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Introduction
The infectious diseases in oral cavity can be divided two legions that it is a gingival and dental disease, and these infectious diseases are related oral biofilm that exists multi-species1). The biofilm of a healthy person is multi-species balance with high percentage of commensal bacteria. However, when this balance is disrobed and certain bacteria increase, it leads to oral disease2). Actinomyces israelii and Actinomyces naeslundii are considered with gingivitis basis on the epidemiological studies3,4). Streptococcus mutans and Streptococcus sobrinus are known to be cariogenic bacteria5). These bacteria have characteristics of acidogenesis and aciduricity, by which dental caries is induced6). Enterococcus faecalis, the predominant human enterococcus, has been related to oral diseases, such as endodontic infections, periodontitis, and peri-implantitis by characteristics of antibiotics resistance7,8).
Streptococcus salivarius is Gram-positive facultative anaerobe9), and act ac commensal bacteria that colonize mucosal surfaces of human10). Furthermore, this bacterium plays important ecological roles that form a barrier against pathogens and reduce their adhesion and colonization11). Also, some strains are probiotic intended for use in the oral cavity12-14). Bacteria produce and use a substance called bacteriocin to compete for habitat between bacteria, and S. salivarius also produce bacteriocin-like inhibitory substances15). To compete better in the oral ecosystem, S. salivarius produce different kinds of lantibiotics such as salivaricin A, salivaricin B, salivaricin 9, and salivaricin G3216-18). The characteristics of these salivaricins contain lanthinonie and methyllanthionine, and S. salivarius is one of lantibiotics17).
In this study, we investigated antimicrobial activity of different strains of S. salivarius isolated from healthy Korean subjects.
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Materials and Methods
1. Bacterial species and cultivation
Streptococcus salivarius used in this study are two isolated, probiotic, and type strain. Isolated stains are S. salivarius G2 and G7 (formerly KCOM 2122 and 2137) and was kindly donated from Green store Inc. (Seongnam, Gyeonggi, Korea). As comparative strains, S. salivarius K12 and ATCC 7073 (type stain) were used.
2. Susceptibility assay
The antibacterial activity of S. salivarius against A. israelii, A. viscosus, E. faecalis, S. mutans, and S. sobrinus was evaluated by a minimum inhibitory concentration using a microdilution methods according to methods recommended by Clinical and Laboratory Standards Institute (CLSI)19). A milliliters of S. salivarius (1×107 bacteria/ml) was inoculated into 10 ml TSBG, and the bacterial suspension was incubated for 24 h in an aerobic condition. The suspension was centrifuged at 5,000 × g for 10 min, and the supernatant was transferred into a new 15 ml conical tube (SPL Life Sciences, Gyeonggi, Korea). The preparation was filtered with 0.22 μm of a polyvinylidene fluoride (PVDF) filter (Millipore, Billerica, MA, USA). The filtered supernatant as a spent culture medium (SCM) was used to susceptibility assay. 180 μl of TSBG was dispensed into 96-well plate (SPL Life Sciences, Gyeonggi, Korea). The SCM was added into 1st well containing the fresh medium and performed 2-fold serial dilution to the 11th column. A. israelii, A. viscosus, E. faecalis, S. mutans, and S. sobrinus were counted with a bacterial counting chamber (Marienfeld Superior, Lauda-Königshofen, Germany) and adjusted 2×106 bacteria/ml with the broth for each stain. The prepared S. mutans suspension (20 μl) was inoculated into the well containing the mixed media. The plate was incubated at 37℃ in an aerobic incubator. The bacterial growth was measured using optical density at 660 nm of wavelength by a microplate reader (BioTek, Winooski, VT, USA).
3. Statistical analysis
The data was obtained through experiment of three times in duplicate and analyzed Kruskal-Wallis test and Mann-Whitney U test using IBM SPSS statistics Ver. 23 (IBM, Armonk, NY, USA). P-values less than 0.05 were considered statistically significant.
