Arch Microbiol DOI 10.1007/s00203-014-0998-7

Original Paper

A comparative study of the effect of probiotics on cariogenic biofilm model for preventing dental caries Sung‑Hoon Lee · Young‑Jae Kim 

Received: 22 April 2014 / Revised: 12 May 2014 / Accepted: 16 May 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Dental caries is induced by oral biofilm containing Streptococcus mutans. Probiotic bacteria were mainly studied for effect on the gastrointestinal tract and have been known to promote human health. However, the information of probiotics for oral health has been lack yet. In this study, we investigated influence of various probiotics on oral bacteria or cariogenic biofilm and evaluated candidate probiotics for dental caries among them. The antimicrobial activity of the spent culture medium of probiotics for oral streptococci was performed. Probiotics were added during the biofilm formation with salivary bacteria including S. mutans. The oral biofilms were stained with a fluorescent dye and observed using the confocal laser scanning microscope. To count bacteria in the biofilm, the bacteria were plated on MSB and BHI agar plates after disrupting the biofilm and cultivated. Glucosyltransferases (gtfs) expression of S. mutans and integration of lactobacilli into the biofilm were evaluated by real-time RT-PCR. Among probiotics, Lactobacillus species strongly inhibited growth of oral streptococci. Moreover, Lactobacillus species strongly inhibited formation of cariogenic biofilm

Communicated by Erko Stackebrandt. S.-H. Lee  Department of Oral Microbiology and Immunology, College of Dentistry, Dankook University, Cheonan, Republic of Korea e-mail: [email protected] Y.-J. Kim (*)  Department of Pediatric Dentistry, School of Dentistry, Seoul National University, 28 Yeongeon‑Dong, Jongno‑Gu, Seoul 110‑749, Republic of Korea e-mail: [email protected] Y.-J. Kim  Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea

model. The expression of gtfs was significantly reduced by Lactobacillus rhamnosus. The integration of L. rhamnosus into the biofilm model did not exhibit. However, L. acidophilus and L casei integrated into the biofilm model. These results suggest that L. rhamnosus may inhibit oral biofilm formation by decreasing glucan production of S. mutans and antibacterial activity and did not integrate into oral biofilm, which can be a candidate for caries prevention strategy. Keywords  Probiotics · Cariogenic biofilm · L. rhamnosus · Biofilm inhibition

Introduction Probiotics, which are live microorganisms, provide a health benefit to the host. Many studies have focused on their roles and effects of probiotics on the maintenance of health for the last two decades (Vanderhoof et al. 1999; Meurman and Stamatova 2007; Ghadimi et al. 2008). Lactobacilli are known to play an important role in the maintenance of human health by stimulating a native immunity and protection against infection of the pathogenic bacteria (Gill and Prasad 2008). This has popularized the consumption of foods containing probiotics such as yogurt, cheese, and milk to promote health. Also, Lactobacillus species are known to be the most effective on intestinal health by producing antibacterial agents and stimulating the immune system (Forestier et al. 2001; Botes et al. 2008). Dental caries is directly associated with oral biofilm including Streptococcus mutans (Liljemark and Bloomquist 1996). The development of oral biofilm is formed by stepwise process. Initial biofilm is formed by early colonizers such as Streptococcus gordonii, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus salivarius on pellicle

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and then S. mutans binds to the initial biofilm (Socransky and Haffajee 2002). S. mutans contributes the development of oral biofilm via production of exopolysaccharide as glucan. S. mutans uptake sucrose to synthesize glucan through three glucosyltransferase such as GtfB, GtfC, and GtfD (Monchois et al. 1999; Kreth et al. 2008), and these glucans play a role in oral biofilm formation (Koo et al. 2010). Therefore, S. mutans is considered the most important contributor to the formation of mature biofilm in oral cavity. Also, S. mutans is an aciduric bacterium. When the development of oral biofilm is formed, the level of pH in the biofilm is reduced about pH 4.0 by bacterial metabolism. Oral bacteria can not grow in lower pH except S. mutans (Marsh and Bradshaw 1997). Eventually, cariogenic biofilm is formed. For the reason of these, control of oral biofilm is important for the care of oral health and the prevention of dental caries. However, oral biofilm is hardly controlled by chemical reagents due to the barrier of oral biofilm with exopolysaccharides, proteins, and cell debris (Socransky and Haffajee 2002). A few studies reported the inhibitory effect of lactobacilli on dental caries, inhibiting the biofilm formation and growth of S. mutans (Testa et al. 2003; Hasslof et al. 2010; Pham et al. 2011), and the antibacterial activity of lactobacilli for planktonic bacteria in the oral cavity has been reported. However, lack of evidence regarding the influence of lactobacilli on oral biofilm has brought controversy about the beneficial effects of lactobacilli against dental caries to date because they have been not studied effect on oral biofilm formed by multi-species bacteria and are acidogenic and aciduric bacteria to provoke cariogenic process (Meurman et al. 1995; Pham et al. 2011; Takahashi and Nyvad 2011). Thus, the aim of this study was to investigate the effects of various probiotics on oral streptococci and oral biofilm using salivary bacteria and to present inhibitory mechanism of biofilm formation. Also, it is to come up with the probiotics for prevention of dental caries.

