Several studies have evaluated the

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1 J Periodontol September 2006 Microbiota of the Dorsum of the Tongue After Plaque Accumulation: An Experimental Study in Humans Marcelo Faveri,* Magda Feres,* Jamil Awad Shibli,* Roberto F. Hayacibara, Mitsue M. Hayacibara, and Luciene Cristina de Figueiredo* Background: The purpose of the present investigation was to determine the effect of the absence of tongue hygiene on the microbiota of the dorsum of the tongue. Methods: Ten volunteers (aged 19 to 22 years) entered the study at baseline and were instructed to abstain from tongue cleaning for 7 days, followed by a period of 3 days without any kind of oral hygiene. Subsequently, a period of 21 days of washout was employed, and this protocol was repeated three times. Microbiological samples were obtained from the dorsum of the tongue at baseline and at the end of the period of coating accumulation and analyzed using the checkerboard DNA-DNA hybridization technique. Results: The species found in highest mean counts at baseline and day 10 were Prevotella melaninogenica and Veillonella parvula. The mean bacterial total counts enhanced significantly during the study (from to ). Proportions of red and blue complexes and levels of 18 species also increased after the period of coating accumulation, including several periodontal pathogens, such as Prevotella intermedia, Prevotella nigrescens, Streptococcus constellatus, Tannerella forsythensis, Porphyromonas gingivalis, Treponema denticola, and P. melaninogenica. Conclusion: The tongue surface could be an important reservoir for periodontal pathogens and may play a role in the recolonization of tooth surfaces and in the etiology of oral halitosis. J Periodontol 2006;77: KEY WORDS Biofilm; DNA probes; hybridization; microbiology; tongue. * Department of Periodontology, Dental Research Division, Guarulhos University, Guarulhos, São Paulo, Brazil. Department of Dentistry, State University of Maringa, Maringa, Parana, Brazil. Several studies have evaluated the presence of specific bacterial species on different regions of the oral cavity. 1-6 These investigations have been motivated by the possible associations between soft tissue bacterial residents and various clinical conditions, such as oral halitosis, dental caries, and periodontal diseases At present, it has been estimated that ;700 species, including phylotypes, could inhabit the human oral cavity. 14 Additionally, the microbiota of the tongue surface is one of the most complex niches in human ecology, and approximately one-third of the bacterial population in the oral cavity is found on the tongue and not on other oral sites Different investigations have shown that the soft tissue surface can harbor periodontal pathogens. 2-4,7,10,11 van Winkelhoff et al. 7 characterized the microbiota of the tongue using culture and phase-contrast microscopy. The authors showed that spirochetes, motile organisms, and black pigmented species, such as Prevotella intermedia, colonized the tongue. They suggested that the presence of these organisms on the tongue surface could serve as an important ecological habitat for periodontal pathogens. In addition, Dahlen et al. 2 examined the tongue of periodontally diseased and non-diseased young adult Kenyan subjects. The periodontal pathogens were doi: /jop

2 Microbial Profile of Tongue Coating Volume 77 Number 9 Figure 1. Experimental design. One week before the beginning of the study, subjects received oral hygiene instructions (OHI). At baseline they were instructed to abstain from mechanical and chemical tongue cleaning procedures for 7 days, followed by a period of 3 days without any kind of oral hygiene. Subsequently, a 21-day washout period was employed. This experimental design was repeated three times (phases 1, 2, and 3). M = microbiological collection. present in both groups, although Porphyromonas gingivalis was detected significantly more frequently in tongue samples from periodontally diseased subjects. Some authors have also shown an association between tongue coating and oral halitosis, which has led to a growing scientific interest in studying the microbiota of the tongue and the factors that could influence this microenvironment. These studies indicated that a wide range of bacterial species, including Treponema denticola, P. gingivalis, Tannerella forsythensis, Prevotella melaninogenica, P. intermedia, Fusobacterium spp., Streptococcus, and Actinomyces spp., could be detected on the tongue surface. 