Distribution of Salivary Lactobacillus and Bifidobacterium Species in Periodontal Health and Disease

Transcription

1 Biosci. Biotechnol. Biochem., 71 (1), , 2007 Distribution of Salivary Lactobacillus and Bifidobacterium Species in Periodontal Health and Disease Kenichi HOJO, 1;2;y Chinami MIZOGUCHI, 1 Naoki TAKETOMO, 1 Tomoko OHSHIMA, 2 Kazuhiro GOMI, 3 Takashi ARAI, 3 and Nobuko MAEDA 2 1 Food Science Institute, Meiji Dairies Corporation, 540 Naruda, Odawara, Kanagawa , Japan 2 Department of Oral Bacteriology, Tsurumi University School of Dental Medicine, Tsurumi, Yokohama, Kanagawa , Japan 3 Department of Periodontics and Endodontics, Tsurumi University School of Dental Medicine, Tsurumi, Yokohama, Kanagawa , Japan Received July 27, 2006; Accepted October 23, 2006; Online Publication, January 7, 2007 [doi: /bbb.60420] We surveyed the distribution of salivary Lactobacillus and Bifidobacterium species in periodontitis patients and healthy subjects. Approximately 700 lactobacilli and 300 bifidobacterial isolates were obtained from 16 young, orally healthy subjects (mean age standard deviation: 21:0 2:0 y), 16 periodontitis patients (51:6 13:8 y), and 14 well-maintained former periodontitis patients (60:2 9:6 y). Among eleven Lactobacillus species detected in saliva, L. salivarius, L. gasseri, and L. fermentum were prevalent, but no species was specifically associated with periodontal health. In contrast, of four Bifidobacterium species, B. adolescentis was specifically (P < 0:05) prevalent in young healthy subjects compared with the other two groups. Furthermore, the bifidobacterial count of the well-maintained subjects was the highest (P < 0:05) among the groups. These results suggest that bifidobacterial count and species might be associated with periodontal health status and/ or age. Key words: Lactobacillus; Bifidobacterium; probiotics; periodontitis; salivary microbiota Lactobacilli and bifidobacteria naturally colonize the vagina and digestive tract, where they are thought to protect against invasive pathogens. 1) Lactobacilli present in the vagina help maintain a low ph environment through their fermentative activities, and strains that produce hydrogen peroxide may be primarily responsible for preventing vaginal colonization by pathogenic species that cause bacterial vaginosis. 2,3) Furthermore, the administration of lactobacilli and bifidobacteria has been reported to prevent and/or help cure intestinal disturbances, infections, and diarrhea. 4 6) In contrast to these beneficial aspects, Lactobacillus and Bifidobacterium species present in the oral cavity have been a focus of concern among dental researchers as a cause of dental caries. As well as the considerable epidemiologic evidence linking them to dental caries, they are often detected in caries lesions. 7 10) Both genera are associated with carious dentine, and Lactobacillus is associated more with the advancing front of caries lesions than with the initiation of the dental caries process. Surprisingly, however, a few recent reports have suggested that lactobacilli and bifidobacteria can prevent oral infectious diseases such as caries 11 13) and periodontal disease ) For example, the administration of a dairy product containing L. rhamnosus appeared to reduce the risk of dental caries in a randomized, double-blind, placebo-controlled trial. 12) This trial demonstrated reduced levels of dental caries and lower mutans streptococcal counts in patients after seven months of administration of L. rhamnosus. It has been suggested that intake of L. salivarius decreases the risk of periodontal disease by eliminating black-pigmented anaerobic rods (BPARs), which have been suspected to play an unfavorable role in periodontal disease. 16) Ishikawa et al. 16) reported that in an in vitro co-culture system, L. salivarius killed BPARs, Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens within 24 h. In the clinical study carried out by these authors, daily intake of tablets containing L. salivarius decreased the number of BPARs in volunteers saliva after 4 weeks of administration. Although these approaches suggest that Lactobacillus and Bifidobacterium species or strains may bring about dental improvement, the available information is limited. In particular, there is no evidence that the lactobacilli and bifidobacteria species naturally present in the oral cavity are associated with periodontal health. In order to determine whether some or all of the indigenous species within these genera exert a protective y To whom correspondence should be addressed. Fax: ; kenichi houjou@meiji-milk.com

