Effects of Streptococcus salivarius K12 on Experimental Periodontitis and Oral Microbiota in Mice

Background: Periodontal diseases comprise a wide range of inflammatory conditions that affect the supporting structures of the teeth, and may lead to severe periodontal destruction and even tooth loss. Streptococcus salivarius K12 (S. salivarius K12), one of oral probiotics, has been reported to be able to inhibit various potentially deleterious bacteria. This study was the first time to investigate the effects of S. salivarius K12 on ligature-induced periodontitis in mice. Methods: A silk ligature was tied around the second left maxillary molar to establish the model of periodontitis, and then, mice in group S. salivarius K12 were administered with S. salivarius K12 (2 × 10 CFU) twice daily for 30 d, the others were treated with isopyknic water. Results: Administration with S. salivarius K12 markedly decreased the anaerobic bacteria accumulation on the teeth, and it also considerably alleviated periodontitis-induced alveolar bone resorption and attachment loss. Moreover, S. salivarius K12 administration increased the relative species abundance of Gram-positive bacterium in oral cavity while decreased Gram-negative bacterium, as well as the diversity of the bacterial community. Conclusion: Oral administration of the probiotic S. salivarius K12 may mitigate the alveolar bone resorption and attachment loss of periodontitis by modulating the oral microbiota.


Introduction
Periodontitis is an inflammatory disease caused by bacterial plaque, the main How to cite this paper: Zhu, L., Li, H., Yang, X.Q., Xue, L., Li, X. and Du, J.R. Journal of Biosciences and Medicines In this study, ligature-induced periodontitis model in mice was adopted to investigate S. salivarius K12's protective ability against periodontitis. Its protective effects were reflected by reduced alveolar bone resorption, attachment loss and osteomorphic integrity. These effects may be mediated through the regulation of S. salivarius K12 on bacterial microenvironment in the oral cavity.

Animals
Male specific-pathogen-free (SPF) C57BL/6 mice, 8 weeks old, were used (Sichuan Provincial People's Hospital Institute of Experimental Animals, Chengdu, China). The mice were housed under a 12 h/12 h light/dark cycle with controlled humidity and temperature. Sterile food and water were available ad libitum. All of the experiments were performed in accordance with the ethical guidelines of the Sichuan University Animal Research Committee.
Fifty-four mice were randomly divided into three groups (n = 18/group): control group (Con; mice without experimental periodontitis [EP], untreated with S. salivarius K12), EP group (mice with EP, untreated with S. salivarius K12), and S. salivarius K12 group (mice with EP, treated with S. salivarius K12). On day 0 of the experiment, silk ligature was placed around the second maxillary molar in the EP group and S. salivarius K12 group.

Ligature-Induced Periodontitis
A 5 -0 silk ligature was tied around the second left maxillary molar to establish the model of periodontitis. Briefly, 5 -0 silk suture was firstly passed through interdentium between first molar and second molar. Then, the suture was passed through interdentium between second molar and third molar. Finally, the suture was tied firmly using a triple-knot. The entire ligature process was performed as gently as possible to avoid secondary injury to the periodontium. The ligatures were inspected once weekly and repositioned if necessary to maintain the ligature in place throughout the experimental period. The contralateral molar remained unligated and served as a baseline control for bone height measurements [19].

Probiotic Treatment
The

Determination of Bacterial Accumulation
One day after the last treatment, the mice were anesthetized by 4% chloral hydrate (i.p.). The ligatures were removed from the second molars and gently washed with phosphate-buffered saline (PBS) to remove food residue and other debris. The ligatures were then placed in Eppendorf tubes with 1 ml of PBS and the bacteria on the ligature were extracted by vortex mixing for 2 min at 3000 rotations per minute [19]. The bacterial suspension was diluted and inoculated on a blood agar plate at 37˚C. After 5 days of anaerobic culture, the living bacterium are predominantly (including facultative anaerobe) anaerobes and the colony forming units (CFU) of living bacterial in each group was calculated [19]. The colony forming unit (CFU/ml) were normalized by dividing CFU by the length (in millimeters) of the corresponding ligature [19].