3,9,14,17 The studies mentioned above suggest that the dorsum of the tongue may serve as a reservoir for infection or reinfection of supragingival and subgingival plaque with periodontal pathogens and could also influence oral halitosis. 18 Even though some studies have evaluated the effect of the absence of oral hygiene in the microbiota of supra- and subgingival plaque, none of them determined the changes that occurred in the composition of the microbiota of the tongue Therefore, the purpose of the present investigation was to determine the effect of the absence of tongue cleaning on the microbiota of the dorsum of the tongue. MATERIALS AND METHODS Subject Population Ten dental students, five men and five women (aged 19 to 22 years), were randomly selected for the study from February 2005 to June All subjects were asked to sign an informed consent form, with which they acknowledged their willingness to participate in the study. The Institutional Committee of Ethics in Clinical Research approved the study protocol. All subjects were submitted to anamnesis and clinical evaluation. Exclusion criteria included subjects with medical disorders or who had taken antibiotic or other antimicrobial therapy in the previous 6 months, smokers, pregnant women, the presence of one or more sites with a probing depth 3 mm, bleeding on probing, presence of carious lesions, and fewer than 20 natural teeth. Study Design One week before the beginning of the study, subjects received oral hygiene instructions and cleaning of the teeth and tongue. At baseline, they were instructed to abstain from all mechanical and chemical tongue cleaning procedures for 7 days, followed by a period of 3 days without any kind of oral hygiene. Subsequently, volunteers were oriented to return to their normal oral hygiene habits for 21 days (washout period). This experimental design was repeated three times (phases 1, 2, and 3) (Fig. 1). The oral hygiene instruction was repeated 1 week before the other two baselines. Volunteers received the same dentifrice without conventional antimicrobial agents, such as triclosan and chlorhexidine. Compliance was assessed by calling the subjects every day during the experimental phase. Microbiological samples were obtained from the posterior dorsum of the tongue at baseline and at the end of the period of coating accumulation (day 10). These collections were carried out at 8:00 am, and the subjects were asked to refrain from toothbrushing, drinking, and eating in the days of sampling. Microbiologic Assessment Sample collection. After removing the excess of saliva, one researcher collected the tongue samples. The coating was taken with only one movement from an area of 10 mm in length (measured with a periodontal probe ) and 5 mm in width (corresponding to Sorriso, Anakol Industria Comercio, Kolynos do Brasil, Colgate Palmolive, São Bernardo do Campo, São Paulo, Brazil. North Carolina periodontal probe, Hu-Friedy, Chicago, IL. 1540

3 J Periodontol September 2006 Faveri, Feres, Shibli, Hayacibara, Hayacibara, de Figueiredo Table 1. Bacterial Species Used for the Development of the Whole Genomic DNA Probe Tested Against Samples of Tongue Coating Species Strain Species Strain Actinobacillus actinomycetemcomitans a 43718* P. acnes 11827* A. actinomycetemcomitans b 29523* Leptotrichia buccalis 14201* A. naeslundii * Eubacterium nodatum 33099* A. naeslundii * M. micros 33270* Actinomyces israelii 12102* P. gingivalis 33277* Actinomyces odontolyticus 17929* P. intermedia 25611* A. gerencseriae 23860* P. nigrescens 33563* T. forsythensis 43037* P. melaninogenica 25845* Capnocytophaga ochracea 33596* Selenomonas noxia 43541* Capnocytophaga gingivalis 33624* S. oralis 35037* Capnocytophaga sputigena 33612* S. sanguinis 10556* Campylobacter rectus 33238* S. constellatus 27823* Campylobacter gracilis 33236* Streptococcus gordonii 10558* C. showae 