Effects of probiotic Lactobacillus salivarius WB21 on halitosis and oral health: an

Effects of probiotic Lactobacillus salivarius WB21 on halitosis and oral health: an open-label pilot trial Tomoyuki Iwamoto, DDS, a Nao Suzuki, DDS, PhD, a Kazunari Tanabe, DDS, PhD, b Toru Takeshita, DDS, PhD, c and Takao Hirofuji, DDS, PhD, a Fukuoka, Japan a Section of General Dentistry, Department of General Dentistry, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan b Tanabe Preservative Dentistry, 2-12-18 Mizutani, Higashi-ku, Fukuoka, 813-0041, Japan c Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan Corresponding author: Nao Suzuki, DDS, PhD. Section of General Dentistry, Department of General Dentistry, Fukuoka Dental College 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan 1

Tel: +81-92-801-0411; Fax: +81-92-801-4909; E-mail: naojsz@college.fdcnet.ac.jp Short title: Effects of probiotics on oral malodor 2

Abstract Objective. To evaluate whether oral administration of lactobacilli alters the degree of halitosis and clinical conditions associated with halitosis. Study design. Twenty patients with genuine halitosis were given 2.0 10 9 Lactobacillus salivarius WB21 and xylitol in tablet form daily. Oral malodor and clinical parameters were evaluated at the same time of day for each patient after 2 and 4 weeks. Results. All 20 patients were positive for L. salivarius DNA in their saliva at 2 weeks, although 12 patients were negative for this organism at baseline. Oral malodor parameters significantly decreased at 2 weeks in the subjects with physiologic halitosis. The scores of an organoleptic test and bleeding on probing significantly decreased at 4 weeks in the subjects with oral pathologic halitosis. Conclusions. Oral administration of probiotic lactobacilli primarily improved physiologic halitosis and, therefore, may have beneficial effects on bleeding in the periodontal pocket. 3

Introduction Probiotics are defined as bacteria with physiological benefits for humans. Required characteristics are scientifically demonstrated beneficial physiological effects; human origin, safety for human use, and stability in acid and bile; and adherence to the intestinal mucosa [1]. The effects of probiotic therapy have been studied extensively for a variety of systemic indications and medical disorders [2]. Previous studies examining the effects of oral probiotics have demonstrated that the consumption of products containing probiotic lactobacilli reduced the caries risk and number of mutans streptococci in the oral cavity [3, 4]. Grudianov et al. [5] reported that the use of probiotic tablets normalized the microbiota in patients with periodontitis and gingivitis as compared with a control group. Ishikawa et al. [6] and Matsuoka et al. [7] found that the administration of Lactobacillus salivarius TI2711 (LS1) to healthy subjects neutralized the ph of saliva and considerably decreased the numbers of black-pigmented anaerobic rods in saliva. Shimauchi et al. [8] reported that probiotic L. salivarius WB21 taken orally improved the periodontal health of healthy volunteers, particularly smokers, but not that of former smokers or those who had never smoked, in 4

a double-blind, placebo-controlled, randomized clinical trial. Taking a tablet containing L. salivarius WB21 also reduced the numbers of periodontal bacteria in subgingival plaque [9] and appeared to have beneficial effects on periodontal health. Although periodontal diseases cause oral malodor, the effects of probiotic bacteria on oral malodor are still unclear. Halitosis is a common problem; oral malodor is caused by periodontitis, tongue debris, poor oral hygiene, deep caries, inadequately fitted restorations, and endodontic lesions [10 12]; it is primarily the result of the microbial metabolism of amino acids in local debris [13]. Many of the compounds that contribute to oral malodor are volatile sulfur compounds (VSCs), such as hydrogen sulfide (H 2 S), methyl mercaptan (CH 3 SH), and dimethyl sulfide (CH 3 SCH 3 ) [11, 14]. Periodontopathic bacteria, including Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, and Fusobacterium nucleatum, produce H 2 S and CH 3 SH [15, 16]. To diagnose halitosis, a simple classification has been developed that includes the categories genuine halitosis, pseudo-halitosis, and halitophobia [17, 18]. Genuine halitosis is subclassified as physiologic or pathologic halitosis, and pathologic halitosis 5