2 effect on periodontopathogens, a systematic comparison of periodontitis patients and healthy subjects is required. The purpose of this study was to determine the salivary counts of Lactobacillus and Bifidobacterium in healthy subjects and periodontitis patients, and to compare the prevalence of the oral bacterial species. Materials and Methods Subjects. Subjects were recruited from dental students at the School of Dental Medicine of Tsurumi University and outpatients at the dental clinic of that university. All of the subjects gave their written informed consent to participation in the study. The Ethical Committee of Tsurumi University and that of Meiji Dairies Corporation approved the study protocol. Young, orally healthy dental students were enrolled in a group consisting of 16 subjects (6 males and 10 females) aged 19 to 27 (mean age standard deviation: 21:0 2:0 years old), who did not exhibit carious teeth or bleeding on probing and had probing depths of less than 3 mm. The outpatients were divided into two groups, a periodontitis group and a well-maintained group, based on the progress of clinical treatment. The periodontitis group consisted of 16 subjects (6 males and 10 females) aged 34 to 70 (51:6 13:8 years old) who had received only oral hygiene instructions or were under treatment with conventional periodontal therapy (scaling and root planing). The mean count of sites with probing depths between 4 mm and 10 mm was 11:9 8:7. The wellmaintained group consisted of 14 subjects (4 males and 10 females) aged 47 to 76 (60:2 9:6 years old) who had completed therapy and were undergoing periodontal maintenance care. Their probing depths were between 2 mm and 5 mm. None of these subjects had received antibiotic medication for at least one month prior to this study. The subjects were asked not to consume products containing lactobacilli or bifidobacteria, such as fermented milk and pickled vegetables, for at least one week before the sampling of saliva. Clinical recording. The Silness and Löe plaque index was used to assess plaque deposits on the gingival areas of the index teeth (teeth nos. 16, 12, 24, 36, 32, and 44). The index was scored on the facial, lingual, mesial, and distal surfaces of the teeth at four sites per tooth. An average score was obtained for each subject by dividing the sum of the individual scores by the number of gradable sites. Scoring criteria were as follows: 0 = no plaque or debris, 1 = film of plaque adherent to the free gingival margin and adjacent tooth area and seen in situ only after probing, 2 = gingival area visibly covered with a thin to moderately thick layer of plaque, 3 = abundant soft matter within the gingival pocket or in the tooth and gingival margin. The Löe and Silness gingival index was recorded on four surfaces of the index teeth with the following parameters: 0 = no gingival inflammation, 1 = mild inflammation without bleeding, 2 = Lactobacilli and Bifidobacteria in Human Saliva 153 moderate inflammation with bleeding, redness, edema, and ulceration, 3 = severe inflammation with spontaneous hemorrhage, redness, edema, and ulceration. Probing depth was measured from the gingival margin to the base of the pocket at six sites of each index tooth. Bacterial culture. Saliva sample from each subject was collected in a dish in the morning, before breakfast and tooth brushing. A dilution series of a sample was made in GAM broth (Nissui, Tokyo), and aliquots (0.1 ml) were plated on three kinds of agar plates: Brucella HK agar (Kyokuto Seiyakukogyo, Tokyo) supplemented with sheep blood (50 ml/l), hemin (5 mg/l), and vitamin K 3 (1 mg/l), to count total bacteria and BPARs; LBS agar (BBL, Cockeysville, MD) to count lactobacilli; and Beerens agar 17) to count bifidobacteria. The plates were incubated anaerobically in a chamber (80% N 2,10% CO 2, 10% H 2 )at37 C for 2 d, except for the Brucella HK agar plates, which were incubated for 7 d. After incubation, the number of colonies appearing