Influence of the long-term use of oral hygiene products containing stannous ions on the salivary microbiome – a randomized controlled trial

Oral hygiene products containing tin are suitable to prevent erosive tooth wear, yet effects on the oral microbiota are not known yet. Therefore, this study determined the salivary microbiome of 16 participants using products with stannous ions for three years (TG) compared with a control group (CG) to assess their influence on the microbiota. Participants were included in a randomized controlled clinical trial (RCT) with biannual visits. Illumina Miseq sequencing revealed as most abundant genera: Streptococcus (TG 14.3%; CG 13.0%), Veillonella (TG 11.3%; CG 10.9%), Prevotella (TG 7.0%; CG 9.8%), Haemophilus (TG 6.6%; CG 7.2%), Porphyromonas (TG 5.9%, CG 5.1%), Leptotrichia (TG 5.8%; CG 4.9%), Actinomyces (TG 4.0%; CG 4.6%) and Neisseria (TG 5.4%; CG 4.2%). Beta-Diversity was not significantly different between groups at both time points, although significant differences between groups were found for certain taxa after three years. The genus Prevotella was found in higher abundance in CG whereas Neisseria and Granulicatella, health-associated taxa, were found more abundantly in TG. Salivary microbiota after three years reflected a composition associated with oral health, hence continual use as a preventive measure for dental erosion can be considered safe and benefitting oral health for patients with a high risk of erosion.

Salivary microbiota, similar to other oral niches, contain commensal microorganisms that -in health-comprise a physiological flora that is beneficial as long as homeostasis is maintained 14 . Environmental disturbances can increase the abundance of potentially pathogenic species leading to oral diseases, such as caries and periodontitis 15,16 . Therefore excessive eradication of the resident flora is not expedient, but it would be desirable to selectively influence the proportion of potentially pathogenic species 17 . In recent years, the salivary microbiome has been studied more extensively. The findings suggest a salivary core microbiome being present in the majority of individuals, accounting for a very large proportion of determined sequences and being fairly stable temporally [18][19][20] . The salivary microbiota resemble various oral sites, e.g. the tongue, the supragingival as well as subgingival plaque and are thought to be shaped by microorganisms deriving from these niches 21 . Certain taxa in saliva have been associated with specific oral diseases, e.g. periodontitis, early childhood caries or with oral health [21][22][23] . Therefore, besides investigating the clinical efficacy of oral hygiene products containing stannous ions, it is essential to investigate their impact on the microbial community. Hence, the aim of our study was to analyze the influence of the long-term use of a mouthrinse and toothpaste containing SnCl 2 /AmF/NaF over three years on the salivary microbiome of an intervention group compared to a control group using products containing AmF/ NaF alone. To achieve this objective, we applied high-throughput DNA amplicon sequencing based on the 16S rDNA on the Illumina MiSeq platform.

Composition of the salivary microbiota in TG and
In order to assess the differences between the salivary microbiome of the two groups, the beta-diversity was analyzed on the basis of the Bray-Curtis dissimilarity. The non-metric multidimensional scaling did not show any distinct clustering in either TG or CG at T0 nor at T3, and there were no significant differences in the calculated beta-diversity (permutational multivariate analysis of variance based on Bray-Curtis dissimilarity; p = 0.447 at T0; p = 0.302 at T3.) (Fig. 2). Consequently, the salivary bacterial community as a whole is not significantly affected by the long-term use of products containing SnCl2/AmF/NaF.
Even though there was no significant difference in beta-diversity, certain taxa showed a significantly different abundance in the two groups at T3, but not at T0. A LEfSe analysis revealed that Prevotella taxa, P. histicola_ jejuni_veroralis, P. salivae and P. loescheii as well as Eikenella sp., Kingella oralis and Haemophilus haemolyticus were found in significantly higher abundance in CG. Neisseria sp., Granulicatella sp., Gemella sanguinis and Kingella denitrificans were found enriched in TG (Fig. 3). Separate Wilcoxon tests confirmed this, showing a significantly higher abundance of the genus Prevotella in CG (p = 0.022) and a significantly higher abundance of www.nature.com/scientificreports www.nature.com/scientificreports/ the genus Neisseria (p = 0.033) as well as Granulicatella (p = 0.042) in TG (Fig. 1d). The analysis of the sequences on the species level revealed that certain Prevotella species were more abundant in CG (e.g. P. histicola_jejuni p = 0.041; P. histicola_jejuni_veroralis, n.s.) ( Fig. 1e; Supplementary Fig. S6). At baseline, these taxa did not show any significant differences ( Supplementary Fig. S7). When the abundance of Prevotella species was related to the distribution of males and females in the two groups, significant gender-related differences were revealed (Fig. 4). Thus, 2 Prevotella taxa were significantly more abundant in males (e.g. P. histicola_jejuni, p = 0.035; P. histicola_ jejuni_veroralis p = 0.015). The genera Neissera and Granulicatella did not show any gender-related differences in their abundance.  As far as the mock community used as positive control, the Illumina MiSeq sequencing detected all taxa included in the mock community. Contaminant reads were ≤5 reads per sequenced sample. The relative abundances of the included taxa were not equal, Tannerella sp. and the two Streptococcus species, which were not differentiated (S. mitis and S. sanguinis) were underrepresented and on the other hand Porphyromonas sp. and Fusobacterium s.p. were somewhat overrepresented. The results of the mock community sequencing are shown in Supplementary Table S3.