is subclassified as an oral or non-oral pathologic halitosis. The treatment of physiologic halitosis primarily involves dental and oral care, oral hygiene instruction, and counseling. Oral pathologic halitosis is caused largely by periodontal disease, and its treatment requires periodontal treatment in addition to the measures used to treat physiologic halitosis. The goal of this treatment regime is to acquire healthy oral conditions, including a normal microflora. The aim of this open-label pilot study was to evaluate whether the oral administration of probiotic L. salivarius WB21 could reduce oral malodor in patients with actual malodor complaining of halitosis and, subsequently, to improve the oral conditions associated with oral malodor. Materials and methods Probiotic product The MINNA NO ZENDAMAKIN WB21 TABLET (Wakamoto Pharmaceutical, Tokyo, Japan) contains 6.7 10 8 colony-forming units (CFU) of L. salivarius WB21 and 280 mg of xylitol per tablet. Strain WB21 is an acid-tolerant 6

lactobacillus that was isolated from L. salivarius WB1004 [19]. The dose throughout the test period was maintained at three tablets per day, taken orally after eating. Subjects and study design The study population consisted of 20 genuine subjects (6 males, 14 females; mean age 50.9 ± 12.1 years, range 30 66 years) who complained of halitosis and presented to the Oral Malodor Clinic of Fukuoka Dental College Medical and Dental Hospital, Japan, between July 2008 and August 2009. None of these patients had received antibiotics within 3 months prior to participating in the study. All of the subjects who participated understood the nature of the research project and provided informed consent. Permission for this study was obtained from the Ethics Committee for Clinical Research of Fukuoka Dental College and Fukuoka College of Health Sciences (approval number 125). The subjects took three tablets of MINNA NO ZENDAMAKIN WB21 TABLET every day, containing a total of 2.01 10 9 CFU L. salivarius WB21 and 840 mg xylitol. They were directed to place a tablet in the mouth for a few minutes and 7

allow it dissolve. They were also instructed not to change their oral hygiene regimens and not to take other probiotic products throughout the study period. Neither professional prophylaxis nor tooth-brushing instruction was performed during or before the experimental period. Maintenance of this regime was confirmed at days 15 and 29 of the study. Malodor was assessed, clinical parameters recorded, and saliva samples obtained from all subjects on days 1, 15 and 29 from ten subjects with oral pathologic halitosis. Malodor assessment For each patient, malodor was assessed and a clinical examination performed at the same time of day at least 5 hours after eating, drinking, chewing, smoking, and brushing or rinsing the mouth. The severity of oral malodor in each individual was determined using an organoleptic test (OLT) and gas chromatography (model GC14B; Shimadzu Works, Kyoto, Japan). For the OLT, each patient was instructed to exhale through the mouth with moderate force into a Teflon sampling bag (GL Science, Tokyo, Japan) for 2 3 s to prevent the dilution of mouth odor with lung and room air. This 8

procedure was repeated until approximately 1 L of breath sample was obtained. Two of the three evaluators (with training and experience in calibration tests) then estimated the odor at a distance of 10 cm from the sampling bag. The OLT scores were estimated on a scale of 0 to 5 (0, no odor; 1, questionable odor; 2, slight malodor; 3, moderate malodor; 4, strong malodor; 5, severe malodor) [18], and mean scores given by the different judges were used. The percentage agreement in the OLT scores among the three evaluators always exceeded 75.0% (κ = 0.50). The threshold level for genuine halitosis was defined as an OLT score of 2 according to the experimental criteria [18]. For the gas chromatographic measurements, the subjects were asked to remain quiet with a closed mouth for 30 s, after which mouth air (10 ml) was aspirated using a gas-tight syringe. These samples were injected onto a gas chromatograph column at 70 C. A glass column was packed with 25% β, β 9-oxydipropionitrile on a 60 80 mesh Chromosorb W AW-DMCS-ST device (Shimadzu, Kyoto, Japan) fitted with a flame photometric detector. The concentration of each sulfur compound was determined based on the values for standard H 2 S, CH 3 SH, and CH 3 SCH 3 gas prepared with a PD-1B permeater (GL Science, Tokyo, Japan). The level of total VSCs was 9