on each plate was counted. Isolation of lactobacilli and bifidobacteria. Lactobacillus and Bifidobacterium colonies grown on LBS and Beerens agar, respectively, from the saliva of each subject were isolated as follows: Plates with approximately 25 to 250 colonies were used; if there were fewer colonies, a plate spread with 0.1 ml of whole saliva and containing approximately 1 to 25 colonies was used. The plates with over about 50 colonies were divided into zones so as to give approximately 25 colonies per zone. One zone was then chosen, and all the colonies in that zone were picked. In this way, approximately 25 colonies were selected at random from each plate. Each colony was purified twice on BL agar (Nissui). Provisional identification of all isolates was based on Gram staining, colony and cell morphology, and biochemical tests. Bifidobacterial isolates from Beerens agar plates were screened by the fructose 6- phosphate phosphoketolase test in order to confirm their classification. 18) Identification of bacterial isolates. Definitive identification of Lactobacillus isolates from LBS agar was performed by multiplex PCR as described by Song et al. 19) To identify Bifidobacterium species, we used species-specific primers and PCR conditions according to the procedure described by Matsuki et al. 20) Bacterial isolates whose species was not successfully identified by PCR were identified by 16S rdna sequencing. Briefly, genomic DNA extracted from a pure culture using a NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany), or from a single colony, was suspended in 50 ml of water and added directly to the PCR mixture. The primers used for PCR amplification were 27F (5 0 -AGAGTTTGATCCTGGCTCAG-3 0 ) and 520R (5 0 -ACCGCGGCTGCTGGC-3 0 ). Amplification reactions were performed in a total volume of 50 ml, con-

3 154 K. HOJO et al. taining 5 ml dissolved DNA (100 ng), 1.25 U Takara Ex Taq (Takara Shuzo, Kyoto, Japan), 5 ml 10 Ex Taq buffer, 4 ml dntp mixture (2.5 mm each), and 10 pmol of each primer. Reactions were carried out in a Perkin Elmer thermal cycler (PE Applied Biosystems, Foster City, CA), as follows: one cycle at 95 C for 5 min was followed by 30 cycles at 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s. Final extension was at 72 C for 5 min. The PCR products were purified with GFX PCR DNA and a Gel Band Purification kit (Amersham Biosciences, Piscataway, NJ), and sequenced directly with an ABI PRISM Dye Terminator Cycle Sequencing Kit (AB Applied Biosystems, Foster City, CA) and a 3100 Genetic Analyzer (AB Applied Biosystems). Cycle sequencing was carried out as recommended by the manufacturer. The results were compared with sequences deposited in the DNA Data Bank of Japan using the BLAST algorithm ( search/blast-j.html). Identification of isolates was based on more than 98% sequence similarity. Statistical analysis. All data are expressed as means standard deviation (SD). A Kruskal-Wallis test and a Mann-Whitney U test with Bonferroni correction were performed to check statistically significant differences between groups. The frequency of occurrence of lactobacilli and that of each species in salivary samples were compared by Fisher s exact probability test. Correlations between lactobacilli and BPARs were determined by Spearman s rank correlation test. Assessment of Bifidobacterium was performed in the same way. A probability (P) value of less than 0.05 was taken to indicate statistical significance. Results Clinical measurements In the present study, we intended to recruit young periodontitis patients and/or orally healthy old subjects, but we could not. Therefore, we set the well-maintained group instead of them. The clinical measurements are given in Table 1. The plaque index of the young, orally healthy group tended (P < 0:10) to be lower than that of the periodontitis group. The gingival index of the young, orally healthy group was significantly (P < 0:05) lower than that of the periodontitis group, and the probing depth of the young, orally healthy group was significantly (P < 0:05) shallower than that of the periodontitis Table 1. Clinical Characteristics of the Subjects Young healthy group (n ¼ 16) Periodontitis group (n ¼ 16) Well-maintained group (n ¼ 14) Plaque Index 0:5 0:3 0:9 0:5 0:6 0:3 Gingival