Suppression of Periodontal Pathogenic Bacteria in the Saliva of Humans by the Administration of Lactobacillus salivarius TI 2711

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1 Suppression of Periodontal Pathogenic Bacteria in the Saliva of Humans by the Administration of Lactobacillus salivarius TI 2711 Hiroki Ishikawa*1, Yuji Alba', Mutsumi Nakanishi*2, Yoshitami Oh-hashi*2 and Yasuhiro Koga*1 Department of Infectious Diseases, Tokai University *1 School of Medicine, Isehara, Kanagawa, *2Marketing Department, Frente Co. Accepted for publication 11 January 2003 The ability of Lactobacillus salivarius TI 2711 (LS 1) to displace periodontopathic bacteria, like Porphyromonas gingivalis and Prevotella intermedia, was studied using humanvolunteers. LS 1 was one thousandfold more susceptible to lactic acid than Lactobacillus acidophilus,a representative acid-resistant Lactobacillus strain frequently found at the sites of caries, when these bacteria were exposed to 50 mm of lactic acid. In an in vitro system, LS 1 completely killed P. gingivalis within 24 hours when these bacteria were cultured together. In a clinical study, 57 subjects took tablets containing 2 ~107 CFU or more of LS 1 daily for 4 or 8 weeks. The number of black-pigmented anaerobic rods, which includes most periodontopathic bacteria, in the saliva decreased to one-twentieth of the initial value after 4 weeks, whereas the numbers of whole bacteria, Streptococcus mutans and lactobacilli did not change. While the saliva ph was widely distributed (ranging from 5.4 to 8.5) before LS 1 treatment, it converged to within a neutral range of around 7.3 after treatment. Thus, the possibility that LS 1 accelerates caries formation by lowering the ph in the oral cavity was excluded. These findings suggest that LS 1 may be, potentially useful probiotic agent against periodontopathic bacteria. J Jpn Soc Periodontol, 45 : , Key words: Porphyromonas gingivalis, Prevotella intermedia, lactobacilli, saliva, probiotics Introduction Accumulating evidence indicates that periodontitis is an infectious disease that requires an initial colonization by etiologic bacteria. Black-pigmented anaerobic rods (BPAR) have been implicated as the major pathogens in the etiology of adult periodontitis, although these organisms are also found in healthy sites". The major BPAR species recovered from active periodontitis sites are Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens2'3). For example, Ishikawa et al.2) reported that the percentages of these three bacterial species among the total number of BPAR were 5.2%, 34.2%, and 24.3%, respectively. The oral microflora of healthy individuals, which consists of hundreds of commensal bacteria, changes into an "unhealthy" community colonized by pathogenic bacteria when pathogenic events develop in the periodontal and dental regions4, 5). Actually, a nonpathogenic or commensal species, mainly Streptococcus, is followed by a succession of species that culminates in the arrival of Gramnegative anaerobic bacteria, like P. gingivalis, a predominant pathogen in periodontitis. These results raise the possibility that maintaining a "healthy" oral microflora or having dominant commensals may inhibit colonization by pathogenic bacteria, thus protecting the hosts from the development of periodontitis. Probiotics are live bacteria belonging to the digestive tract flora with either low or no pathogenecity, and are beneficial to the health and well being of the

2 host6). The maintenance of these microbes is important in preventing infectious diseases. Therefore probiotics have been considered to be effective tools for controlling the overgrowth of pathogenic bacteria. This role of probiotics as an alternative to antibiotics has been named "microbial interference treatment"7). Lactobacilli are indigenous bacteria colonizing the oral cavity and digestive tracts. A large body of evidence shows that exogenous lactobacilli play a positive role in the prevention and treatment of gastrointestinal disorders8). Among the lactobacillus species that colonize the oral cavity, Lactobacillus acidophilus is the most frequently detected species in both young children and elderly people5,9). However, this species is acidogenic and aciduric; consequently a possible role in the development of