33612* Streptococcus mitis 49456* Eikenella corrodens 23834* Streptococcus intermedius 27335* Fusobacterium periodonticum 33693* S. anginosus 33397* Fusobacterium nucleatum ss. nucleatum 25586* V. parvula 10790* F. nucleatum ss. polymorphum 10953* T. denticola B1 F. nucleatum ss. vincentii 49256* Treponema socranskii S1 N. mucosa 19696* * American Type Culture Collection, Rockville, MD. Forsyth Institute, Boston, MA. the tip of the Gracey curet i ) of the dorsum of the tongue from a region posterior to the sulcus terminalis. The samples were placed in separate Eppendorf tubes containing 0.15 ml TE (10 mm Tris-HCl and 1 mm EDTA, ph 7.6). A total of 0.10 ml 0.5 M NaOH was added to each tube, and the samples were dispersed using a vortex mixer. Checkerboard DNA-DNA Hybridization DNA isolation and preparation of DNA probes. Table 1 presents a list of the 39 bacterial species strains used for the preparation of DNA probes. The growth from 3- to 7-day cultures was harvested and placed in tubes containing 1 ml TE buffer (ph 7.6). Cells were washed twice and lysed at 37 C for 1 hour with either 10% sodium dodecyl sulfate (SDS) and proteinase K # (20 mg/ml) (for Gram-negative strains) or lysozyme** (15 mg/ml) and achromopeptidase (5 mg/ml) (for Gram-positive strains). DNA was isolated and purified using the method described by Smith et al. 22 Whole-genomic DNA probes were prepared for each species by labeling 1 mg DNA with digoxigenin using a random primer technique. 23 i Hu-Friedy. Sigma-Aldrich, St. Louis, MO. # Sigma-Aldrich. ** Sigma-Aldrich. Sigma-Aldrich. Sigma-Aldrich. 1541

4 Microbial Profile of Tongue Coating Volume 77 Number 9 Figure 2. Microbial profiles of mean counts ( 10 6 ) of 39 species evaluated in the tongue coating at baseline and day 10 in phases 1, 2, and 3. Counts of individual species were computed in each subject and averaged across subjects for each time point in each experimental phase. Species are ordered according to complexes described by Socransky et al. 23 Significant differences among experimental phases were determined using the Friedman test (P >0.05). Before the microbial detection in clinical samples, the specificity and sensitivity of the 40 DNA probes were assessed, hybridizing each DNA probe against individual pure cultures of all the test species adjusted to 10 4,10 5,10 6, and 10 7 cells. The sensitivity of the assay was set to allow the detection of ;10 4 cells of given species by adjusting the concentration of each individual DNA probe. DNA-DNA hybridization. Counts of 39 species were determined in each sample using the checkerboard DNA-DNA hybridization technique. 24 The samples were boiled for 10 minutes and neutralized using 0.8 ml of 5 M ammonium acetate. The released DNA was then placed into individual lanes of a device, ii concentrated onto a cm positively charged nylon membrane, and fixed to the membrane by baking at 120 C for 20 minutes. Two lanes on each membrane contained standards consisting of a mixture at 10 5 and 10 6 cells of each bacterial species tested. The membrane was placed in a second device ## with the sample lanes rotated 90 to the channels of the apparatus. The probes were diluted to ;20 ng/ml in hybridization solution (45% formamide,*** 5 standard saline citrate [SSC] [1 SSC = 120 mm NaCl and 15 mm Na citrate, ph 7.0], 1 Denhardt s solution, 20 mm Na phosphate [ph 6.5], 0.2 mg/ml yeast RNA, iii 10% dextran sufate, and 1% casein ### ), placed in individual channels of the device, and hybridized overnight at 42 C. The membranes were washed twice at high stringency for 20 minutes each time at 68 C in phosphate buffer (0.1 SSC and 0.1% SDS). Detection and enumeration of taxa. Membranes were blocked by a 1-hour incubation in blocking buffer containing 1% casein in maleate buffer (100 mm maleic acid**** and 150 mm NaCl, ph 7.5). Hybrids were detected by exposing the membranes to a 1:50,000 dilution of anti-digoxigenin antibody conjugated to alkaline phosphatase for 30 minutes, using a modification previously described. 25 Signals were detected by chemiluminescence. In brief, membranes were incubated in a chemiluminescent agent for 5 minutes at room temperature and exposed to films in autoradiographic cassettes for 10 minutes. Signals were converted to absolute counts by comparison with the standard lanes on the membrane. Sigma-Aldrich. ii Minislot-30, Immunetics, Cambridge, MA. Boehringer Mannheim, Indianapolis, IN. ## Miniblotter-45, Immunetics. *** Sigma-Aldrich. Sigma-Aldrich. Sigma-Aldrich. Sigma-Aldrich. iii Roche Diagnostics, Mannheim, Germany. Sigma-Aldrich. ### Sigma-Aldrich. **** Sigma-Aldrich. Roche Diagnostics. CDP-Star, Roche Diagnostics. 1542