defined as the total concentrations of H 2 S, CH 3 SH, and CH 3 SCH 3. The threshold levels for genuine halitosis were defined as 2.5 ng/10 ml of total VSCs in mouth air by gas chromatography, according to previous reports [20, 27]. The OLT score was given priority over the VSC levels for the evaluation of malodor. Clinical examinations The oral health of each patient was evaluated based on the numbers of teeth, caries, and fillings, the no-good margin, plaque control, mobility of the teeth, periodontal pocket depth (PPD), bleeding on probing (BOP), degree of tongue coating, volume of stimulated salivary flow, and the presence of occult blood in the saliva. PPD and BOP were measured at six points around each tooth in all of the subjects. The tongue coating score (TCS) was assessed using conventional criteria, with simple modifications: 0, no tongue coating; 1, thin tongue coating covering less than one-third of the tongue dorsum; 2, thick tongue coating covering approximately one-third of the tongue dorsum or thin tongue coating covering one-third to two-thirds of the tongue dorsum; 3, thick tongue coating covering one-third to two-thirds of the tongue dorsum 10

or thin tongue coating covering more than two-thirds of the tongue dorsum; and 4, thick tongue coating more than two-thirds of the tongue dorsum [21, 22]. Oral pathologic halitosis was determined based on the presence of teeth with a PPD 5 mm or TCS 3 as these threshold values showed a linear correlation with any oral malodor test by logistic regression analysis, and these values have been utilized in previous studies concerning oral malodor [21, 22]. Plaque control was evaluated using the Silness & Löe Plaque Index [23]. The volume of stimulated salivary flow was measured using the chewing gum test. The patient was asked to pool saliva in the oral cavity and spit into a vessel every minute throughout the 5-min collection period. The presence of occult blood in the saliva was tested using Perioscreen (Sunstar, Osaka, Japan). Microbiological examination The prevalence of L. salivarius, periodontopathic bacteria producing VSCs, including P. gingivalis, P. intermedia, and T. denticola, and cariogenic bacteria, including Streptococcus mutans and Streptococcus sobrinus, was determined by polymerase chain reaction (PCR) and the amount of L. salivarius in the saliva of the 11

subjects was determined by real-time PCR. The chromosomal DNA from P. gingivalis ATCC 33277, P. intermedia ATCC 25611, T. denticola ATCC 35405, S. mutans Xc, S. sobrinus 6715, and L salivarius WB21 were used as positive controls. Table 1 shows the sequences of the oligonucleotide primers used in this study [24 27]. The chromosomal DNA of the bacteria in the saliva was obtained from 500 µl of stimulated whole saliva mixed with an equal amount of phosphate-buffered saline. This was centrifuged at 12,000 g for 10 min. Subsequently, 300 µl of lysis buffer (50 mm Tris/HCl, ph 8.0; 1 mm EDTA; and 1% SDS), 0.3 g of zirconium beads, and a 3-mm-diameter tungsten carbide bead were added to the precipitate, which was then boiled at 100 C for 10 min. The oral bacteria in the saliva samples were ruptured using a Disruptor Genie cell disrupter (Scientific Industries, Bohemia, NY, USA) at room temperature for 3 min [28]. The samples were then denatured at 70 C for 10 min by the addition of 300 µl of 1% SDS, and the DNA was purified by repeated phenol/chloroform extraction, precipitated with 100% ethanol, and resuspended in 100 µl TE (10 mm Tris/HCl, ph 8.0 and 1 mm EDTA). For conventional PCR, each 10 µl volume of the PCR mixture contained 0.25 mm deoxynucleotide triphosphates, 10 12