Bone Loss Measurement
Mice were sacrificed by perfusing ice-cold saline after removing its ligature. The maxilla was detached and soft tissue was removed. The maxilla was fixed in 4% paraformaldehyde for 48 h and then stained with 1% methylene blue [20]. Periodontal bone heights were assessed under a Nikon SMZ1270 microscope (Tokyo, Japan) using a 40× objective. The periodontal bone height was measured from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC). The measurements were performed on the palatal and buccal surfaces of the maxillae as described previously [19]. Five sites of the first molar, three sites of the second molar, and the palatal or buccal cusp of the third molar were evaluated. To calculate the total bone loss, the six-site total CEJ-ABC distance on the ligated side was subtracted from the six-site total CEJ-ABC distance on the contralateral unligated side of the same mouse [19].

Histomorphometric Analysis
The maxillae were decalcified in 10% ethylenediaminetetraacetic acid (pH 7.2) at 4˚C for approximately 4 weeks. The decalcification was accomplished when the needle of a syringe could insert unimpededly into the bone tissue around the teeth. The decalcified maxillae were dehydrated in sequentially increased concentration of ethanol. After that, the maxillae were embedded in paraffin according to standard methods and cut into 4-mm-thick mesio-distal serial sections. Hematoxylin eosin (HE) staining and tartrate resistant acid phosphatase (TRAP) staining were performed with the Hematoxylin-Eosin/HE Staining Kit (G1005, Wuhan Sercicebio Technology, Wuhan, China) and Acid Phosphatase Journal of Biosciences and Medicines Leukocyte Kit (G1050, Wuhan Sercicebio Technology, Wuhan, China), respectively. A panorama of the maxillae, sections of the furcation area of the first maxillary molar, and sections of the alveolar bone between the first molar and second molar were captured by light microscopy (Nikon Eclipse CI, Nikon DS-U3, Tokyo, Japan) [21]. Similar anatomical locations were selected for quantitative measurements in order to avoid the deviation. The distance between the first and second molar was measured of the enamel from the first molar to the second molar at 200× magnification. And the distance of CEJ-ABC, that is, the vertical distance from the midpoint of the two molar enamel to the alveolar crest, was measured at 200× magnification. Multinuclear TRAP-positive cells (i.e., osteoclasts) in the interproximal bone and the furcation of the maxillary first molar were counted at 200× magnification and osteoclast density expressed as number of osteoclasts per mm 2 (IPP).

16S Ribosomal RNA Gene Sequencing
Saliva, obtained from the oral cavity of all mice, were collected by sterile cotton swabs and stored in sterilized equipment at −80˚C. Genomic DNA was extracted using a DNA extraction kit (Sangon, Shanghai, China) according to the manufacturer's instructions. DNA quantification was performed using a Nanodrop 2000 device (Thermo Scientific, Waltham, Massachusetts, USA). Based on the measured concentration, DNA was diluted to 1 ng/ml with sterile water. 16S ribosomal RNA (rRNA)/18S rRNA/ITS genes of distinct regions (16SV4/16SV3/16SV3-V4/16SV4-V5, 18S V4/18S V9, ITS1/ITS2, and Arc V4) were amplified using specific primers with a barcode. All of the polymerase chain reactions (PCRs) were performed with Phusion High-Fidelity PCR Master Mix (New England Biolabs). PCR products were divided by electrophoresis and samples with bright main strip between 400 -450 bp were isolated and purified for further analysis. Sequencing libraries were generated using a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, California, U.S.) The library was then sequenced on an Illumina HiSeq 2500 platform, and 250 bp paired-end reads were generated. Paired-end reads from the initial DNA fragments were merged using FLASH [22]. Sequence analysis was performed using UPARSE software. Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). To understand the diversity and composition of microbial community of the samples, alpha diversity (e.g. richness), beta diversity (weighted and un-weighted UniFrac, namely, principal component analysis) and relative abundance profiles of microbial community were further analyzed.

Statistical Analysis
The data were analyzed using Student's t-test for comparison of two groups, one-way analysis of variance (ANOVA) followed by LSD test and Tamhane's T2 test for multiple groups and KruskalWallis tests when the variance is uneven. Journal of Biosciences and Medicines The data expressed as mean ± SEM. SPSS 20.0 software was used for the statistical analyses. Values of p < 0.05 were considered statistically significant.