5 J Periodontol September 2006 Faveri, Feres, Shibli, Hayacibara, Hayacibara, de Figueiredo Data Analysis Microbiological data were available from all subjects at all visits. Mean counts 10 6 of each species were computed for each subject in each visit and then averaged across subjects. In a similar way, proportions of bacterial complexes 23 were computed at each phase. The significance of differences among the three baselines (phases 1, 2, and 3) of the 10 subjects was evaluated using the Friedman test. The significance of differences between baseline and day 10 in mean levels of the test species was determined using the Wilcoxon signed-rank test. Adjustments were made for multiple comparisons as described by Socransky et al. 26 Statistical significance was reached at a 5% level. RESULTS Figure 2 presents the results for each time point of the three experimental phases separately. There were no significant differences among the three baselines or among the three day-10 time points from phases 1, 2, and 3 (P >0.05). Therefore, the results of the three experimental phases (10 samples per phase) were averaged at each visit, providing 30 samples per time point. Mean total DNA probe counts ( 10 6 SD) observed at baseline and after 10 days of coating accumulation are presented in Figure 3. Total counts of organisms present on the tongue surface at the beginning of the experimental phase averaged and increased to after 10 days without tongue hygiene (P <0.001). Mean counts of the 39 species evaluated during the study are presented in Figure 4. The species were ordered according to the complexes described by Socransky et al. 27 The two species present in highest levels at baseline were P. melaninogenica and Veillonella parvula. These organisms comprised almost 50% of the total bacterial counts at baseline ( ). Mean counts of 19 species were significantly changed (P <0.05). Actinomyces gerencseriae, Actinomyces naeslundii 1, Actinomyces naeslundii 2, Streptococcus oralis, Streptococcus sanguinis, Campylobacter showae, Micromonas micros, P. intermedia, Prevotella nigrescens, Streptococcus constellatus, T. forsythensis, P. gingivalis, T. denticola, Eubacterium saburreum, Neisseria mucosa, Streptococcus anginosus, P. melaninogenica, and V. parvula increased significantly after the phase of absence of tongue cleaning (P <0.05). Propionibacterium acnes was the only species that showed a significant reduction in levels after 10 days of coating accumulation (P <0.05). The proportions of the microbial complexes are presented in Figure 5. Areas of the pies were adjusted to reflect the mean total counts at each time point. As Figure 3. Bar charts of total bacterial mean counts ( 10 6 ) of tongue coating samples at baseline and after 10 days of bacterial accumulation. Total bacterial counts were computed for each subject and averaged in the group in each experimental phase. Subsequently, mean bacterial counts of the three phases at both time points were calculated (N = 30). The significance of differences between experimental phases was determined using the Wilcoxon signed-rank test (P <0.001). described previously, an increase in mean total bacterial counts was observed at the end of the period of tongue-coating accumulation. The orange complex and purple complex represented the highest proportion at baseline (26.1% and 22.4%, respectively) and at the end of the experimental phase (25.9% and 25.7%). A significant increase in the proportions of the red complex (0.4% to 1.2%) and the Actinomyces species (2.2% to 4.8%) was observed at the end of the study (P <0.01). The green complex showed a reduction in proportions at day 10, although this change was not statistically significant. DISCUSSION It has been shown that oral bacteria demonstrate specific tropism toward the different biologic surfaces of the oral cavity. The microorganisms have distinct receptors and adhesion molecules that dictate the development of different biofilms on the oral surfaces. 4,5 The non-shedding surfaces of the teeth offer a far different habitat than the continually shedding surfaces of the oral musosa. 4 Changes in the microbial composition that occur on the tooth surface after a period of absence of oral hygiene have been studied; 1543