PCR buffer, 5 U Ex Taq polymerase (TaKaRa Bio, Shiga, Japan), 2 µm of each primer, and 2 µl of template DNA. Amplification was conducted using a TaKaRa PCR thermal cycler with the following parameters: 94 C for 2 min, followed by 30 cycles of 94 C for 10 s, 58 C for 15 s, and 72 C for 1 min, with a final extension at 72 C for 2 min. The amplified products were electrophoresed, stained with 0.5 µg/ml ethidium bromide, and photographed under ultraviolet light. Quantitative real-time PCR for L. salivarius DNA was performed using a QuantiFast SYBR Green PCR Kit (QIAGEN, Hilden, Germany) in a StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer s instructions. The cycling conditions were 95 C for 10 min followed by 40 cycles of 95 C for 3 s and 60 C for 30 s. DNA melting curves for the target amplicons were assessed for any putative PCR artifacts or non-specific PCR products. The bacterial cell numbers per sample were calculated using a standard curve constructed from the diluted chromosomal DNA of L. salivarius WB21 (1.0 10 8 CFU/ml). The periodontopathic bacteria producing VSCs, including P. gingivalis, P. intermedia, T. denticola, and F. nucleatum, and other common oral bacteria were quantified using a PCR-invader assay performed by the manufacturer 13

(BML, Tokyo, Japan). Statistical analysis Statistical comparisons of the demographic and baseline characteristics between the subjects with physiologic and oral pathological halitosis were performed using either a chi-square test, unpaired t-test, or a Wilcoxon-Mann-Whitney test. The differences in the oral malodor levels, clinical parameters, and bacterial parameters on days 1, 15, and 29 according to the oral administration of L. salivarius WB21 were performed using the chi-square test and Wilcoxon-Mann-Whitney test. The SPSS statistical software package was used for all analyses (release 11.0 J; SPSS Japan). Results Baseline characteristics of subjects All 20 subjects were diagnosed with genuine halitosis (OLT score 2), consisting of nine subjects with physiologic halitosis (PPD <5 mm, TCS <3) and 11 subjects with oral pathologic halitosis (PPD 5 mm or TCS 3). Table 2 shows the 14

demographic and baseline characteristics of the study subjects. The average age and proportion of males for the subjects with oral pathologic halitosis were higher than for the subjects with physiologic halitosis (P=0.043 and P=0.008, respectively). Most subjects (10/11) with oral pathologic halitosis had periodontitis and significantly higher numbers of PPD 5 mm (P<0.001) and average PPD (P=0.004), as compared to the patients with physiologic halitosis. The percentage of BOP in the subjects with oral pathologic halitosis was also greater than that in the subjects with physiologic halitosis, but the difference was not significant (P=0.078). The prevalence of P. gingivalis, which is implicated as a major pathogen in periodontitis, was notable in saliva from the subjects with oral pathologic halitosis (90.9%) compared with the subjects with physiologic halitosis (33.3%, P=0.007). More than half of the subjects (55.6%) with physiologic halitosis were positive for L. salivarius DNA versus only 27.3% of the subjects with oral pathologic halitosis. There was no difference in the oral malodor levels of the two groups. Changes in the clinical and oral malodor parameters over 2 weeks with the 15

administration of WB21 Table 3 shows the differences in the clinical and oral malodor parameters between days 1 and 15 with the administration of L. salivarius WB21. In both groups, inflammatory factors (occult blood in saliva and percentage of BOP) decreased and stimulated salivary flow increased. In the oral pathologic halitosis group, the percentage of BOP decreased notably (P=0.008). The oral malodor parameters were reduced in both groups. The physiologic halitosis group had a significant decrease in the OLT score, and in the concentrations of H 2 S, CH 3 SH, and total VSCs, while the oral pathologic halitosis group showed no significant decreases. In particular, the OLT score (1.6 ± 1.8) at day 15 in the physiologic halitosis group was less than 2, which is the threshold level of genuine halitosis. The prevalence of L. salivarius DNA in saliva determined using PCR was 100% in the subjects in both groups at day 15. The detection of the other oral bacteria had not changed. Changes in the clinical and oral malodor parameters over 4 weeks in the patients with oral pathologic halitosis 16