Index 0:5 0:4 a 1:2 0:7 b 0:7 0:4 Probing Depth 2:4 0:4 a 3:6 1:4 b 2:7 0:8 Means in the same row with different letters subscripts are different at p < 0:05. group. Likewise, the gingival index and the probing depth of the well-maintained group tended (P < 0:10) to be lower and shallower than those of the periodontitis group. There were no significant differences between the clinical measurements of the young, orally healthy group and those of the well-maintained group. Numbers of oral bacteria The viable counts of lactobacilli, bifidobacteria, BPARs, and total bacteria in each group were determined. The well-maintained group had a significantly (P < 0:05) higher number of total bacteria than the young, orally healthy group (log 10 9:1 0:6 vs. 8:5 0:4 cfu ml 1 ). BPARs counts were significantly (P < 0:05) lower in the young, orally healthy group than in the periodontitis group: log 10 6:3 1:0 and 7:3 0:7 cfu ml 1 respectively. Lactobacilli were detected in 75% of the young, orally healthy group, 63% of the periodontitis group, and 86% of the well-maintained group. The mean count of lactobacilli in the young, orally healthy group (log 10 2:8 1:5 cfu ml 1 ) was also the lowest (P < 0:05) among the groups (periodontitis group: log 10 4:8 1:4 cfu ml 1, well-maintained group: log 10 4:8 1:4 cfu ml 1 ). Bifidobacteria were detected in 80% of the young, orally healthy group, 63% of the periodontitis group, and 64% of the well-maintained group. The bifidobacterial count was significantly (P < 0:05) higher in the well-maintained group (log 10 5:4 1:0 cfu ml 1 ) than in the periodontitis group (log 10 3:7 1:5 cfu ml 1 ) or the young, orally healthy group (log 10 4:1 0:8 cfu ml 1 ). There were no significant differences among the frequencies of occurrence of either lactobacilli or bifidobacteria in the three groups. We analyzed the results of viable cell counts in order to find whether a correlation existed between BPARs and either lactobacilli or bifidobacteria, but we detected no significant relationship. Distribution of Lactobacillus and Bifidobacterium species In this study, we obtained approximately 700 isolates of lactobacilli and 300 isolates of bifidobacteria. Analyses by multiplex PCR or 16S rdna sequence of Lactobacillus isolates showed that several Lactobacillus species inhabit the human oral cavity (Table 2). L. salivarius, L. gasseri, and L. fermentum were the most prevalent species in the saliva, but this finding was not restricted to the healthy subjects. The frequency of occurrence of Lactobacillus species in the three groups was similar (Table 2); for example that of L. salivarius was not significantly different, at 25%, 50%, and 50% in the salivary samples from the young, orally healthy group, the periodontitis group, and the well-maintained group respectively. Table 3 shows the distribution of Bifidobacterium species isolated from the human saliva samples. We detected fewer Bifidobacterium species than Lactobacillus species. B. dentium occurred most frequently in all

4 Table 2. Distribution of Lactobacillus Species Found in Saliva Young healthy group (n ¼ 16) Periodontitis group (n ¼ 16) Well-maintained group (n ¼ 14) Cell counts 1 No. of subjects Cell counts No. of subjects Cell counts No. of subjects Total Lactobacillus 2:8 1:5 a 12 (75%) 4:8 1:4 b 10 (63%) 4:8 1:4 b 12 (86%) L. salivarius 3:8 1:0 4 (25%) 4:0 1:5 8 (50%) 4:8 0:9 7 (50%) L. fermentum 3:1 1:6 4 (25%) 3:7 1:6 6 (38%) 4:3 1:5 8 (57%) L. gasseri 3:5 0:8 5 (31%) 4:1 1:2 8 (50%) 4:1 1:3 6 (43%) L. paracasei 2:6 1:3 5 (31%) 3:2 1:5 3 (19%) (7%) L. oris 4:4 0:7 2 (13%) (6%) 5:1 0:3 2 (14%) L. vaginalis 3:4 0:7 2 (13%) 3:9 0:4 2 (13%) N.D. L. crispatus (6%) (6%) 5:2 0:3 2 (14%) L. parakefiri (6%) N.D. 4:1 1:6 2 (14%) L. plantarum (6%) N.D. 4:0 1:0 3 (21%) L. rhamnosus N.D. 2 N.D (7%) L. delbrueckii N.D. N.D (7%) 1 Cell counts are expressed as means log 10 cfu SD/ml. Means in the same row with different letters subscripts are different at p < 0:05. 2 N.D., not detected. Lactobacilli and Bifidobacteria in Human Saliva 155 Table 3. Distribution of Bifidobacterium Species Found in Saliva Young healthy group (n ¼ 10) 1 Periodontitis group (n ¼ 16) Well-maintained group (n ¼ 14) Cell counts 2 No. of subjects Cell counts No. of subjects Cell counts No. of subjects Total Bifidobacterium 4:1 0:8 a 8 (80%) 3:7 1:5 a 10 (63%) 5:4 1:0 b 9 (64%) B. dentium 4:1 0:4 4 (40%) 3:7 1:5 a 10 (63%) 5:4 1:0 b 9 (64%) B. adolescentis 3:9 1:1 4 (40%) N.D. 3 N.D. B. urinalis (10%) N.D. N.D. B. longum (10%) N.D. N.D. 1 Analysis of bifidobacteria in young healthy group was performed in 10 out of 16 subjects. 