Materials and methods Bacterial strain and cultivation Bifidobacterium bifidum ATCC 29512, Lactobacillus acidophilus ATCC 4356, Lactobacillus brevis ATCC 14869, Lactobacillus casei ATCC 334, and Lactobacillus rhamnosus GG (ATCC 53103) were cultured with Lactobacilli De Man Rogosa Sharpe (MRS) broth at 37 °C in anaerobic condition. Lactococcus lactis and Streptococcus thermophilus were cultivated into M17 medium (BD bioscience, San jose, CA, USA). S. gordonii ATCC 10558, S. oralis ATCC 9811, S. sanguinis 804, and S. mutans ATCC 25175 were cultured with trypticase soy broth (TSB) at 37 °C in an anaerobic atmosphere (H2 5 %, CO2 10 %, and N2

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Arch Microbiol

85 %). For formation of oral biofilm, salivary bacteria were cultured in brain–heart infusion (BHI) broth supplemented with 1 % sucrose and 1 % mannose anaerobically. Preparation of spent culture medium of probiotics Probiotics were cultivated in each specific broth at 37 °C for 36 h, and then cultured probiotics were centrifuged at 5,000×g for 10 min at 4 °C. The supernatant was transferred into a new tube and filtrated through polyvinylidene fluoride (PVDF) filter with a pore size of 0.22 μm (Millipore Co., Billerica, MA, USA). Antimicrobial activity of probiotics against oral streptococci The antimicrobial activity of spent culture medium of probiotics against S. gordonii, S. mutans, S. oralis, and S. sanguinis as the member of cariogenic biofilm was performed Kirby–Bauer disk diffusion test according to the recommendations of Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS). Briefly, oral streptococci were cultivated into TSB at 37 °C in an anaerobic chamber for 24 h. The bacterial suspension was plated on trypticase soy agar, and the soaked cotton disk in probiotics—cultured medium—was then placed the agar. The plate was incubated at 37 °C for 36 h in anaerobic chamber. Also, the minimum inhibitory concentration assay was carried out using 96-well plate. In total, 180 μl of fresh BHI broth was put into a sterile 96-well round-bottomed polystyrene microtitre plate (SPL Lifescience, Gyeonggi, Korea). In total, 180 μl of spent culture medium of lactobacilli was added to the first row of the plate, and then, twofold serial dilutions were performed by multi-channel micropipette. Control wells contained of MRS broth only (negative control). The number of bacteria was counted by Petroff–Hasser counting chamber (Hausser Scientific) and then diluted to 2 × 106 cells/ml with BHI broth. The bacterial suspensions (20 μl; 1 × 105 cells) were inoculated into the plates. The plates were incubated at 37 °C overnight, and the optical density was measured at 660 nm by microplate reader (SpectraMax M2; Molecular devices, Sunnyvale, CA). Preparation of conditioning plate for biofilm formation Pooled saliva of twelve healthy donors was centrifuged at 8,000× for 10 min 4 °C, and then, the supernatant was filtrated with 0.22 μm polyvinylidene fluoride (PVDF) membrane. The saliva was diluted to twofold using phosphatebuffered saline (PBS; pH 7.2) and dispensed to 8-well glass slip and polystyrene 12-well plate. The slip and the plate were dried on 37 °C and sterilized in UV sterilizer. These procedures were repeated seven times.