caries, especially root caries, has been suggested5). On the other hand, Lactobacillus. salivarius, an obligate homofermentative lactobacillus, is far less aciduric than L. acidophilus and is sometimes isolated from the saliva of healthy subjects10). The acid-susceptible property of L. salivarius also raises the possibility that this lactobacillus species could be used as a non-cariogenic probiotic for maintaining a healthy ecosystem of oral microflora, thus preventing the colonization of periodontopathic bacteria. To investigate this possibility, we administered L. salivarius to human volunteers and examined the suppressive effect of this probiotic bacteria on periodontopathic bacteria in the saliva. Bacteria Materials and Methods L. salivarius TI 2711 (LS 1) was isolated from the saliva of a healthy human volunteer and identified using the API 50 CHL Kit (Biomeriux, Marcy- 'Etoile 1, France). Lactobacillus gasseri OLL 2716 and Lactobacillus brevis WB 1005 were provided by Meiji Dairy Products (Tokyo, Japan) and Wakamoto Pharmaceu. Co. (Tokyo, Japan), respectively. L. acidophilus JCM 1123 T, and Lactobacillus rharnnosus GG ATCC were obtained from the Japan Collection of Microorganisms (JCM), Wako, Saitama and the American Type Culture Collection (ATCC), Rockville, Maryland, respectively. Lactobacilli were grown in MRS broth (Difco Lab., Detroit, Michigan) in 10% H2, 10% CO2 and 80% N2 for one day. The number of colony-forming units (CFU) of lactobacilli was counted on modified LBS agar (Becton Dickinson, Sparks, MD). To examine the resistance of the lactobacillus species to acidity, around 1010 CFU of lactobacilli were suspended in 10 ml of a ph 2 anaero dilution buffer and incubated for 2 hours at 37 Ž. Next, 0.1 ml of the bacterial suspension was neutralized by adding it to ph 7 anaero dilution buffer and then spread over agar plates for counting the bacterial number. To test the susceptibility of the bacteria to lactic acid, the bacteria were suspended at a concentration of 107 CFU/ml in an anaero dilution buffer in which 0 to 100 mm of lactic acid were included and adjusted to ph 4.0 by the addition of NaOH or HC1. Porphyrornonas gingivalis JCM 8525, Prevotella interrnedia JCM 7365 and Prevotella nigrescens ATCC 33563T were obtained from JCM and ATCC, respectively. The number of periodontopathic bacteria was counted on EG agar (Nissui, Tokyo, Japan) plates containing 5% defibrinated horse blood and 10 pg/ml of gentamycin 3 days after incubation at 37 Ž in a jar equipped with an AnaeroPack (Mitsubishi Gas Chemical, Tokyo, Japan), a disposable 02 absorbing and CO2 generating agent. The number of mutant streptococci was counted on TS agar (Becton Dickinson) plates containing sucrose, cystine, yeast extract and 10 units/ml of bacitracin 3 days after incubation at 37 Ž in a jar equipped with an AnaeroPack. For mixed cultures, 108 CFU of LS 1 were cocultured with 108 CFIT of each pathogenic bacteria in 10 ml of GAM broth (Nissui) containing 0. 7% glucose in a jar equipped with an AnaeroPack. At 0, 6, 12 and 24 hours after incubation, 0.1 ml of the broth were remoned from the coculture and the number of pathogenic bacteria were counted. As controls, cultures of pathogenic bacteria that did not include LS 1 were also made. To count the number of BPAR in the saliva, a series of saliva dilutions were spread on EG agar (Nissui) plates containing 5% defibrinated horse blood and 10,ug/m/ of gentamycin.after incubation for 3 days at 37 Ž in a jar equipped with an AnaeroPack, the colonies with black/dark brown pigmentation were counted. For confirmation, some of these blackpigmented colonies were randomly selected and

3 Suppression of periodontopathic bacteria by lactobacilli 107 examined using both Gram staining and the API Rapid 20 A System. As a result, all of colonies were identified as species belonging to the Porphyromonas and Prevotella genera. In cultures with less than Statistics The paired Wilcoxon signed rank test was used for the statistical analysis, and a value of p<0.05 was considered significant. 