1.Background

Streptococcus mutans is a gram-positive facultative anaerobic¹⁾ and acidogenic bacterium that produces lactic acid in the final stage of sugar fermentation²⁾. S. mutans degrades sucrose into glucose by producing the enzyme glucosyltransferase³⁾, and utilizes glucose to produce exo-polysaccharide, which helps in the formation of biofilm⁴⁾. Insoluble glucan is the main component of biofilm and allows bacteria to attach to the tooth surface⁵⁾. S. mutans produces acid through fermentation of sucrose within the biofilm to create an acidic environment. This initiates and promotes enamel demineralization, eventually resulting in dental caries¹⁾. Under homeostasis, only 2% of S. mutans exist in biofilm, however in an environment with addi-tional sucrose, the activity and growth of acidogenic and aciduric bacteria, mainly S. mutans and L. casei are enhanced⁶⁾. Lactobacillus is a genus of acid-resistant bac-teria that accounts for 1% of cultivatable human oral microflora⁷⁾. It includes L. casei, L. paracasei, L. plan-tarum, Lrhamnosus, L. fermentum, and L. acidophilus⁸⁾. Members of genus Lactobacillus colonize the oral tissues and coagulates with other bacteria adjacent to it, forming a biofilm⁹⁾, further acidifying the surrounding environment and accelerating dental caries. They are known to be involved in advanced dental caries¹⁰⁾. Additionally, some species belonging to Lactobacillus demonstrate inhibitory effects on S. mutans, and have anti-caries potential¹¹⁾, thus, interest in Lactobacillus has increased¹²⁾. Representative pathogens associated with periodontitis are Porphyro-monas gingivalis, Treponema denticola, Tannerella forsy-thia and A. actinomycetemcomitans⁷⁾. A. actinomycetem-comitans is known to inhabit the subgingival region and has various virulent characteristics that can damage perio-dontal tissues⁷⁾.

Dental plaque is a pathogenic biofilm deposited on the tooth surface and hosts a complex oral flora in which S. mutans and caries-related bacteria produce acids³⁾. The removal of dental plaque is the most effective way to control dental caries and periodontitis¹⁾. The most com-monly recommended method for dental plaque removal is mechanical plaque control, tooth brushing, however adju-nctive methods using anti-plaque agents such as chlor-hexidine, zinc chloride and antibiotics may also be used¹⁾. When it is difficult to expect successful results with scaling or root planning alone, adjunctive therapies using antibiotics or probiotics are useful¹³⁾. However, long-term use of chlorhexidine, zinc chloride and antibiotics are not harmless to the human body and is associated with side effects such as tooth staining, adverse effects on the beneficial microflora, and the development of antibiotic- resistant bacteria¹⁴⁾. Recently, interest in using probiotics which have no side effects, are beneficial to health and have inhibitory effects on oral pathogenic bacteria, is increasing¹⁵⁾. Probiotics were defined by the World Health Organization (2014) as live microorganisms that confer a health benefit to human when ingested in adequate amou-nts¹³⁾. They are added to commercial dairy products such as milk, yogurt, and cheese¹⁾. Most probiotics are gram- positive bacteria belonging to the genus Lactobacillus or Bifidobacterium, and the species that mainly founded in the oral cavity are L. acidophilus, L. casei, L. fermentum, L. rhamnosus and L. salivarius⁷⁾. Although Lactobacillus has been reported to reduce the number of S. mutans in saliva and dental plaque^15,16), the effect is not always confirmed in vitro and in clinical studies^17,18), rather Lactobacillus has been identified as a risk factor for dental caries^19,20). However, the findings remain ambiguous and show a lacks of sufficient understanding. Additionally, the effect of S. mutans, L. casei and A. actinomycetemc-omitans based on the sucrose concentration and their interrelationship has not been studied previously.

2.Objectives

Our in vitro study aimed to reveal microbial and biochemical changes in single cultures of S. mutans, L. casei and A. actinomycetemcomitans and co-cultures of S. mutans and L. casei or A. actinomycetemcomitans according to sucrose concentration. Additionally, we attempted to confirm the anti-oral bacterial and anti-biofilm activities of L. casei and A. actinomycetemcomitans against S. mutans according to sucrose concentration.