6 Microbial Profile of Tongue Coating Volume 77 Number 9 Figure 4. Mean counts ( 10 6 ) of the 39 species evaluated in the tongue-coating samples at baseline and after 10 days of bacterial accumulation. Counts of individual species were computed for each subject and averaged in the group in each experimental phase. Subsequently, mean bacterial counts of the three phases at both time points were calculated (N = 30). Species are ordered according to complexes described by Socransky et al. 23 Significant differences over time were determined using the Wilcoxon signed-rank test (*P <0.05). however, the results of this bacterial accumulation in the oral mucosa and tongue are still not well established. 20,21 The present investigation described the microbial profiles on the dorsum of the tongue before and after a period of absence of oral hygiene. At baseline, the tongue coating was found to be colonized predominantly by Gram-negative species. P. melaninogenica and V. parvula were the species detected in highest mean counts before and after the coating accumulation. Other authors have observed high levels of these species on the tongue using different microbiological techniques. 4,5,13,28 Several periodontal pathogens from the red and orange complexes, including P. gingivalis, T. denticola, T. forsythensis, P. intermedia, 1544 and P. nigrescens, were detected on the surface of the tongue at baseline and increased significantly, in counts, after the phase of hygiene withhold. These results agree and extend the results of other studies that suggested that the dorsum of the tongue may be an important reservoir for periodontal pathogens and could be a major factor in the recolonization of tooth surfaces after periodontal therapy. 2-4,7 The morphology of the dorsum of the tongue provides additional irregularities, such as fissures, grooves, and depapillated areas, that may facilitate the accumulation of microorganisms. These anatomic niches may create an environment where microorganisms are well protected from flushing action of the saliva and where oxygen levels are low, thus promoting the development of anaerobic microbiota. 15,16,28,29 The development of a predominant anaerobic microbiota associated with the tongue coating has been considered an ideal microenvironment to produce malodorous compounds, such as volatile sulfur compounds (VSCs). There is very little information in the literature regarding the microbiota associated with oral halitosis. 16 P. intermedia, P. gingivalis, T. denticola, T. forsythensis, and other bacterial species have showed a high capability of producing VSCs Even though the purpose of this study was not to determine the microbiota associated with halitosis, the significant increase of the total bacterial counts and of 18 individual species, including the pathogens mentioned above (Fig. 3), may suggest that the lack of tongue cleaning could contribute to oral halitosis. Hartley et al. 33 found that malodor was related to higher number and proportions of Gram-negative anaerobe species on the tongue surface. Another interesting result in this study was the composition and the changes observed in the proportions