2 Cell counts are expressed as means log 10 cfu SD/ml. Means in the same row with different letters subscripts are different at p < 0:05. 3 N.D., not detected. Significant differences in prevalence (P < 0:05). three groups. B. adolescentis, B. urinalis, and B. longum were detected only in the young, orally healthy group, at frequencies of 40%, 10%, and 10% respectively. The difference in the frequency of occurrence of B. adolescentis was statistically significant (P < 0:05), suggesting that this species is specific to young, orally healthy adults. Discussion Recent clinical studies using molecular biological techniques have revealed the bacterial profiles associated with periodontal health and disease. Socransky et al. 21,22) examined subgingival microbial profiles in periodontal disease and health using checkerboard DNA DNA hybridization, a new molecular approach that permits the characterization of a diverse bacterial community. Their data pointed to microbial complexes related to clinical measurements, and may be useful as a basis for the diagnosis and treatment of periodontal diseases. Kumar et al. 23) used quantitative 16S clonal analyses to identify candidate periodontal pathogens and beneficial bacterial species, and found that Streptococcus and Veillonella ssp. were a significantly larger component of the microbial community in healthy subjects than in those with periodontitis. Other studies have also suggested that Streptococcus and Veillonella are associated with periodontal health. 24,25) But in the present study we focused on lactobacilli and bifidobacteria, which have not been listed among the candidates for promotion of periodontal health. This study was prompted by recent studies 14 16) suggesting that lactobacilli and bifidobacteria might prevent periodontal disease. Whereas previous researchers analyzed subgingival plaque, we collected the saliva of 46 individuals to compare healthy oral microbiota and those present in cases of periodontitis. We considered that saliva was suitable for our study since Sakamoto et al. 26,27) have shown that saliva, like subgingival plaque, is effective to analyze the composition of oral bacterial communities. In addition, saliva contains the bacteria from several oral sites, and is easily obtained. Contrary to our expectations, the mean lactobacillus cell count of the young, orally healthy group was the lowest of the three groups. The analysis of Lactobacillus species revealed that no particular species was associated with periodontal health (Table 2). Ishikawa et al. 16) reported that daily intake of L. salivarius isolated from healthy humans decreased the number of BPARs in the saliva. In our study, L. salivarius was one of the most prevalent and predominant Lactobacillus species in saliva, but it is unlikely that salivary cell counts of

5 156 K. HOJO et al. L. salivarius are related to periodontal health, since neither the numbers of this species nor the frequencies of its occurrence differed significantly among the three groups. We also focused on bifidobacteria in the oral cavity. Bifidobacteria are one of the predominant bacterial groups in the human intestine, but constitute a minor population in the oral cavity, where Actinomyces, Streptococcus, and Veillonella predominate. Therefore, little attention has been paid to bifidobacteria as commensal bacteria of the human oral cavity. Our results showed that bifidobacteria were common in the human oral cavity, although they did not predominate. The frequencies of occurrence of bifidobacteria were 80%, 63%, and 64% in the young, orally healthy group, the periodontitis group, and the well-maintained group respectively. The bifidobacterial mean count of the wellmaintained group was significantly (P < 0:05) the highest among the groups, which suggests that the difference in bifidobacterial counts might be related to treatment progress. It is not clear whether the indigenous bifidobacterial count is associated with