1.Study design

S. mutans, L. casei, and A. actinomycetemcomitans were purchased from Korea Microbiological Conservation Center and Korean Collection for Type Cultures (Table 1). Bacteria were cultured in a selective medium at 36.5°C for 24 hours in an incubator (Daihan Scientific Co., Daegu, Korea) and diluted to 5×10⁶ CFU/ml. Single culture groups of S. mutans, L. casei and A. actinomycetemcomitans and co-culture groups in which 10%, 20%, 30% and 40% of L. casei or A. actinomycetemcomitans were applied to S. mutans were used for the study. In order to confirm the changes according to the concentration of sucrose, 0%, 1%, and 5% sucrose (Samchun Chemicals, Pyeongtaek, Korea) was added to each selective medium, and then incubated at 36.5°C for 24 hours. Values of bacterial growth were measured at a wavelength of 600 nm using a UV-Vis spectrophotometer (Human Corp., Seoul, Korea).

Table 1

Bacterial Strains and Culture Conditions

Oral bacteria	Strain	Culture media	Aero condition
Streptococcus mutans	KCCM 40105	BHI agar and broth (MB Cell Ltd., Seoul, Korea)	Facultative anaerobic
Lactobacillus casei	KCCM 12452	MRS broth (MB Cell Ltd., Seoul, Korea)	Facultative anaerobic
Aggregatibacter actinomycetemcomitans	KCTC 2581	BHI broth (MB Cell Ltd., Seoul, Korea)	Microaerophilic

KCCM: Korea Microbiological Conservation Center, KCTC: Korean Collection for Type Cultures, BHI: brain heart infusion.

Using the modified method of Somanah et al.²¹⁾, the single and the co-culture groups with oral bacteria diluted 5×10⁶ CFU/ml were cultured in a selective medium containing 0%, 1% and 5% sucrose at 36.5°C for 24 hours. The pH of the media were measured using a pH meter (TTBH Pte Ltd., Petro Centre, Singapore).

According to the method of O’Toole et al.²²⁾, S. mutans, L. casei and A. actinomycetemcomitans diluted to 5×10⁶ CFU/ml were single cultured, and S. mutans and L. casei or A. actinomycetemcomitans were applied at concentra-tions of 10%, 20%, 30% and 40% to S. mutans and were co-cultured with selective medium containing 0%, 1% and 5% sucrose in 96-well plates at 36.5°C for 24 hours. After removing the medium from the 96-well plate, the wells were washed twice with distilled water and stained with Crystal violet (MB Cell Ltd., Seoul Korea) diluted to 0.1% in H₂O for 15 minutes at room temperature. This was followed by treatment with 95% ethanol and measurement at 570 nm using a microplate reader (Sunrise TM, Tecan, Grödig, Austria).

To measure the antibacterial activity of L. casei and A. actinomycetemcomitans against S. mutans, the modified method of disk diffusion assay by Jeong et al.²³⁾ was used. After spreading 100 µL of S. mutans on the brain heart infusion agar plate, the paper disks were treated with L. casei and A. actinomycetemcomitans in specific ratios was placed on the agar plate and incubated at 36.5°C for 24 hours. The diameter of the formed clear zone was measured using a Vernier caliper (Mitutoyo Co., Kana-gawa, Japan). Penicillin G antibiotic discs (Oxoid Ltd., Hampshire, United Kingdom) were used as control.

2.Statistical analysis

Experiments were independently performed in triplicate. Results were analyzed using SPSS 25.0 (SPSS Inc., Chi-cago, IL, USA) and a one-way analysis of variance was performed. The post-hoc analysis was performed using Tukey’s test, and the significance level was 0.0073.

1.Microbial and biochemical changes in single oral bacterial culture according to sucrose concentration

The growth value, biofilm formation and acid production by S. mutans, L. casei and A. actinomyce-temcomitans, each single cultured in selective medium containing 0%, 1%, and 5% sucrose were compared and analyzed (Fig. 1). The growth value of S. mutans signi-ficantly increased with the increase in the sucrose concen-tration, while that of L. casei was not affected by the concentration of sucrose. The growth of A. actinomycete-mcomitans was significantly increased in the medium containing 1% sucrose, and decreased in 5% sucrose compared with that in 1% (Fig. 1A). Biofilm formation of S. mutans and L. casei was the highest in the medium containing 1% sucrose and decreased in 5% sucrose, however that of A. actinomycetemcomitans wase not affected by the concentration of sucrose (Fig. 1B). The pH due to acid production by S. mutans was 6.0 in a medium containing 0% sucrose, however, those in media con-taining 1% and 5% sucrose were 4.2 and 4.3, respectively (Fig. 1C). The addition of sucrose induced rapid acid production by S. mutans independent of sucrose concen-tration. L. casei and A. actinomycetemcomitans showed a constant pH value regardless of the sucrose concentration (Fig. 1C).