104 CFU of BPAR in one milliliter of the sample, the BPAR colonies sometimes could not be counted became the number of other colonies on the agar plates was overwhelming. In such cases, the number of BPAR in the whole saliva sample was designated as 104 CFU/ml. Subject population Seventy-eight subjects were randomly selected from the volunteers. Informed consent and approval were obtained from all the subjects and the Ethical Committee of our university, respectively. The subjects were divided into three groups. The first group consisted of 21 subjects (16 males and 5 females) aged 22 to 46 (mean age, 30.4 years) who did not take LS 1. The second group consisted of 28 subjects (20 males and 8 females) aged 22 to 62 (mean age, 35.1 years) who took 2 ~107 CFU of LS 1 a day. The third group consisted of 29 subjects (22 males and 7 females) aged 22 to 62 (mean age, 35.2 years) who took 1 ~108 CFU of LS 1 a day. None of these subjects had been treated with antibiotics for at least one month nor had they used any oral rinse product for at least 2 weeks prior to the start of the study. No oral hygiene instructions were given, but each subject was instructed to continue his or her usual oral hygiene regimen. Study outline Each subject placed 5 tablets in their mouth and let the tablets dissolve without chewing. This intake was performed 5 times a day and was continued for 8 weeks. The tablets (0.14 g/tablet) consisted of 80% sorhitol, 20% erythritol and frozen-dry LS 1 (2 ~107 or 1 ~108 CFU/25 tablets). One day after the last intake of LS 1, the saliva was collected in the morning from subjects who had refrained from any oral intake or tooth brushing that morning. To collect the saliva, the subjects were asked to chew on a piece of paraffin wax for one minute and then expectorate all their saliva into a tube with a gauge. The ph of the saliva was measured using a ph meter (phboy-p 2 ; Shindengen Co., Tokyo, Japan) immediately after collection. Results Inhibitory effect of LS 1 on periodontopathic bacteria in vitro P. gingivalis, P. intermedia and P. nigrescens grew exponentially in the GAM broth under anaerobic conditions. However, cocultures of these periodontopathic bacteria with a L. salivarius strain, LS 1, resulted in a drastic killing of the pathogenic bacteria around 12 hours after the start of the coculture and almost complete eradication after 24 hours in three independent experiments. A representative result is shown in Fig. 1. On the other hand, the number of LS 1 increased by about 1 log number in the cultures after 24 hours (data not shown). In addition to LS 1, we examined the inhibitory effect of eleven other L. salivarius strains, including L. salivarius ATCC and L. salivarius ATCC using coculture assays. All eleven L. salivarius strains completely killed P. gingivalis after 24hours, but they were not as efficient as LS 1 at killing P. intermedia (data not shown). The amount of lactic acid that accumulated in the 24-hour culture containing LS 1 was around 100 mm. The lactic acid released from LS 1 was thus considered to be a major bactericidal factor because the addition of 100 mm to a single culture of P. gingivalis completely killed the bacteria within 6 hours (Fig. 2). Resistance of LS 1 to acidity and lactic acid Among the Lactobacillus strains examined for this susceptibility to acidity in the present study, L. salivarius TI 2711 (LS 1), OLL 2658 (data not shown), OLL 2676 (data not shown) and No. 11 (data not shown) were the most susceptible to acidity and L. acidophilus strains like L. gasseri and L. acidophilus were the least susceptible (Table 1). Indeed, the surviving bacterial number was just 0.004% of the initial bacterial number in LS 1, whereas 0.48% of L. acidophilus JCM 1132 T survived after being exposed to ph 2.0 for 2 hours. In addition, LS 1 was far more sensitive to lactic acid than L. acidophilus ; lactic acid concentrations of

4 108 Fig. 1 Coculture of periodontopathic bacteria with LS 1 Periodontopathic bacteria at a concentration of 108 CFU werecocultured with (closed circles) Jr without (open circles) 108 CFU of LS 1 in 10 ml of the broth.at 0, 6, 12 and 24 hrs after the ;tart of the coculture, the viable number of periodontopathic bacteria was counted. Fig. 2 Treatment of bacteria with lactic acid Zero to 100 mm of lactic acid was added to cultures in which 107 CFU/ml of each species of bacteria, as indicated at the top of the figure, were suspended in broth. At 0, 6, 12 and 24 hours after the addition of lactic acid, the viable number of bacteria was counted.

