Mira, 2017) are some examples of the beneficial bacteria within the oral cavity that inhibit the pathobionts, restoring the microbial ecological balance. In Colombia, there are no reports available on the determination of beneficial bacteria with oral probiotic characteristics and the possible host conditions that determine their presence.
S. dentisani belongs to the Mitis group and has been isolated from the dental plaque of caries-free Spanish individuals (Camelo-Castillo, Benítez-Páez, Belda-Ferre, Cabrera-Rubio, & Mira, 2014). This coccusshaped bacterium grows in colonies of approximately 1.5 mm in diameter, is a facultative anaerobe, and has an optimum pH of 7, although it  (Table 1). The inclusion criterion was an age between 6 and 12 years, and the exclusion criteria were having basic systemic pathologies, having received antibiotics in the last 3 months, and having teeth brushed at least 8 hr prior to sample collection. Before starting the study, informed consent was requested from the parents of the children and also a questionnaire that requested personal information (geographic origin, sex, and age), diet information (frequency of food intake per day and frequency of fermentable carbohydrates intake per day), and oral hygiene habits (brushing frequency and use of fluoride-containing products). This study was approved by the ethics subcommittee of the Universidad Cooperativa de Colombia, Villavicencio (21102015).

| International Caries Detection and Assessment System
For the diagnosis and selection of children, the ICDAS was used to determine carious activity. The dentists who made the diagnosis and collected the samples were calibrated in the ICDAS system, obtaining satisfactory inter-and extra-examiner reproducibility (Kappa value ≥0.7).

| Sample collection
Prior to sample collection, the dental plaque index was determined using the modified Silness and Löe index (Mombelli, Van Oosten, Schürch, & Lang, 1987). Subsequently, supragingival plaque was collected with a sterile curette, scraping over tooth surfaces (buccal, lingual, and occlusal) in Quadrants 1 and 3 for temporary and permanent dentition, without touching the gums. Samples were collected in microtubes containing 200 μl of sterile saline solution, to which the corresponding code was assigned. The samples were kept frozen at −20°C until molecular analysis.

| DNA extraction and qPCR
For DNA extraction, 200 μl of each sample was used, to which 5 μl of lysozyme from chicken egg white (10 mg/ml, Sigma-Aldrich,

| RESULTS
One hundred dental plaque samples from children divided into three groups were evaluated: ICDAS 0 with 36 samples analyzed, ICDAS 1 and 2 with 32 samples, and ICDAS >3 with 32 samples. Table 2 shows the distribution according to each characteristic evaluated.  (17)  >3 49 (49) Grouped by age according to stages of mixed dentition.
3.1 | Identification and quantification of S. dentisani in dental plaque samples and 2.29, respectively. No statistically significant differences were found between caries-free children and those with caries (Table 3).
When comparing the quantification of S. dentisani according to the groups related to characteristics such as sex, origin, age, plaque index, oral hygiene, and eating habits, statistically significant differences were found in the origin of children (P < 0.01), the use of fluoride-containing products (P < 0.01), and the frequency of food intake (P < 0.05). Samples from children from Bogotá, Pasto, and  Children whose parents did not report the use of fluoride products had a significantly higher median of S. dentisani (1.12 cells/ng DNA, IQR 0.03-6.39) than those that used these products regularly (0.38 cells/ng DNA, IQR 0. 002-7.20). Similarly, children who had a lower food intake (Jiang et al., 2016;Kilian et al., 2016;Zaura, Nicu, Krom, & Keijser, 2014) presented higher levels than those with higher intakes (1.04 cells/ng DNA, IQR 0.009-6.39 vs. 0.40 cells/ng DNA, IQR 0.002-7.20, respectively; Table 3).