Fig. 1. Microbial and biochemical changes in single oral bacterial culture according to sucrose concentration. (A) Growth value. (B) Biofilm formation. (C) Acid production. *p<0.05, **p<0.01 compared with 0% sucrose group. ^#<0.05, ^##<0.01, ^###<0.001 compared with 1% sucrose group.

2.Microbial and biochemical changes in oral bacterial co-culture according to sucrose concentration

Comparative analysis results of the growth value, bio-film formation and acid production by co-culture groups with S. mutans and L. casei or A. actinomycetemcomitans applied at 10%, 20%, 30%, and 40% to S. mutans in selective meda containing 0%, 1%, and 5% sucrose are shown in Fig. 2. In media containing 0% sucrose, 30% and 40% of L. casei significantly reduced the growth of S. mutans, and it was confirmed that L. casei had anti-bacterial activity against S. mutans, however, bacterial growth in a co-culture of S. mutans and L. casei with medium containing 1% and 5% sucrose increased as the sucrose concentration increased. In the co-culture of S. mutans and A. actinomycetemcomitans, bacterial growth increased as the ratio of A. actinomycetemcomitans increased in the medium containing 1% sucrose, however, the value decreased and did not change in the medium containing 5% sucrose (Fig. 2A). Biofilm formations were decreased in the co-culture group with 30% and 40% of L. casei of S. mutans in medium containing 0% and 1% sucrose, however, no change was observed in 5% sucrose medium (Fig. 2B). In the co-culture of S. mutans and A. actinomycetemcomitans, the biofilm formations were reduced by 20% and 30% of A. actinomycetemcomitans in the medium containing 1% sucrose (Fig. 2B). The value of acid production in S. mutans and L. casei or A. actino-mycetemcomitans co-cultures were pH 5.9 in the medium containing 0% sucrose, however, the values gradually decreased and acidified as the ratio of L. casei to S. mutans increased. That of S. mutans and A. actinomycete-mcomitans did not change in even when the ration A. actinomycetemcomitans to S. mutans was increased (Fig. 2C). In contrast, co-culture of S. mutans with L. casei and A. actinomycetemcomitans in medium containing 1% sucrose acidified to the lowest pH 4.2 did not change regardless of ratio of L. casei and A. actinomyce-temcomitans to S. mutans (Fig. 2C). The clear zone formed around paper disks treated with 10%, 20%, 30% and 40% L. casei and A. actinomycetemcomitans to S. mutans on an agar plate containing 0% sucrose was confirmed (Fig. 3, Table 2). Only 30% and 40% of L. casei showed susceptible anti-oral microbial activity against S. mutans, however, A. actinomycetemcomitans did not demo-nstrate anti-oral bacterial activity against S. mutans (Fig. 3, Table 2).

Table 2

Anti-oral Bacterial Activity of Lactobacillus casei and Aggregatibacter actinomycetemcomitans against Streptococcus mutans according to Bacterial Ratio

S. mutans	L. casei ratio (%)					Penicillin G (10 Units)c
	0a	10a	20a	30b	40b
	A. actinomycetemcomitans ratio (%)
	0a	10a	20a	30a	40a

^aresistant (<5 mm), ^bsusceptible (6∼14 mm), ^cmore susceptible (15∼24 mm).

Fig. 2. Microbial and biochemical changes in oral bacterial co-culture according to sucrose concentration. (A) Growth value. (B) Biofilm formation. (C) Acid production. **p<0.01, ***p<0.001 compared with the 0% sucrose group. ^##p<0.01 compared with 1% sucrose group of 20% L. casei. ⁺⁺⁺p<0.001 compared with the 1% sucrose group of 10% A. actinomycetemcomitans.

Fig. 3. Anti-oral bacterial activity of Lactobacillus casei (Lc) and Aggre-gatibacter actinomycetemcomitans (Aa) against Streptococcus mutans according to bacterial ratio. P: Pe-nicillin G.

1.Key results and Interpretation

In single cultures, S. mutans was sensitive to the sucrose in the medium. Both bacterial growth and biofilm formation were increased in a medium containing 1% sucrose com-pared to that in one with 5% sucrose, and the presence of sucrose in the medium rapidly acidified the S. mutans-cul-ture, regardless of the concentration of sucrose. The growth and biofilm formation of L. casei and A. actinomycete-mcomitans were increased in a medium containing 1% sucrose, and those of 5% sucrose were similar or decreased. The results of single culture show that an environment containing sucrose can increase pathogenic bacterial activity and initiate oral diseases such as dental caries and perio-dontitis. In the S. mutans and L. casei co-culture, 30% and 40% of L. casei in the medium containing 0% sucrose showed anti-oral bacterial activity against S. mutans, and those in the medium containing 0% and 1% sucrose demo-nstrated anti-biofilm activity. In the S. mutans and A. acti-nomycetemcomitans co-culture, 20% and 30% of A. actino-mycetemcomitans in the medium containing 1% sucrose showed anti-biofilm activity against S. mutans. In contrast, in the co-culture of S. mutans and L. casei or A. actinomyce-temcomitans in a medium containing sucrose, bacterial growth and biofilm formation increased.