Ten patients from the oral pathologic halitosis group (five males and five females; mean age 56.9 ± 6.0 years, range 48 65 years) continued to take the MINNA NO ZENDAMAKIN WB21 TABLET for 4 weeks. One subject discontinued the regime as the patient malodor levels were reduced 2 weeks following the beginning of the study and the subject considered further examination unnecessary. Table 4 shows the changes in the clinical and oral malodor parameters of 10 patients at days 1, 15, and 29. The percentage of BOP significantly decreased at days 15 (5.7 ± 5.0) and 29 (7.2 ± 5.8) compared with day 1 (11.4 ± 8.1, P=0.004 and P=0.006, respectively). The OLT score fell below 2, which is the threshold level of genuine halitosis, at day 29 (1.9 ± 0.5, P=0.013 versus day 1). The VSC concentrations measured using gas chromatography were maintained between days 15 and 29. The numbers of L. salivarius at day 15 [2.9 ± 0.4 (log 10 ± SE)] and day 29 (2.5 ± 0.9) were significantly greater than that at day 1 (1.0 ± 1.3, P=0.002 and P=0.004, respectively); however, no significant difference between days 15 and 29 were evident. The proportion of L. salivarius in the saliva was lower than those of periodontal bacteria during the intervention period. The number of P. intermedia at day 15 (4.7 ± 0.9) was 17

significantly greater than at day 1 (4.6 ± 1.0, P=0.030). The administration of WB21 did not change the amount of other periodontal bacteria producing VSCs or the total amount of bacteria in saliva. Discussion We found that in those patients with physiologic halitosis receiving probiotic tablets containing L. salivarius WB21, the OLT score and the H 2 S, CH 3 SH, and total VSC concentrations decreased significantly. In those subjects with oral pathologic halitosis, the percentage of BOP decreased at 2 weeks. In addition, at 4 weeks the OLT score and the percentage of BOP were significantly lower in the subjects with oral pathologic halitosis, as compared with day 1. The oral malodor parameters showed a greater improvement in those subjects with physiologic halitosis, as compared to those with oral pathologic halitosis (Table 3). Physiologic halitosis is not associated with organic lesions, suggesting that malodor is caused primarily by an imbalance of microbiota in the oral cavity. Probiotic therapy may thus provide an effective approach for the control of oral microbiota, and inducing a beneficial shift away from 18

pathogens [29]. This may explain the improved results observed in the subjects with physiologic halitosis. Since, in subjects with oral pathologic halitosis, the administration of L. salivarius WB21 decreased the inflammation in the periodontal pocket, the use of probiotic bacteria as an adjunct to mechanical debridement may provide an effective therapeutic approach for the treatment of periodontitis as well as oral malodor. We postulated that the amount of periodontopathic bacteria, which produce H 2 S and CH 3 SH, in the saliva of the patients with oral pathologic halitosis, would decrease with the administration of L. salivarius WB21. However, we observed that the levels of both periodontal bacteria and the total oral bacteria in saliva, without P. intermedia at day 15, did not change during days 1, 15, and 29. Given that the target of probiotic therapy may be not a specific organism but the entire microflora, each bacterial number might not differ dramatically. Mayanagi et al. [9] reported that using a tablet containing L. salivarius WB21 reduced the numbers of periodontal bacteria in subgingival plaque, but did not reduce those in supragingival plaque. Washio et al. [30] reported that H 2 S-producing bacteria in the tongue biofilm of patients without 19

periodontitis were mainly Veillonella and Actinomyces species, and the numbers of both H 2 S-producing bacteria and total bacteria were higher in the odor group. Although the TCS did not change in our study, the effects of administering WB21 on the numbers and activity of these H 2 S-producing bacteria requires elucidation in the future. On the other hand, following oral administration of L. salivarius WB21, the number of P. intermedia significantly increased at day 15 compared with baseline. In a previous study, we reported that the prevalence of P. intemedia was lower in subjects with salivary β-galactosidase activity than in subjects without enzyme activity [31]. In addition, oral malodor levels were significantly higher in subjects with β-galactosidase activity than in subjects without enzyme activity. Although P. intermedia has the ability to produce VSCs, this organism may be associated with a healthy oral condition. The number of L. salivarius in saliva was higher on day 15, but did not continue to increase until day 29. Shimauchi et al. [8] reported that the proportion of L. salivarius in saliva specimens from healthy volunteers in both test and placebo groups tended to decrease during the intervention period. These data suggest that overgrowth and superinfection by L. salivarius WB21 in the oral cavity is not of concern. 20