S. salivarius K12 Relieves Ligature-Induced Bone Resorption
The appearance of tissue loss and deep periodontal "pocket" are the hallmark of  Figure   1(B)) compared with the EP group. In addition, alveolar bone loss on the palatal side was generally more severe than that on the buccal side in both the S. salivarius K12 group (0.383 mm vs. 0.135 mm) and EP group (0.664 mm vs. 0.301 mm) compared with the Con group (Figure 1(C)).

S. salivarius K12 Reduces Local Accumulation of Anaerobes
To determine the local longitudinal accumulation of anaerobically-grown bacteria in the ligature, bacteria were extracted from the sutures and counted under anaerobic conditions. After 30 d of treatment with S. salivarius K12, anaerobic bacteria in the S. salivarius K12 group decreased by 75% compared with the EP group (7 × 10 4 vs. 28 × 10 4 CFU/mm, p = 0.002, Figure 2), which means that the releasing of harmful substances by pathogens may also be controlled.

S. salivarius K12 Alleviates Ligature-Induced Teeth Pathological Changes
To gain further insights into the modulation effects of S. salivarius K12 on Journal of Biosciences and Medicines   Tartrate-resistant acid phosphatase (TRAP) is a specific marker enzyme of osteoclasts. Through the TRAP staining of the teeth, the alveolar bone resorption and the expression of osteoclasts were observed. As shown in Figure 4, ligature placement significantly increased bone resorption and osteoclast density, while the administration of S. salivarius K12 notably reduced bone resorption and osteoclast density (Figure 4(A) and Figure 4(B)). In addition, TRAP-positive cells were counted in the interproximal bone (Figure 4(C)) and furcation of the maxillary first molar (Figure 4(D)). The amount of osteoclast in the S. salivarius K12 group was 1.39/mm 2 in interproximal bone (p = 0.004, F = 24.642) Figure 4(C)) and 1.85/mm 2 in furcation of the maxillary first molar (p = 0.012, F = 27.770, Figure 4(D)), both lower than those in EP group (2.80/mm 2 and 2.61/mm 2 , respectively).

S. salivarius K12 Changes the Composition of Oral Bacterial Community
The bacterial in saliva was analyzed using 16s rRNA gene sequencing technology EP group. Journal of Biosciences and Medicines to explore the possible mechanism of S. salivarius K12 on periodontitis. The analysis of 16s rRNA gene copy number revealed marked differences between the EP and S. salivarius K12 group, the copy number of the S. salivarius K12 group was 3.9 × 10 4 copies/ng DNA and the EP group was 4.5 × 10 4 copies/ng DNA (p = 0.025, F = 6.021, Figure 5(B)). According to the sequencing results, the content of gram-positive bacteria, such as Lactobacillus, Streptococcus et al., in the Con group and the S. salivarius K12 group was 30%, which was much higher than 13% in the EP group. While the content of gram-negative bacteria, such as Pseudomonas, Paracoccus, Escherichia-Shigella et al., in the EP group was 75.4%, which was much higher than 45.8% in the Con group, whereas S. salivarius K12 dramatically decreased its content to 30% ( Figure 5(C)).

S. salivarius K12 Modulates the Microbial Community Diversity
The microbial diversity within each group (α-diversity) was assessed by calculating the Chao 1 index, and there was a significant decrease in S. salivarius K12-treated periodontitis mice compared with that in EP group (280.4 vs. 369.2, Figure 6(A)), which means the diversity of communities in group S. salivarius K12 has reduced. Principal component Analysis (PCA) was utilized to assess the dissimilarity of individual microbial communities between different groups (β-diversity). The more similar the colony composition of samples, the closer their distances on the PCA map were, thereby demonstrating the validity of the experimental grouping design. In the present study, PCA plotting of weighted β-diversity dissimilarity matrix data, revealed a distinct clustering effect on mice in each group ( Figure   6(B)), as well as the un-weighted β-diversity PCA dissimilarity matrix data ( Figure 6(C)).
LEfSe analysis was used to find species with significant difference between groups. When the LDA value was 4, LEfSe could identify significant differences between the EP and S. salivarius K12 group. It can be clearly seen from Figure  6(D) that the amount of streptococci in the S. salivarius K12-treated group was much higher than that of the EP group.