periodontal health, because we found no significant difference in bacterial cell counts between the young, orally healthy group and the periodontitis group. However, the result shown above encourages us to study more about bifidobacteria in oral environments. The number of Bifidobacterium species identified in our saliva study was low compared to that of Lactobacillus species: eleven Lactobacillus species were detected compared with four bifidobacterial species, viz., B. dentium, B. adolescentis, B. urinalis, and B. longum. Among these, B. dentium occurred most frequently in all three groups, whereas the others were detected only in the young, orally healthy group. In the present study, the frequency of occurrence of B. adolescentis was higher (P < 0:05) in the young, orally healthy group than in the periodontitis group or the well-maintained group. It is possible that this finding reflects the periodontal health status and/or the age of the donors although we cannot rule out the other possibilities (e.g., immunosuppresion and dietary habits) often present in clinical studies. To the best of our knowledge, this is the first report suggesting that the composition of Bifidobacterium species in the oral cavity might be influenced by periodontal status and/or age. The growth of BPARs, typical periodontal pathogens, is stimulated by vitamin K produced by Veillonella, 28) and this substance is also reported to be an essential growth factor for one strain of Bifidobacterium (formerly Lactobacillus bifidus strain). 29) We anticipate that bifidobacteria might suppress BPARs through competition for vitamin K. Hence, the analysis of vitamin K auxotrophy of Bifidobacterium isolates is of interest and requires further research. In summary, it is interesting that B. adolescentis, B. urinalis, and B. longum were detected only in the young, orally healthy group although B. dentium was prevalent in all groups. In particular, B. adolescentis was frequently observed in the young, orally healthy group, but it is not clear whether periodontal status or age is related to this result. In addition, the mean bifidobacterial count of the well-maintained group was highest among the groups. The data presented in this study should serve to shed light on the ecology of oral commensal bacteria, and highlights a need for further clarification of the role of bifidobacteria in human oral microbiota. Acknowledgments The authors thank Noboru Nakamichi and Takashi Sasaki for critical reading of the manuscript. We thank Hiroko Takamatsu, Tomoko Nishio, Saori Kurama, Mika Shibazaki, and Tomonobu Konno for technical assistance. Thanks are also due to Takaji Yajima and Hajime Tsuno for encouragement. References 1) Orrhage, K., and Nord, C. E., Bifidobacteria and lactobacilli in human health. Drugs Exp. Clin. Res., 26, (2000). 2) Eschenbach, D. A., Davick, P. R., Williams, B. L., Klebanoff, S. J., Young-Smith, K., Critchlow, C. M., and Holmes, K. K., Prevalence of hydrogen peroxideproducing Lactobacillus species in normal women and women with bacterial vaginosis. J. Clin. Microbiol., 27, (1989). 3) Hawes, S. E., Hillier, S. L., Benedetti, J., Stevens, C. E., Koutsky, L. A., Wolner-Hanssen, P., and Holmes, K. K., Hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections. J. Infect. Dis., 174, (1996). 4) Marteau, P. R., de Vrese, M., Cellier, C. J., and Schrezenmeir, J., Protection from gastrointestinal diseases with the use of probiotics. Am. J. Clin. Nutr., 73, 430S 436S (2001). 5) Saavedra, J. M., Bauman, N. A., Oung, I., Perman, J. A., and Yolken, R. H., Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus. Lancet, 344, (1994). 6) Koebnick, C., Wagner, I., Leitzmann, P., Stern, U., and Zunft, H. J., Probiotic beverage containing Lactobacillus casei Shirota improves gastrointestinal symptoms in patients with chronic constipation. Can. J. Gastroenterol., 17, (2003). 7) Becker, M. R., Paster, B. J., Leys, E. J., Moeschberger, M. L., Kenyon, S. G., Galvin, J. L., Boches, S. K., Dewhirst, F. E., and Griffen, A. L., Molecular analysis of bacterial species associated with childhood caries. J. Clin. Microbiol., 40, (2002). 8) Byun, R., Nadkarni, M. A., Chhour, K. L., Martin, F. E., Jacques, N. A., and Hunter, N., Quantitative analysis of diverse Lactobacillus species present in advanced dental caries. J. Clin. Microbiol., 42, (2004). 9) Marchant, S., Brailsford, S. R., Twomey, A. C., Roberts,