Discussion
Oral hygiene products containing stannous ions are suitable for continuous use as preventive measures 24 , however, their long-term influence on the oral microbiota has not been studied yet. Therefore, this study aimed to analyze the salivary microbiome of individuals with elevated risk for dental erosion that used products containing SnCl 2 /AmF/NaF over a period of three years in comparison to a control group. Since the salivary microbiota contains bacteria shed from different oral sites its composition is thought to reflect the oral microbiota as a whole 21,25 . Therefore there is an increased interest in sampling saliva to determine oral health, and to our knowledge this is the first study that investigated the effect of a long-term use of such products on the salivary microbiota in vivo. The most noteworthy outcome was that no significant difference could be observed in the microbial composition in TG compared to CG at both time points, even though certain taxa showed significantly different abundances in the two groups at T3. Overall, the microbiome of TG after the three-year treatment period revealed a composition of microbial taxa that can be interpreted as health-associated.
Previous studies have proven the ability of stannous ions to reduce plaque and salivary bacterial count. Addy However, in contrast to our study, the focus of these authors was the short-term effect of these products to reduce the bacterial load until plaque regrowth occurred. They failed to take into account the microbial  www.nature.com/scientificreports www.nature.com/scientificreports/ community as a whole in vivo reacting to outward influences as an ecological framework with specific interactions between the taxa present. Our study used comprehensive high-throughput sequencing revealing the influence of the long-term use of stannous chloride on the whole microbial community. The samples of both groups, TG and CG revealed a high abundance of the genera Streptococcus, Veillonella, Prevotella, Granulicatella, Gemella, Haemophilus, Neisseria, Porphyromonas, Actinomyces and Rothia, corresponding to the taxa found as salivary core microbiome in many recent studies 20,28,29 . Regarding the species diversity, both groups did not differ from each other at T3 which is indicative that the treatment groups' microbiota represents a healthy status. Kumar et al. argue that similar levels of diversity are important, since any deviation from the controlled diversity in the oral ecosystem associated with a healthy status appears to result in a dysbiotic condition, eventually leading to disease 30 . On the other hand, the species richness was lower in TG, which is also indicative of a healthy status 20,31 .
As for differentially abundant taxa found in the two groups at T3, the genera Prevotella, Neisseria and Granulicatella stand out. The genus Prevotella was found in significantly higher abundances in CG (Fig. 1d). In part this could be attributed to the fact that the control group included more men and P. histicola-jejuni and P. histicola-jejuni-veroralis were significantly more abundant in males. Zaura et al., analyzing the salivary microbiome of 268 individuals also reported a higher abundance of Prevotella spp. in males, whereas females showed a higher abundance of certain Streptococcus species 29 . Yet, since the whole genus Prevotella, showed a significantly higher abundance in CG it can be assumed that stannous chloride might suppress the growth of many representatives of this taxon.
Interestingly, Yang et al., studying salivary microbiomes of caries-active and healthy individuals, found an association of Prevotella sp. (P. histicola among others) with caries-active individuals which was confirmed by Teng et al., who reported an increased abundance of various Prevotella species in the saliva of children with early childhood caries 22,23 . According to their assumption, this taxon could be indicative of a caries-active status. Belstrom et al., comparing saliva of healthy individuals with caries and periodontitis patients, showed that traditional periodontal pathogens and cariogenic species, were found significantly more often in the saliva from patients with the respective condition 21 . As for Prevotella taxa, P. intermedia, P. baroniae and P. falsenii were enriched in periodontitis patients, P. denticola in caries patients, yet P. scopos and P. bergensis were associated with oral health. Regarding the genera Neisseria and Granulicatella being more abundant in TG, both taxa can be regarded as health-associated. Yamashita et al. were able to differentiate several types of salivary microbiomes according to periodontal health and found a high abundance of Neisseria flavescens, Granulicatella adiacens and some other taxa enriched in periodontally healthy individuals. On the other hand, individuals with a high abundance of P. histicola, P. melaninogenica, several Veillonella and Streptococcus species exhibited poorer oral health 31 . Correspondingly, Sanz et al. infer from recent high-throughput sequencing studies that Neisseria and Granulicatella among other taxa can be regarded as genera normally associated with oral health 32 . Taken all together, we can conclude that the differential abundances of the genera Prevotella, Neisseria and Granulicatella, i.e. a lower abundance of Prevotella spp. and a higher abundance of the latter two after the long-term use of products containing stannous ions can be seen as a beneficial effect for the entirety of the oral microbiota.
In conclusion, the analysis of the salivary microbiome after a long-term use of oral hygiene products containing SnCl 2 /AmF/NaF reflects a healthier status of the microbial community. This result was also confirmed by the clinical data on the oral health status demonstrated by the biannual caries assessment by the ICDAS-II-System for both groups 33 . The clinical data on erosion showed that the treatment group yielded a better result after four years with less increment in erosion 34 . In this respect the continual use of these products as a preventive measure for tooth erosion can be recommended for patients with a high risk of erosion and can be considered as contributing to oral health in general.