Discussion
Plenty of evidence has suggested that the colonization of probiotics in biofilms can protect against oral disease [8] [29]. The most famous probiotics, Lactobacilli and Bifidobacteria, have shown obvious advantage in both clinical trials and preclinical studies. However, according to some studies, they may have difficulty in colonizing the mouth [30]. Facing these problems, seeking for substitutes of conventional probiotics is a necessity. Streptococci is known to be a pioneer colonizer of oral surface which would predominantly occupy the biofilm. Teughels et al. demonstrated that some species of Streptococci prominently inhibited periodontopathogens A. actinomycetemcomitans from the colonization of (D) LDA value distribution. Red, taxa in EP group. Green, taxa in S. salivarius K12 group. The data are expressed as mean ± SEM. n = 8. *p < 0.05. epithelial cells in vitro [31]. Van Hoogmoed CG and co-workers found that pre-adhering of Streptococcus strains successfully antagonized adhesion of P. gingivalis [32].
S. salivarius K12 has been marketed internationally by the New Zealand Company BLIS Technologies Ltd (Dunedin, New Zealand) and consumed as a probiotic for more than a decade [ The relative abundance of bacterial species is often related to its function. In the healthy oral cavity, there is a relatively coordinated, dynamic and stable micro-ecology. Imbalance of oral flora will induce chronic inflammatory, such as periodontitis. Since periodontitis is an infective disease that is predominantly caused by gram-negative anaerobes, the proliferation of those bacteria in periodontium results in a host inflammatory response and then leads to alveolar bone loss, which is the most basic phenotypes of periodontitis [38] [40] [41]. In this study, after evaluating the therapeutic effects of S. salivarius K12 on periodontitis, we envisaged the possible mechanism of action may be achieved by the adjustment of oral microbiota and its composition. 16s rRNA sequencing technology was used to analyze salivary flora to explore the possible mechanism, and the results showed that the amount of each kind of bacteria in the three groups was different, but the relative abundance of gram-positive anaerobes in the Con and S. salivarius K12 group were markedly higher than that of the EP group, while the relative abundance of gram-negative anaerobes decreased. This explained in a certain sense that S. salivarius K12 did have a regulatory effect on the oral flora.
Combining with the analysis of microbial diversity, we found that the bacterial diversity of S. salivarius K12-treated mice was dramatically reduced compared However, there is one thing to point out, Lactobacillus, Streptococcus and Bifidobacterium are generally considered as beneficial bacteria, In EP group, the relative abundance of Lactobacillus and Streptococcus decreased as expected, while Bifidobacterium increased a lot. Besides, in S. salivarius K12 group, although S. salivarius K12 treatment reduced the amount of almost all gram-negative bacteria, it inhibited some beneficial bacterial, such as Lactobacillus. That is to say, the introduction of S. salivarius K12 didn't recover the oral microbiota to its original homeostasis, instead, it led the oral microbiota to another constitution. In fact, the oral environment is a homeostasis including both probiotics and pathogens. The efficacy of probiotic therapy on oral disease, sometimes, cannot be illustrated by changes in a single strain, instead, it should be based on changes in the entire microbial environment. This is the charm that microbiology possesses, and it is also a difficult point in this filed.
It should be admitted that the complex interactions between bacteria have yet to be further studied. As far as the current technology is concerned, the RNA sequencing technology can only be accurate to the genus level. If one day, it can be accurate to species or even strains, the authors believe that it would be useful to analyze the role of probiotics on oral diseases.

Conclusion
In conclusion, S. salivarius K12 does have beneficial effects on preventing and alleviating periodontitis, and it may be achieved by adjusting the proportion of gram-positive and gram-negative bacteria in the oral cavity. In short, S. salivarius K12 will have good prospects as an adjuvant for nonsurgical periodontal therapy.