However, further studies may be necessary in order to improve the stability of L. salivarius WB21 in the complex oral microflora, allowing the maintenance of its activity. Although some species of lactobacilli have been reported to occur in high numbers in both superficial and deep caries [24, 32], several Lactobacillus species have been isolated from healthy mouths [33, 34]. Byun et al. [24] detected higher L. gasseri and L. ultunensis than the other prevalent Lactobacillus species on carious dentine. Colloca et al. [33] predominantly isolated L. fermentum, L. plantarum, L. salivarius, and L. rhamnosus from healthy mouths. In an examination based on periodontology, the prevalent strains in healthy subjects were L. gasseri and L. fermentum versus L. plantarum in chronic periodontitis patients [34]. In our study, the prevalence of L. salivarius DNA in the physiologic halitosis group was greater than in the oral pathologic halitosis group. These analyses indicate that the pathological characteristics of the different Lactobacillus species should be elucidated in more detail. Different findings regarding probiotic lactobacilli and cariogenicity have also been reported. Haukija et al. [35] reported that the probiotic species used in commercial 21

products, including L. rhamnosus, L. casei, L. reuteri, and Bifidobacterium lactis, bound to saliva-coated hydroxyapatite and reduced the adhesion of S. mutans in vitro. Stahinic et al. [36] demonstrated that the isolate L. salivarius BGHO1 derived from a healthy oral cavity antagonized the growth of other bacteria, including S. mutans. Conversely, Matsumoto et al. [37] reported that L. salivarius LR1952R with S. mutans MT8148 significantly increased the caries scores compared with S. mutans MT8148 alone in rat experimental models. Mayanagi et al. [9] observed no adverse effects during an intervention using L. salivarius WB21 until the 8-week follow-up, and our patients who have continued to take L. salivarius WB21 for 3 months have also not developed new caries (data not shown). The limitations of this study include the small number of participants and the short intervention period. Therefore, further studies including a large-scale randomized clinical trial are necessary to determine the effectiveness of probiotics for the treatment and prevention of oral malodor and periodontal diseases. The current study highlights the need to fully understand the functions of probiotics in the oral cavity. 22

Acknowledgments We thank Dr. H. Hirata and Dr. S. Nakaya, Sagami Research Laboratories, Wakamoto Pharmaceutical Co., Ltd., for support and assistance during the study. We are grateful to Dr. M. Yoneda (Section of General Dentistry, Department of General Dentistry, Fukuoka Dental College), Dr. T. Naito (Section of Gerodontology, Department of General Dentistry, Fukuoka Dental College), and Prof. Y. Yamashita (Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University) for their critical discussion of the study and amending of the manuscript. This study was supported by Grants-in-Aid for Scientific Research (C) 20592249 (to M.Y.) and 20592444 (to T.N.), Grants-in-Aid for Young Scientists 21890187 (to T.T.), and Grants-in-Aid for Scientific Research (Strategic Research Promotion) from the Ministry of Education, Culture, Sports, Technology and Science, Japan. 23

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Table 1. Sequences of the primers used in this study. Species Sequence (5 3 ) Product size (bp) References Lactobacillus salivarius CGA AAC TTT CTT ACA CCG AAT GC 332 24 GTC CAT TGT GGA AGA TTC CC Porphyromonas gingivalis TGT AGA TGA CTG ATG GTG AAA ACC 211 25 ACG TCA TCC CCA CCT TCC TC Prevotella intermedia TTT GTT GGG GAG TAA AGC GGG 575 26 TCA ACA TCT CTG TAT CCT GCG T Treponema denticola TAA TAC CGA ATG TGC TCA TTT ACA T 316 26 TCA AAG AAG CAT TCC CTC TTC TTC TTA Streptococcus mutans ACT ACA CTT TCG GGT GGC TTG G 517 27 CAG TCT AAG CGC CAG TTT CAT C Streptococcus sobrinus GAT AAC TAC CTG ACA GCT GAC T 711 27 AAG CTG CCT TAA GGT AAT CAC T