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Results
1. Antimicrobial activity against A. israelii
The antimicrobial activity of S. salivarius against A. israelii was investigated by minimum inhibitory concentration using a microdilution method according to recommended by CLSI. The SCM of S. salivarius type strain significantly inhibited the growth of A. israelii at 2-fold dilution (P<0.05). The SCM of S. salivarius G2 and G7 significantly inhibited the growth of A. israelii at 8-fold dilution and completely inhibited above 4-fold dilution (P<0.05). Finally, the SCM of S. salivarius K12 significantly inhibited the growth of A. israelii at 4-fold dilution and completely inhibited at 2-fold dilution (P<0.05) (Fig. 1).
 | Fig. 1Susceptibility assay of A. israelii for the spent culture medium of S. salivarius. A. israeliis was cultured in BHI broth and inoculated into TSB. After cultivating overnight, susceptibility test of A. israelii for the SCM of S. salivarius was performed according to the protocol of CLSI. The experiments were performed three times in duplicate and the representative data express mean and standard deviation. *Significance compared to untreated control bacteria (P<0.05).
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2. Antimicrobial activity against A. viscosus
Next, in experiment of the susceptibility test of A. viscosus, The SCM of type strain significantly inhibited the growth of A. viscosus at 2-fold dilution (P<0.05). The SCM of G2 and G7 strain significantly inhibited the growth of A. viscosus at 8-fold dilution and completely inhibited above 4-fold dilution (P<0.05). Finally, the SCM of K12 strain significantly inhibited the growth of A. viscosus at 4-fold dilution and completely inhibited at 2-fold dilution (P<0.05) (Fig. 2), As shown Fig. 2, the antimicrobial activity was strong in the order of G7, G2, K12 and type strain.
 | Fig. 2Antimicrobial activity of the SCM of S. salivarius against A. vicosus. A. viscosus was cultivated in BHI broth and inoculated into TSB. After cultivating overnight, antimicrobial activity of the SCM of S. salivarius against A. viscosus was examined according to the protocol of CLSI. The experiments were performed three times in duplicate and the representative data express mean and standard deviation. *Significance compared to untreated control bacteria (P<0.05).
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3. Antimicrobial activity against E. faecalis
When the SCM was investigated antimicrobial activity against E. faecalis as apical periodontitis related bacteria, The SCM of S. salivarius ATCC 7073 as type strain significantly reduced the growth of E. faecalis at 4-fold dilution and completely inhibited the growth of at 2-fold dilution (P<0.05), and the SCM of S. salivarius K12 significantly reduced the growth at 8-fold dilution and completely inhibited at 2-fold dilution (P<0.05). Finally, The SCM of S. salivarius G2 and G7 strain significantly reduced the growth of E. faecalis at 16-fold dilution and completely inhibited above 8-fold dilution (P<0.05) (Fig. 3).
 | Fig. 3Susceptibility assay of E. faecalis for the spent culture medium of S. salivarius. E. faecaliss was cultured in BHI broth and inoculated into TSB. After cultivating overnight, susceptibility test of E. faecalis for the SCM of S. salivarius was performed according to the protocol of CLSI. The experiments were performed three times in duplicate and the representative data express mean and standard deviation. *Significance compared to untreated control bacteria (P<0.05).
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4. Antimicrobial activity against S. mutans
In antimicrobial experiment using the SCM against S. mutans as a cariogenic bacterium, the antimicrobial activity of the SCM showed strong in the order of G2, G7, K12 and type strain (Fig. 4). The SCM of G2 and G7 showed similar antimicrobial activity against S. mutans growth. The SCM of S. salivarius ATCC 7073 as type strain significantly reduced the growth of S. mutans at 8-fold dilution and completely inhibited the growth of at 2-fold dilution (P<0.05), and the SCM of S. salivarius K12 significantly reduced the growth of S. mutans at 8-fold dilution and completely inhibited at 2-fold dilution (P<0.05). Finally, The SCM of S. salivarius G2 and G7 strain significantly reduced the growth of S. mutans at 16-fold dilution and completely inhibited above 8-fold dilution (P<0.05).
 | Fig. 4Antimicrobial activity of the SCM of S. salivarius against S. mutans. S. mutans was cultivated in TSB. After cultivating overnight, antimicrobial activity of the SCM of S. salivarius against S. mutans was examined according to the protocol of CLSI. The experiments were performed three times in duplicate and the representative data express mean and standard deviation. *Significance compared to untreated control bacteria (P<0.05).