| DISCUSSION
In the present pilot study, S. dentisani was detected in the dental plaque samples of all the children evaluated, with a median of 0.46 cells/ng DNA (IQR 0.13-1.02), without finding significant differences between caries-free children and those with some caries index level. nutrient supplements, and pH in microbial diversity and quantification (Wake et al., 2016).
Therefore, the lifestyle can influence in the concentration of S. dentisani in the children. In this study, one condition that significantly influenced the quantification of S. dentisani is the amount of daily food intake. Children with a maximum frequency of food intake of 3 showed significantly higher quantification of S. dentisani than children with higher food intake. This difference could occur due to changes in the oral environment after food intake, resulting in differences in the multiplication and permanence of S. dentisani. One example would be the decrease in salivary pH. Because the optimum pH range for this bacterium is between 7 and 7.5, its growth would be expected to decrease as conditions reached an acidic pH. However, in this study, no significant quantification differences in S. dentisani were detected in children with higher or lower frequencies of fermentable carbohydrate intake, which are the type of carbohydrates that have the greatest effect in terms of pH reduction; however, it has been shown that a high dietary content of starches or fruits also reduces plaque pH (Moynihan, 2005) and the repeated acidic challenge imposed by multiple meals has been proposed to represent an important selective pressure against nonacidophilic microorganisms (Rosier, Marsh, & Mira, 2018).
Another aspect that may influence the quantification of S. dentisani in this variable is that after food intake, some microorganisms are metabolically activated, and through different interactions during biofilm formation (Benítez-Páez, Belda-Ferre, Simón-Soro, & Mira, 2014), they may influence the presence of S. dentisani. This influence is mediated through the competition of nutrients and/or space or by the production of substances toxic to bacteria, such as bacteriocins.
This finding could indicate that diet is influencing the presence of this bacterium and, therefore, the relationship with the children's place of origin, because the altitude of the city (Villavicencio) that was home to the children with the lowest levels of S. dentisani is very different from those of the other cities. Altitude can determine the type of food that is grown and therefore more frequently consumed. This possible relationship with diet could be influenced by the endogenous nutritional environment (saliva, tissues, crevicular fluid, microbial metabolites, etc.) through systemic circulation. In an exploratory study involving metagenomic sequencing of 16S ribosomal RNA, Kato et al. (2017) reported an association between intake of a specific nutrient (saturated fat acids, vitamin C, and glycemic load) and microbial diversity. Saturated acid was correlated with relative abundance of Betaproteobacteria and Fusobacteria, vitamin C exhibited positive correlations with abundance in fusobacteria class and Leptotrichiaceae and Lachnospiraceae families, and finally, the glycemic load was positively correlated with Lactobacillaceae abundance (Kato, Vasquez, & Moyerbrailean, 2017 Finally, children whose parents did not report the use of fluoridecontaining products showed a significantly higher quantification of S. dentisani than those who used the products. This finding could indicate an inhibitory effect of fluoride on this bacterium. Few studies have analyzed the effect of fluoride within the diversity of the human oral microbiome, and the results indicate only a minimal effect. However, these studies did not control for the use of water and fluoride products. A study conducted in mice by Yasuda et al., (2017) compared the effects of water and fluoride products and found a selective impact on oral microbiota, especially acidogenic bacteria (Yasuda et al., 2017). Fluoride has been shown to affect the metabolism of S. mutans and showed low bacterial adhesion strength by force spectroscopy (Loskill et al., 2013).
In conclusion, the presence of S. dentisani was identified and quantified using the qPCR technique in dental plaque samples of children from four Colombian cities. The study found no significant differences in the quantification of bacteria in caries-free children with respect to those classified with some caries index level. This could be due to the large variability in S. dentisani levels with a limited sample size or to a lack of association with the oral health parameters studied. Regarding other variables studied in the quantification of this bacterium, a possible relationship was found between the use of fluoride-containing products, origin, and the frequency in daily food intake. In the future, a potential relationship between the different factors studied in the present work with other potentially beneficial (e.g., ammonia producers) or cariogenic (e.g., acid producers) organisms should be investigated.
The design of new in vitro or in vivo experiments to confirm the possible relationships between S. dentisani and variables such as the use of fluoride-containing products and the frequencies of food intake could determine the ideal conditions for using this bacterium as a probiotic. Additionally, understanding this relationship would add knowledge of the behavior of this bacterium as part of the oral microbiome and its possible interactions in the oral cavity.