Table 2. Baseline levels (day 1) of the clinical parameters and oral malodor in the patients. Physiological halitosis (n=9) Oral pathologic halitosis (n=11) P Age (average ± SE) 48.3 ± 11.9 56.5 ± 5.9 0.043 Female (%)* 100 (9/9) 45.5 (5/11) 0.008 Clinical parameters Number of teeth 26.6 ± 2.5 26.4 ± 3.0 0.880 Number of caries 0.2 ± 0.4 0.0 0.169 Number of filling 13.7 ± 3.9 15.5 ± 4.4 0.332 Inadequate restoration* (%) 0.0 (0/9) 0.0 (0/11) NA Tooth mobility* (%) 11.1 (1/9) 18.2 (2/11) 0.660 Occult blood in saliva (%)* 44.4 (4/9) 45.5 (5/11) 0.964 % of BOP 6.0 ± 3.4 11.0 ± 11.9 0.078 Average of PPD 2.2 ± 0.2 2.6 ± 0.3 0.004 Number of 5mm PPD 0.0 2.1 ± 2.3 <0.001 TCS 1.3 ± 0.5 1.5 ± 0.9 0.642 Plaque Index 0.2 ± 0.2 0.3 ± 0.2 0.105 Stimulated salivary flow (ml/5 min) 6.1 ± 3.5 8.7 ± 3.9 0.755 Oral malodor parameters OLT score 2.4 ± 0.7 2.8 ± 0.8 0.336 Gas chromatography (ng/10 ml mouth air) H 2 S 4.1 ± 2.9 3.7 ± 2.9 0.989

CH 3 SH 2.7 ± 1.7 2.9 ± 2.0 0.695 CH 3 SCH 3 0.6 ± 0.6 0.7 ± 0.6 0.738 total VSCs 7.4 ± 4.5 7.4 ± 5.2 0.996 Bacterial parameters (%)* Porphyromonas gingivalis 33.3 (3/9) 90.9 (10/11) 0.007 Prevotella intermedia 11.1 (1/9) 45.5 (5/11) 0.095 Treponema denticola 55.6 (5/9) 72.7 (8/11) 0.423 Streptococcus mutans 88.9 (8/9) 72.7 (8/11) 0.369 Streptococcus sobrinus 11.1 (1/9) 27.3 (3/11) 0.369 Lactobacillus salivarius 55.6 (5/9) 27.3 (3/11) 0.199 NA, not available; BOP, bleeding on probing; PPD, periodontal pocket depth; TCS, tongue coating score; OLT, organoleptic test. * Chi-square test. Wilcoxon-Mann-Whitney test. The other parameters were evaluated by t-test. Significant difference (P<0.05) between physiologic halitosis and oral pathologic halitosis.

Table 3. Changes in the clinical and oral malodor parameters between days 1 and 15. Physiologic halitosis group (n=9) P Oral pathologic halitosis group (n=11) P Day 1 Day 15 Day 1 Day 15 Clinical parameters with regard to oral malodor Occult blood in saliva (%)* 44.4 (4/9) 11.1 (1/9) 0.114 45.5 (5/11) 27.3 (3/11) 0.375 % of BOP 6.0 ± 3.4 3.9 ±3.9 0.234 11.0 ± 7.8 5.8 ± 4.7 0.008 Average of PPD 2.2 ± 0.2 2.2 ± 0.1 0.232 2.6 ± 0.3 2.5 ± 0.3 0.141 Number of 5mm PPD 0.0 0.0 NA 2.1 ± 0.8 2.1 ± 0.9 1 TCS 1.3 ± 0.5 1.0 ± 1.1 0.371 1.5 ± 0.9 1.5 ± 0.7 1 Plaque Index 0.2 ± 0.2 0.1 ± 0.1 0.859 0.3 ± 0.2 0.3 ± 0.2 0.306 Stimulated salivary flow (ml/5 min) 6.1 ± 3.5 7.1 ± 7.3 0.139 8.7 ± 3.9 9.1 ± 4.3 0.422 Oral malodor parameters OLT score 2.4 ± 0.7 1.6 ± 1.8 0.020 2.8 ± 0.8 2.2 ± 0.9 0.057 Gas chromatography (ng/10 ml mouth air) H 2 S 4.1 ± 2.9 2.0 ± 2.2 0.009 3.7 ± 2.9 2.5 ± 2.6 0.068 CH 3 SH 2.7 ± 1.7 0.9 ± 1.0 0.004 2.9 ± 2.0 2.1 ± 1.8 0.154 CH 3 SCH 3 0.6 ± 0.6 0.2 ± 0.3 0.108 0.7 ± 0.6 0.6 ±0.6 0.155 total VSCs 7.4 ± 4.5 3.1 ± 3.5 0.004 7.4 ± 5.3 5.3 ± 4.7 0.102 Bacterial parameters (%)*