5 Suppression of periodontopathic bacteria by lactobacilli 109 Table 1 Resistance to acidity (ph 2) Table 2 Effect of LS 1 treatment on the bacterial number in the saliva more than 50 mm rapidly killed LS 1 but not did affect L. acidophilus even 24 hours after treatment (Fig.2). These results indicate that LS 1 is highly susceptible to both acidity and lactic acid. Clinical examination of LS 1 using saliva Two out of the 57 subjects who took LS 1 complained of soft stools and abdominal discomfort after taking the LS 1 for about 1 week; these two subjects were withdrawn from the study. After 4 weeks of treatment with LS 1, the mean of the total BPAR number in one milliliter of saliva significantly decreased in both the group treated with 2 x 107 CFU of LS 1 a day (106.5 to CFU/m/, p<0.005, n=27) and the group treated with 1 ~108 CFU of LS 1 a day (106.7 to 105.4/CFU/ml, p<0.005, n=28) (Table 2). Figure. 3 shows the detailed changes in the number of BPAR colonies before and after the LS 1 treatment in each of the 55 subjects. In this figure, we can also see that the number of subjects whose BPAR was undetectable (<104 CFU/ml) increased from 8 (14%) to 30 (53%) 4 weeks after LS 1 treatment. The control group did not show any significant change in the number of BPAR colonies from their saliva (107.1 to p>0.05, n=21). These findings suggest that LS 1 exerts a suppressive effect on BPAR in saliva. On the other hand, the number of Mutans streptococci did not decrease in the saliva after the LS 1 treatment. Neither the overall number of bacteria nor the number of lactobacilli changed significantly after LS 1 treatment for 4 weeks. Considering that the number of lactobacilli was around CFU/ml to 104.7CFU/m/ in the subjects, even though they had taken more than 2 ~107 CFU of LS 1 lactobacil-

6 Fig. 3 Number of BPAR in whole saliva Saliva samples were obtained from 55 subjects before and 4 weeks after LS 1 treatment, and the number of BPAR in the whole saliva samples was counted. The circles indicate the value for each subject. The values for the same subjects are connected by lines. li just 24 hours prior to sampling, almost all of the administered LS 1 was thought to pass through the oral cavity and/or guickly die there. Before the LS 1 treatment, the ph value of the saliva samples varied between 5.4 and 8.5, as shown in Fig. 4. After 4 weeks of treatment with LS 1, however, the ph values fell within a small neutral range of around 7.3. For example, case No.20, No.42 and No. 44 showed acidic values of 5.6,5.4 and 5.5 at 0 weeks, respectively, but these values increased to neutral values of 7.3, 7.2 and 7.1 after 4 weeks of treatment, respectively. Furthermore, while case No. 4, No. 15 and No. 16 exhibited moderately alkaline values of 8.4, 8.1 and 8.5 at 0 weeks, respectively, these values decreased to neutral values of Fig. 4 Saliva ph Saliva samples were obtained from 55 subjects in the same manner as that used in Fig. 3. The ph values of the samples measured immediately after collection. were 7.5, 7.6 and 7.4 after 4 weeks, respectively. As a result, the mean ph value of all the subjects shifted from 7.0 to 7.3 after LS 1 treatment. Thus, the oral intake of LS 1 probably dose not accelerate the progress of dental caries by lowering the ph in the oral cavity through acid production. Discussion A significant correlation has been reported between the number of pathogenic bacteria colonizing the tooth surface, the dorsum of the tongue or periodontal tissue, and the salivary level of these bacteria11,12) Furthermore the sampling technique in the present study, included the chewing of paraffin to increase the shedding of bacteria from those

7 Suppression of periodontopathic bacteria by lactobacilli sites in the oral cavity. Therefore, the saliva samples employed in the present study to analyze oral microflora appear to accurately reflect the properties of such microflora. Since early morning saliva samples have been reported to contain higher levels of bacteria than samples obtained at other times of the day13), we collected the saliva samples in the morning. BPAR is strongly associated with adult periodontitis. Ishikawa et al.2) reported a clinical evaluation in which the total number of BPAR and the percentage of BPAR out of the total number of bacteria at active gingivitis sites were significantly higher than in healthy subjects and in healthy sites of gingivitis. Among BPAR, P. gingivalis, P. intermedia and P. nigrescens had the highest colony numbers in samples from active sites of gingivitis. Because the number of BPAR was higher in healthy sites of gingivitis than in healthy subjects, they proposed that these periodontopathic bacteria may be transmitted from active to healthy sites in subjects with gingivitis. Susceptible healthy sites are thought to require colonization by pathogenic bacteria derived from periodontitis lesions before periodontal diseases can be initiated, since the microflora of plaque at healthy