Arch Microbiol

Formation and observation of cariogenic biofilm model Salivary biofilm was formed according to the method of described by Lee et al. (2012) Briefly describing, unstimulated saliva was collected from 12 healthy donors, pooled in equal proportions and mixed with BHI broth supplemented with 1 % sucrose and 1 % mannose. The mixture was centrifuged at 1,500×g for 10 min at 4 °C to remove oral debris. The supernatant of the mixture was transferred into three new tubes, and then, total bacteria in the supernatant were counted with Petroff–Hasser counting chamber (Hausser Scientific, Horsham, PA, USA) and added S. mutans (1/400 or 1/800 of total bacteria) to generate cariogenic biofilm because saliva in adults scarcely had S. mutans. The mixture was vortexed for 15 s. In total, 1 ml and 400 μl of the mixture were inoculated into a salivacoated polystyrene 12-well plate and a saliva-coated 8-well glass chamber with or without L. acidophilus, L. casei, or L. rhamnosus in various concentrations, respectively, and incubated at 37 °C for 48 h anaerobically. Thereafter, the saliva-coated 12-well polystyrene plate was washed three times with (PBS) to remove non-adherent bacteria and subjected to mechanical disruption, serial dilution, and plating on BHI agar plate and Mitis-salivarius bacitracin (MSB) agar plate to count whole bacteria and S. mutans in the biofilm, respectively. The plates were incubated at 37 °C for 36 or 48 h in anaerobic chamber, and CFU was counted. In order to observe biofilm formation, 8-well glass chamber was washed three times with PBS and stained with SYTO® 9 dye (Invitrogen, Eugene, OR, USA) according to the manufacturer’s instructions. The biofilm was visualized by LSM 5 Pa confocal laser scanning microscope (Zeiss, CarlZeiss, Oberkochen, Germany) using z-stack scans from 0 to 30 μm. In order to measure depth of biofilm, the z-stack scans of six randomly chosen location were taken per sample to decide the average depth of biofilm (Lee et al. 2012). Effect of lactobacillus on glucan expression of S. mutans Glucan production of S. mutans was indirectly analyzed using comparison of gtfs expression. S. mutans was cultivated into BHI including 1 % sucrose with or without spent medium of L. acidophilus, L. casei, or L. rhamnosus in nonkilling concentration (1.56 %) of S. mutans for 18 h. The bacteria were washed three times with PBS. Total RNA was then isolated with a TRIzol® Max bacterial RNA isolation kit (Invitrogen Life Tech, Carlsbad, CA) according to the manufacturer’s instructions. cDNA was synthesized by mixing total RNA (1 μg) and Maxime™ RT Premix (Random primer; iNtRON, Gyeonggi, Korea) in a 20 μl reaction volume and incubating the mixture at 45 °C for 1 h. cDNAs were mixed with 10 μl of SYBR Premix Ex Taq and 0.4 μM of each primer pair in 20 μl final volume and subjected to

40 PCR cycles (95 °C for 15 s, 60 °C for 15 s, and 72 °C for 33 s) with ABI-Prism 7500 real-time PCR (Applied Biosystems, Foster City, CA, USA). The PCR products were investigated for each amplification product using a dissociation curve of amplification. 16S rRNA, a housekeeping gene, was used as a reference to normalize expression levels and to quantify changes of gtfs expression between non-treated and treated the spent medium. The sequences of primers for real-time RT-PCR were as follows : 5′-AGC AAT GCA GCC AAT CTA CAA AT-3′ and 5′-ACG AAC TTT GCC GTT ATT GTC A-3′ for the GtfB gene; 5′-CTC AAC CAA CCG CCA CTG TT-3′ and 5′-GGT TAA CGT CAA AAT TAG CTG TAT TAG C-3′ for the GtfC gene; 5′-CAC AGG CAA AAG CTG AAT TAC A-3′ and 5′-GAT GGC CGC TAA GTC AAC AG-3′ for the GtfD gene; and 5′-GAA AGT CTG GAG TAA AAG GCT A-3′ and 5′-GTT AGC TCC GGC ACT AAG CC-3′ for 16S rRNA gene. Detection of probiotics in cariogenic biofilm model In order to investigate integration of probiotics in cariogenic biofilm model, quantitative real-time PCR was performed using specific primer of probiotics. Salivary bacteria including S. mutans were formed on 12-well plate with probiotics and then extracted DNA of total bacteria in the biofilm by G-spin Genomic DNA extraction kit (iNtRON Biotech., Gyeonggi, Korea). DNA was mixed with 10 μl of SYBR Premix Ex Taq and 0.4 μM of each primer pair in 20 μl final volume. The condition of real-time PCR was 40 PCR cycles at 94 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 33 s, and performed with ABI-Prism 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The sequences of primers for real-time RT-PCR were as follows : 3′-GAA AGA GCC CAA ACC AAG TGA TT-5′ and 3′-CTT CCC AGA TAA TTC AAC TAT CGC TTA-5′ for L. acidophilus gene; 3′-CTA TAA GTA AGC TTT GAT CCG GAG ATT T-5′ and 3′-CTT CCT GCG GGT ACT GAG ATG T-5′ for L. casei gene; 3′-GCA GTC GTT AGC CAC CCG AA-5′ and 3′-ACT CCT GTA ATT GCC AGC CG-5′ for L. rhamnosus gene; and 5′-TGG AGC ATG TGG TTT AAT TCG A-3′ and 5′-TGC GGG ACT TAA CCC AAC A-3′ for Universal primer. The standard curve was generated by using given each lactobacilli spp count in different concentrations (1 × 103–107) and cycle threshold (Ct) of the amplified DNA of the lactobacilli. The count of L. acidophilus, L casei, and L. rhamnosus colonization in the biofilm model was then calculated from the standard curve. Statistical analysis The statistical analysis was performed with Kruskal–Wallis test and Mann–Whitney test using SPSS 10 (SPSS Inc,

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Arch Microbiol

Chicago, IL, USA). p values