Porphyromonas gingivalis 33.3 (3/9) 33.3 (3/9) 1 90.9 (10/11) 81.8 (9/11) 0.534 Prevotella intermedia 11.1 (1/9) 11.1 (1/9) 1 45.4 (5/11) 45.4 (5/11) 1 Treponema denticola 55.6 (5/9) 55.6 (5/9) 1 72.7 (8/11) 63.6 (7/11) 0.647 Streptococcus mutans 88.9 (8/9) 100 (9/9) 0.303 72.7 (8/11) 72.7 (8/11) 1 Streptococcus sobrinus 11.1 (1/9) 11.1 (1/9) 1 27.3 (3/11) 27.3 (3/11) 1 Lactobacillus salivarius 55.6 (5/9) 100 (9/9) 0.023 27.3 (3/11) 100 (11/11) <0.001 BOP, bleeding on probing; PPD, periodontal pocket depth; ND, not available; TCS, tongue coating score; OLT, organoleptic test. * Chi-square test. The other parameters were evaluated by Wilcoxon-Mann-Whitney test. Significant (P<0.05) difference versus day 1.

Table 4. Changes in the clinical and oral malodor parameters in the patients with oral pathologic halitosis over 4 weeks (n=10). Day 1 Day 15 P Day 29 P Clinical parameters with regard to oral malodor Occult blood in saliva (%)* 40.0 (4/10) 30.0 (3/10) 0.647 20.0 (2/10) 0.338 % of BOP 11.4 ± 8.1 5.7 ± 5.0 0.004 7.2 ± 5.8 0.006 Average of PPD 2.6 ± 0.3 2.5 ± 0.3 0.106 2.5 ± 0.3 0.136 Number of 5mm PPD 2.0 ± 0.8 2.0 ± 0.9 1 1.9 ± 1.1 1 TCS 1.5 ± 1.0 1.6 ± 0.7 0.766 1.4 ± 0.5 0.766 Plaque Index 0.3 ± 0.2 0.3 ± 0.2 0.474 0.3 ± 0.2 0.557 Stimulated salivary flow (ml/5 min) 9.2 ± 3.9 9.5 ± 4.3 0.506 9.8 ± 4.2 0.191 Oral malodor parameters OLT score 2.9 ± 0.7 2.3 ± 0.9 0.057 1.9 ± 0.5 0.013 Gas chromatography (ng/10 ml mouth air) H 2 S 3.5 ± 2.9 2.5 ± 2.7 0.131 2.3 ± 2.9 0.111 CH 3 SH 2.9 ± 2.1 2.2 ± 1.9 0.283 2.2 ± 2.2 0.557 (CH 3 ) 2 S 0.8 ± 0.6 0.6 ± 0.6 0.326 0.7 ± 0.8 0.402 total VSCs 7.2 ± 5.5 5.4 ± 4.9 0.193 5.2 ± 5.7 0.232 Amount of bacteria (log 10 ± SE)(copies/ml) Porphyromonas gingivalis 4.7 ± 0.8 4.7 ± 0.8 1 4.7 ± 0.7 0.722 Prevotella intermedia 4.6 ± 1.0 4.7 ± 0.9 0.030 4.7 ±0.9 0.363 Treponema denticola 4.5 ± 0.8 4.5 ± 0.9 0.800 4.5 ± 0.9 0.944

Fusobacterium nucleatum 6.5 ± 0.6 6.5 ± 0.7 1 6.4 ± 0.7 0.695 Lactobacillus salivarius 1.0 ± 1.3 2.9 ± 0.4 0.002 2.5 ± 0.9 0.004 Total bacteria 9.6 ± 0.3 9.6 ± 0.3 0.906 9.6 ± 0.3 0.375 BOP, bleeding on probing; PPD, periodontal pocket depth; TCS, tongue coating score; OLT, organoleptic test. * Chi-square test. The other parameters were evaluated by Wilcoxon-Mann-Whitney test. Significant (P<0.05) difference versus day 1.