6 G. J., and Beighton, D., The predominant microflora of nursing caries lesions. Caries Res., 35, (2001). 10) Munson, M. A., Banerjee, A., Watson, T. F., and Wade, W. G., Molecular analysis of the microflora associated with dental caries. J. Clin. Microbiol., 42, (2004). 11) Ahola, A. J., Yli-Knuuttila, H., Suomalainen, T., Poussa, T., Ahlstrom, A., Meurman, J. H., and Korpela, R., Short-term consumption of probiotic-containing cheese and its effect on dental caries risk factors. Arch. Oral Biol., 47, (2002). 12) Nase, L., Hatakka, K., Savilahti, E., Saxelin, M., Ponka, A., Poussa, T., Korpela, R., and Meurman, J. H., Effect of long-term consumption of a probiotic bacterium, Lactobacillus rhamnosus GG, in milk on dental caries and caries risk in children. Caries Res., 35, (2001). 13) Nikawa, H., Makihira, S., Fukushima, H., Nishimura, H., Ozaki, Y., Ishida, K., Darmawan, S., Hamada, T., Hara, K., Matsumoto, A., Takemoto, T., and Aimi, R., Lactobacillus reuteri in bovine milk fermented decreases the oral carriage of mutans streptococci. Int. J. Food Microbiol., 95, (2004). 14) Caglar, E., Kargul, B., and Tanboga, I., Bacteriotherapy and probiotics role on oral health. Oral Dis., 11, (2005). 15) Grudianov, A. I., Dmitrieva, N. A., and Fomenko, E. V., Use of probiotics Bifidumbacterin and Acilact in tablets in therapy of periodontal inflammations. Stomatologiia, 81, (2002). 16) Ishikawa, H., Aiba, Y., Nakanishi, M., Oh-Hashi, Y., and Koga, Y., Suppression of periodontal pathogenic bacteria in the saliva of human by the administration of Lactobacillus salivarius TI2711. J. Jap. Soc. Periodontol., 45, (2003). 17) Beerens, H., An elective and selective isolation medium for Bifidobacterium spp. Lett. Appl. Microbiol., 11, (1990). 18) Scardovi, V., Genus Bifidobacterium Orla-Jensen 1924, 472 AL. In Bergey s Manual of Systematic Bacteriology Vol. 2, eds. Sneath, P. H. A., Mair, N. S., Sharpe, M. E., and Holt, J. G., Williams and Wilkins, Baltimore, pp (1986). 19) Song, Y., Kato, N., Liu, C., Matsumiya, Y., Kato, H., and Watanabe, K., Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays Lactobacilli and Bifidobacteria in Human Saliva 157 using group- and species-specific primers derived from the 16S-23S rrna intergenic spacer region and its flanking 23S rrna. FEMS Microbiol. Lett., 187, (2000). 20) Matsuki, T., Watanabe, K., Tanaka, R., Fukuda, M., Takada, T., and Oyaizu, H., Distribution of bifidobacterial species in human intestinal microfrola examined with 16S rrna-gene-targeted species-specific primers. Appl. Environ. Microbiol., 65, (1999). 21) Socransky, S. S., Smith, C., and Haffajee, A. D., Subgingival microbial profiles in refractory periodontal disease. J. Clin. Periodontol., 29, (2002). 22) Socransky, S. S., Haffajee, A. D., Cugini, M. A., Smith, C., and Kent, R. L., Microbial complexes in subgingival plaque. J. Clin. Periodontol., 25, (1998). 23) Kumar, P. S., Griffen, A. L., Moeschberger, M. L., and Leys, E. J., Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis. J. Clin. Microbiol., 43, (2005). 24) Haffajee, A. D., Cugini, M. A., Tanner, A., Pollack, R. P., Smith, C., Kent, R. L., and Socransky, S. S., Subgingival microbiota in healthy, well-maintained elder and periodontitis subjects. J. Clin. Periodontol., 25, (1998). 25) Tanner, A., Maiden, M. F., Macuch, P. J., Murray, L. L., and Kent, R. L., Microbiota of health, gingivitis, and initial periodontitis. J. Clin. Periodontol., 25, (1998). 26) Sakamoto, M., Takeuchi, Y., Umeda, M., Ishikawa, I., and Benno, Y., Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis. J. Med. Microbiol., 52, (2003). 27) Sakamoto, M., Umeda, M., Ishikawa, I., and Benno, Y., Comparison of the oral bacterial flora in saliva from a healthy subject and two periodontitis patients by sequence analysis of 16S rdna libraries. Microbiol. Immunol., 44, (2000). 28) Marcotte, H., and Lavoie, M. C., Oral microbial ecology and the role of salivary immunoglobulin A. Microbiol. Mol. Biol. Rev., 62, (1998). 29) Glick, M. C., Zilliken, F., and Gyorgy, P., Supplementary growth promoting effect of 2-methyl-1,4-naphthoquinone of Lactobacillus bifidus var. pennsylvanicus. J. Bacteriol., 77, (1959).