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5. Antimicrobial activity against S. sobrinus
The antimicrobial activity of S. salivarius against S. sobrinus was investigated by minimum inhibitory concentration using a microdilution method according to recommended by CLSI. The SCM of S. salivarius type strain significantly reduced the growth of S. sobrinus at 4-fold dilution and completely inhibited the growth at 2-fold dilution (P<0.05), and the SCM of S. salivarius K12 significantly reduced the growth of S. sobrinus at 8-fold dilution and completely inhibited at 2-fold dilution (P<0.05) (Fig. 5). The SCM of S. salivarius G2 and G7 significantly inhibited the growth of S. sobrinus at 16-fold dilution and completely inhibited above 8-fold dilution (P<0.05).
 | Fig. 5Susceptibility assay of S. sobrinus for the spent culture medium of S. salivarius. S. sobrinus was cultured in TSB. After cultivating overnight, susceptibility test of S. sobrinus for the SCM of S. salivarius was performed according to the protocol of CLSI. The experiments were performed three times in duplicate and the representative data express mean and standard deviation. *Significance compared to untreated control bacteria (P<0.05).
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Discussion
S. salivarius is commensal bacteria in oral cavity and detected at high proportions in oral biofilm of healthy person. Also, this bacterium plays an important role in bacterial balance for healthy condition in oral cavity that form a barrier against pathogens and reduce their adhesion and colonization11). Furthermore, some strains are probiotic intended for use in the oral cavity12-14). In this study, the antimicrobial isolated S. salivarius was investigated against Gram-positive bacteria in oral cavity.
In this study, S. salivarius G2 and G7 are previously named KCOM 2122 and 2137, and ownership was changed from Korean collection for Oral Microbiology to Green Store Inc. These stains were isolated strains from healthy person of Korean. Also, comparative strains were used S. salivarius ATCC 7073 as a type strain and K12 as a known probiotic strain. All strains in this study showed the antimicrobial activity against A. israelii, A. viscosus, E. faecalis, S. mutans, and S. sobrinus. In comparing the antimicrobial activity against Gram-positive strains, S. salivarius G7, G2, K12, and ATCC 7073 showed strong in order. These data can be proven that the previous researchers predicted that the characteristics of S. salivarius may be different for each strain.
Most probiotics with antimicrobial activity are Lactobacillus species20). Among Lactobacillus spp, L. rhamnosus, L. acidophilus, and L. casei inhibit the growth of S. mutans20). The inhibition of S. mutans growth by SCM of Lactobacillus spp was several times higher than that of S. salivarius, indicating that Lactobacillus spp might secret stronger bacteriocins. Also, Lactococcus lactis inhibits cariogenic biofilm containing S. mutans21). However, in practice, it is difficult to apply to the oral cavity because it can cause dental caries due to the aciduricity of these bacteria.
The antimicrobial activity of most isolated S. salivarius was investigated using Streptococcus pyogenes as upper a respiratory disease related bacterium15), and their antimicrobial activity was compared. Furthermore, this antimicrobial activity of S. salivarius is appeared by producing bacteriocin-like inhibitory substances which is called salivaricin22). The production of salivaricin type is different for each S. salivarius strain, and the antimicrobial activities and mechanisms of each salivaricin are also different23). The lantibotic nisin A and salivaricin 9 has antimicrobial activity through pore formation on bacterial surface. However, the salivaricin B inhibits formation of peptidoglycan layer22). The antimicrobial activity of S. salivarius was changed by culture condition23). In this study, TSBG was used for cultivating S. salivarius. The antimicrobial activity of S. salivarius was reduced by high glucose and low sucrose condition15). Also, extreme pH reduction inhibits producing salivaricin of S. salivarius21).
In this study, isolated S. salivarius G2 and G7 satisfied one result to be probiotics for oral cavity. It is considered that additional examination such as antibiotic resistance and cytotoxicity should be investigated.
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