sites contains few of these periodontal pathogens14,15). Such transmission of bacteria is thought to be predominantly mediated through the saliva. In the present study, LS 1 administration significantly decreased the number of BPAR in the saliva, suggesting that LS 1 may exert an inhibitory effect on the initiation and progression of periodontal diseases. The effect of LS 1 treatment on the number of pathogenic bacteria at gingival sites will be studied in the future. The major factors associated with caries development are thought to include dietary carbohydrate consumption, the action of saliva, the microbial flora of dental plaque, and the ph lowering ability of dental plaque16). Regarding the plaque flora/ caries relationship, evidence indicates that an increase in caries activity is associated with an enrichment of plaque with organisms that have a relatively high capacity for acid tolerance17,18). The present in vitro study demonstrated that LS 1 was far less tolerant to acidity and lactic acid than the L. acidophilus group, including L. gasseri and L. acidophilus. Indeed, L. acidophilus is one of the predominant lactobacillus species found in aciduric oral microflora9). In addition, this acid-resistant lactobacillus is thought to be involved in the initiation and progression of root caries, which are prevalent in patients with both treated and untreated periodontal disease). Therefore, the addition of acid-sensitive LS 1 to oral microflora will displace these acid-tolerant bacteria and render the microflora less-tolerant to acidity, thus leading to an inhibition in the increase of such aciduric pathogenic bacteria as Streptococcus mutans. Regarding the ph lowering ability of plaque, the administration of LS 1 neutralized the ph of the saliva, thus suggesting that the participation of LS 1 in the oral microflora decreased the acid production of plaque flora. The reason why the ph value was adjusted to a neutral range remains uncertain. The administration of LS 1 might "freshen" the oral microflora so that it consists predominantly of commensal bacterial species with less capacity for acidogenesis. Taken together, these findings suggest that LS 1 is unlikely to cause caries formation, either by rendering plaque flora more acid tolerant acid generation. Reference or by promoting 1) Slots J, Rams TE: Microbiology of periodontal disease, p , In J Slots and MA Taubman, Contemporary oral microbiology and immunology, Mosby Year Book, St Louis, ) Ishikawa H, Okamoto M: Incidence and distribution of Prevotella intermedia, Prevotella nigrescens and Porphyromonas gingivalis isolated from gingivitis. J Jpn Soc Periodontol, 41: , ) Teanpaisan R, Douglas CWI, AR Eley, Walsh TF: Clonality of Porphyrornonas gingivalis, Prevotella nigrescens isolated from periodontally diseased and healthy sites. J Periodont Res, 31: , ) Socransky SS, Haffajee AD, Cugini MA, Smith C, Jr RL Kent: Microbial complexes in subgingival plague. J Clin Periodontol, 25: , ) Tanner ACR, Milgrom PM, Kent R, Jr, Mokeem SA, Page RC, Riedy CA, Weinstein P, Bruss J : The microbiota of young children from tooth and tongue samples. J Dent Res, 81: 53-57, 2002.

8 6) Fuller R: Probiotics in man and animals. J Appl Bacteriol, 66: , ) Bengmark S: Ecological control of the gastrointestinal tract. The role of probiotic flora. Gut, 42: 2-7, ) Reid G, Bruce AW, McGroarty JA, Cheng KJ, Costerton JW: Is there a role for Lactobacilli in prevention of urogenital and intestinal infection. Clin Microbiol Rev, 3: , ) van Houte J: Bacterial specificity in the etiology of dental caries. Int Dent J, 30: , ) Salminen S: Lactic Acid Bacteria. In L Axelsson Classification and Physiology. Marcel dekker, Inc. New York, ) Lindquist B, Emilson CG, Wennerholm K: Relationship between mutans streptococci in saliva and their colonization of the tooth surface. Oral Microbiol Immunol, 4: 71-76, ) Togelius J, Kristoffersson K, Anderson H: Streptococcus mutans in saliva: intraindividual variations and relation to the number of colonized sites. Acta Odontol Scand, 42: , ) Carlsson J: Effect of diet on presence of Streptococcus salivarius in dental plaque and saliva. Odontol Rev, 16: , ) Smith GLF: Diagnosis of periodontal disease activity by detection of key microbial antigen. J Clin Periodontol, 21: , ) Socransky SS, Haffajee AD: Microbial mechanism in the pathogensis of destructive periodontal diseases: A critical assessment. J Periodont Res, 26: , ) van Houte J: Role of micro-organism in caries etiology. J Dent Res, 73: , ) Lingstrom P, van Ruyven FOJ, van Houte J, Kent R: The ph of dental plaque in its relation to early enamel caries and dental plaque flora in humans. J Dent Res, 79: , ) van Houte J, J Lopman, Kent R: The final ph of bacteria comprising the predominant flora on sound and carious human root and enamel surfaces. J Dent Res, 75: , Corresponding author: Yasuhiro Koga Department of Infectious Diseases, Tokai University School ofmedicine, Bosei-dai, Isehara, Kanagawa, , Japan