2.Comparison with previous studies

Changes in the oral environment caused by frequent intake of carbohydrates enhance the growth and activity of acidogenic and aciduric bacteria, mainly S. mutans and Lactobacillus⁶⁾. In S. mutans, acid production and biofilm accumulation are maximized in an environment with the range of 1∼5% sucrose²⁴⁾. Probiotics such as L. sali-varius, L. plantarum, L. rhamnosus, and L. fermentum have better antimicrobial activity in an environment with 1∼2% glucose¹³⁾. According to our in vitro study, bacterial growth and biofilm formation increased in single and co-cultures of S. mutans, L. casei, and A. actinomycete-mcomitans in meda containing sucrose. Our study first suggested that the growth and biofilm formation by A. actinomycetemcomitans were affected by sucrose. In pre-vious in vitro studies, L. plantarum⁹⁾, L. acidophilus¹²⁾ and L. reuteri²⁵⁾ inhibited the biofilm formation by S. mutans. L. fermentum²⁶⁾ and L. reuteri²⁵⁾ reduced the growth of S. mutans according to the ratio of application. Moreover, L. reuteri, when cultured to a concentration of approximately three times that of S. mutans, has been reported to kill more than 90% of S. mutans²⁷⁾. Previous in vivo studies indicated that the risk of initiation and progression of early dental caries decreased in elementary school students who consumed milk supplemented with L. rhamnosus and Bifidobacterium longum²⁸⁾. Intake of tablets²⁵⁾ and yogurt supplemented with L. reuteri²⁷⁾ were also reported to reduce the number of S. mutans and risk of dental caries. The oral probiotic Lactobacillus was reported to inhibit bacterial growth and biofilm formation against S. mutans through the production of acid or bacteriocin-like poly-peptides or both²⁹⁾ and the low pH environment, by modu-lating acid production and/or oxidation-reduction potential, that were reported to be important factors in the establishment and inhibition of growth of pathogens^7,12). In our study, 30% and 40% of L. casei in a medium containing 0% sucrose showed both anti-oral bacterial and anti-biofilm activities against S. mutans, similar to previous findings. However, in our study, the acid produ-ction of single culture and co-culture of S. mutans, L. casei and A. actinomycetemcomitans increased with addition of sucrose, regardless of the ratio of L. casei and A. actino-mycetemcomitans to S. mutans and the concentration of sucrose. Therefore, it can be suggested that the anti- bacterial and anti-biofilm activities of L. casei against S. mutans are due to other anti-microbial mechanisms than the acidic environment caused by the production of orga-nic acid and hydrogen peroxide by L. casei. The inhibitory effect of L. casei against S. mutans may be due to the production of bacteriocin or bacteriocin-like polypeptides, which destroy the cell membrane of S. mutans^9,12) and production of biosurfactant³⁰⁾, as well as the auto-aggre-gation and co-aggregation ability to compete with S. mutans for nutrition or adhesive surface^31,32) and the ability of anti-biofilm formation through the inhibition of sucrose decomposition of S. mutans⁹⁾.

3.Limitations

This study was conducted in vitro and had limitations in that our findings cannot reflect the diverse and complex bacterial ecosystem of the oral cavity. Further studies to unravel not only the mechanisms by which L. casei or A. actinomycetemcomitans induce anti-oral bacterial and anti-biofilm activities against S. mutans, but also the relationship between probiotics and periodontal disease- causing bacteria must be conducted.

4.Suggestion

In a medium containing sucrose, the growth and biofilm formation by S. mutans, L. casei and A. actinomycetemco-mitans increased. Contrastingly, 30% and 40% of L. casei in the medium containing 0% sucrose showed both anti- oral bacterial and anti-biofilm activities. This indicates that in a 0% sucrose environment, L. casei can be used as probiotic therapy to reduce S. mutans. The concentration of sucrose in the oral environment is important for the control of bacteria causing dental caries and periodontal disease. To apply probiotic therapy using L. casei for S. mutans reduction, the concentration of sucrose must be considered.

This work was supported by Youngsan University Research Fund of 2022.

No potential conflict of interest relevant to this article was reported.

This article dose not require IRB screening, because commercially available bacterial strains were used.

Raw data is provided at the request of the corresponding author for reasonable reason.