7 J Periodontol September 2006 Faveri, Feres, Shibli, Hayacibara, Hayacibara, de Figueiredo Figure 5. Pie charts of mean proportions of microbial complexes (Socransky et al. 27 ) at baseline and after 10 days of bacterial accumulation. Colors represent different complexes (blue = Actinomyces; gray = other). The significance of differences in mean proportions between baseline and day 10 for each complex was tested using the Wilcoxon signed-rank test (*P < 0.01). of the microbial complexes. The profile of the complexes is quite different from that observed in the subgingival or supragingival biofilm in periodontally healthy subjects. The tongue is colonized with higher proportions of purple complex and a group named others, probably due to the high levels of P. melaninogenica and V. parvula observed. Conversely, the Actinomyces spp. and the green complex are present in lower proportions than in the tooth biofilm. The proportion of red complex at baseline was very low, similar to the proportions observed in subjects with periodontal health. Interestingly, after the period of bacterial accumulation, the proportion of this complex increased significantly, as well as the proportion of Actinomyces species. CONCLUSIONS The analysis made in the present study of 39 bacterial species, including those difficult to cultivate, allowed a systematic evaluation of the tongue coating. It was observed that the absence of tongue hygiene increased the total bacterial counts and the counts and proportions of individual species and complexes, including several anaerobic virulent microorganisms. These findings suggest that the tongue surface could be an important reservoir for periodontal pathogens and may play a role in the recolonization of tooth surfaces and in the etiology of oral halitosis. REFERENCES 1. Danser MM, van Winkelhoff AJ, de Graaff J, van der Velden U. Putative periodontal pathogens colonizing oral mucous membranes in denture-wearing subjects with a past history of periodontitis. J Clin Periodontol 1995;22: Dahlen G, Manji F, Baelum V, Fejerskov O. Putative periodontopathogens in diseased and non-diseased persons exhibiting poor oral hygiene. J Clin Periodontol 1992;19: De Boever EH, Loesche WJ. Assessing the contribution of anaerobic microflora of the tongue to oral malodor. J Am Dent Assoc 1995;126: Mager DL, Ximenez-Fyvie LA, Haffajee AD, Socransky SS. Distribution of selected bacterial species on intraoral surfaces. J Clin Periodontol 2003;30: Socransky SS, Haffajee AD. Periodontal microbial ecology. Periodontol ;38: Danser MM, Timmerman MF, van Winkelhoff AJ, van der Velden U. The effect of periodontal treatment on periodontal bacteria on the oral mucous membranes. J Periodontol 1996;67: van Winkelhoff AJ, van der Velden U, Winkel EG, de Graaff J. Black-pigmented Bacteroides and motile organisms on oral mucosal surfaces in individuals with and without periodontal breakdown. J Periodontal Res 1986;21: Dahlen G, Slots J. Use of bacteriology in the campaign against caries. Phillip J Restaur Zahnmed 1986;1: Bosy A, Kulkarni GV, Rosenberg M, McCulloch CA. Relationship of oral malodor to periodontitis: Evidence of independence in discrete subpopulations. J Periodontol 1994;65: Tyrrell KL, Citron DM, Warren YA, Nachnani S, Goldstein EJ. Anaerobic bacteria cultured from the tongue dorsum of subjects with oral malodor. Anaerobe 2003;9: Lee KH, Tanner AC, Maiden MF, Weber HP. Pre- and post-implantation microbiota of the tongue, teeth, and newly placed implants. J Clin Periodontol 1999;26: Tanaka M, Yamamoto Y, Kuboniwa M, et al. Contribution of periodontal pathogens on tongue dorsa analyzed with real-time PCR to oral malodor. Microbes Infect 2004;6: Donaldson A, McKenzie D, Riggio M, et al. Microbiological culture analysis of the tongue anaerobic microflora in subjects with and without halitosis. Oral Dis 2005;11: Kazor CE, Mitchell PM, Lee AM, et al. Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients. J Clin Microbiol 2003;41: Roldan S, Winkel EG, Herrera D, Sanz M, van Winkelhoff AJ. The effects of a new mouthrinse containing chlorhexidine, cetylpyridinium chloride and zinc lactate on the microflora of oral halitosis patients: A dualcentre, double-blind placebo-controlled study. J Clin Periodontol 2003;30: Roldan S, Herrera D, Sanz M. Biofilms and the tongue: Therapeutical approaches for the control of halitosis. Clin Oral Investig 2003;7: Kato H, Yoshida A, Awano S, Ansai T, Takehara T. Quantitative detection of volatile sulfur compoundproducing microorganisms in oral specimens using real-time PCR. Oral Dis 2005;11: Quirynen M, Avontroodt P, Peeters W, Pauwels M, Coucke W, van Steenberghe D. Effect of different 1545

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