Materials and Methods
Study design. The study was performed as an RCT in accordance with guidelines of Good Clinical Practice and conforming to the declaration of Helsinki. Ethical approval was obtained from the local ethics committee (S-566/2012; University of Heidelberg). The study was registered at the German Clinical Trials Registry Platform (DRKS00005019, registration 2013/05/27). Previous studies have shown that endurance athletes are at a higher risk of erosive tooth wear than controls 34 . For this reason, 54 participants who performed at least five hours of endurance training per week were recruited between March and October 2013 and included in the study. Inclusion criteria were: good general health, written informed consent. Exclusion criteria were: cumulative weekly training under five hours, restrictions in oral hygiene, pregnancy or lactation, participation in another clinical study within the last 30 days, use of antibiotics within the last 30 days prior to the study and each recall time point, affiliation with the dentistry department as student or staff. The full details of the study are given elsewhere 33 . Randomization of the participants into treatment group (TG) and control group (CG) was performed by block randomization (using sequentially numbered envelopes). The numbers of participants withdrawing from the study were documented in the CONSORT flow diagram (Fig. S1).
Clinical oral examinations were performed every 6 months with assessment of dental erosion (BEWE), caries index (ICDAS II) and a professional tooth cleaning at the Department of Conservative Dentistry, University Hospital Heidelberg (Heidelberg, Germany) by one blinded calibrated examiner 33,34 . For the analysis of the salivary microbiota stimulated saliva was sampled from each participant at baseline and after the three-year treatment period using sterile paraffin gum. Each participant refrained from eating and drinking, smoking and brushing their teeth for at least 2 hours before sampling.
The participants in the TG received mouth rinse containing F/Sn (AmF/NaF/SnCl 2 ; 800 ppm Sn 2+ , 500 ppm F -; Elmex Erosion Protection Mouthrinse, CP GABA, Hamburg, Germany) and toothpaste containing F/Sn (NaF/ Sn 2+ ; 3500 ppm Sn 2+ , 1450 ppm F − ; Elmex Erosion Protection Toothpaste; CP GABA, Hamburg, Germany) and were instructed to use mouthrinse 1 × 30 s per day and toothpaste twice a day for routine oral hygiene. The participants in the CG did not receive any oral hygiene products but were given instructions to use conventional www.nature.com/scientificreports www.nature.com/scientificreports/ fluoridated toothpaste (1500 ppm) but no products containing stannous compounds. During the follow-up visits the compliance of the participants of both groups was verified, the brands of the oral hygiene products used by the CG were retrieved and all the instructions were repeated.

16S rDNA Illumina MiSeq high-throughput sequencing.
To analyze the salivary microbiome we applied high-throughput DNA amplicon sequencing based on the 16S rDNA on the Illumina MiSeq platform. For DNA extraction the DNA PowerSoil Kit (Qiagen, Hilden, Germany) was used according to the manufacturer's protocol. The microbial community was analyzed using Illumina MiSeq paired-end-sequencing with two times 300 bp read length. The amplicon library was constructed using the primers 5′-CCTACGGGNGGCWGCAG-3′ and 5′-GACTACHVGGGTATCTAATCC-3′ 35 for the variable regions v3-v4 including the recommended adaptors for Illumina Sequencing (MiSeq Reagent Kit v3, Illumina, Eindhoven, Netherlands). Amplification, indexing, library quantification, pooling and sequencing were performed according to the Illumina MiSeq protocol for amplicon sequencing. Filtering, denoising, merging and the taxonomic classification based on the Silva database (v132 36 ; was done with the Divisive Amplicon Denoising Algorithm 2 (DADA2) pipeline 37 . For comparison, taxonomic annotation was performed using Uclust (Qiime v. 1.9.1) based on a different database (Zymo Research, Irvine, USA) to achieve further taxonomic assignment on the species level. Statistical analysis. All statistical analyses were performed in R (V.3.6.0). As taxa abundances are not normally distributed, non-parametric tests (Wilcoxon Rank Sum Test) were used with multiple testing correction when applicable. P-values < 0.05 were regarded as statistically significant. The species richness was determined using Chao1 (richness) and alpha diversity was analyzed using Shannon Diversity (OTU-based diversity). Beta diversity was assessed by computing weighted Bray-Curtis distances to compare microbial communities based on relative abundance for each microbial community. NMDS was performed to compare beta diversity among groups. To determine features most likely to explain differences between TG and CG, we applied the linear discriminant analysis (LDA) effect size (LEfSe) method for differential abundance analysis 38 .

Data availability
The datasets supporting the conclusion of this article are available through GenBank (SRA accession: PRJNA577839; SUB6429482; accession numbers SRX7007447 -SRX7007521 and can be found at https://www. ncbi.nlm.nih.gov/sra/PRJNA577839 after the release date.