University of Groningen
Tongue coating
Seerangaiyan, Kavitha
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Seerangaiyan, K. (2018). Tongue coating: It’s impact on intra-oral halitosis and taste. Rijksuniversiteit Groningen.
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CHAPTER
6
General discussion, conclusions, and future
perspectives
CHAPTER 6
General discussion, conclusions, and future perspectives
The overarching goal of this thesis was to study the role of tongue coating (TC) in intra-oral halitosis (IOH) and taste. A PubMed search revealed that since 1992, a total of 196 reports on TC have been published, indicating a scarcity of studies (Figure 1). Much remains to be learned about the role of TC in the etiology of IOH because many questions are unanswered. It is generally assumed that the microbiome of the TC plays a crucial role in IOH. Relatively new is the interest in the microbial metabolites that could be responsible for IOH. Studies on the composition of the tongue microbiome in relation to the metabolome have, until recently, not been performed.
Fig. 1. Using MeSH term “Halitosis AND (tongue coating OR microbiome)” in
PubMed search
Our starting point was the assumption that the formation of TC changes the composition of the tongue microbiome and metabolome in IOH (Figure 2). We also hypothesized that removal of TC would improve the taste perception.
Tongue coating
TC, a thin “white coat” present in healthy people, is relatively thick in people with IOH. TC might be the predominant cause of IOH in individuals with healthy periodontium. In traditional Chinese and Ayurvedic medicine, diagnosis of gastrointestinal and other organ disorders is based on the presence and certain characteristics of the TC (color, moisture, and texture). In these medical traditions, the
GENERAL DISCUSSION
tongue is the mirror of the body, reflecting changes in other organs and aiding in disease diagnosis. Advances in medical instrumentation for diagnosing systemic disease have superseded TC-based diagnosis in general. Recently, an epidemiological study on TC showed that TC is more prevalent (28%) among conditions such as geographic tongue (16.4%) and fissured tongue (14.9%) [1]. Renewed interest in the meaning of TC in systemic disease diagnosis may arise because some aspects of TC recently have been linked to diabetes mellitus [2].
Fig. 2. Overview of this study
Methods for evaluating tongue coating
IOH is associated with increased TC. Therefore, evaluating the quantity of TC is useful in developing a treatment strategy and can serve as a biomarker for systemic diseases. Evaluation of the TC based on quantity is mainly done by visual indices and criteria involving thickness, covered area, and discoloration of the coating. An alternative method includes collection of a tongue scraping and determining its wet weight. Of these methods, the visual index is a simple, quick, and reliable approach. Several visual index options are the Kojima, Miyazaki, Gomez, and Winkel (WTCI: Winkel Tongue Coating Index). Among these, the WTCI is the most commonly used.
General discussion, conclusions, and future perspectives
The overarching goal of this thesis was to study the role of tongue coating (TC) in intra-oral halitosis (IOH) and taste. A PubMed search revealed that since 1992, a total of 196 reports on TC have been published, indicating a scarcity of studies (Figure 1). Much remains to be learned about the role of TC in the etiology of IOH because many questions are unanswered. It is generally assumed that the microbiome of the TC plays a crucial role in IOH. Relatively new is the interest in the microbial metabolites that could be responsible for IOH. Studies on the composition of the tongue microbiome in relation to the metabolome have, until recently, not been performed.
Fig. 1. Using MeSH term “Halitosis AND (tongue coating OR microbiome)” in
PubMed search
Our starting point was the assumption that the formation of TC changes the composition of the tongue microbiome and metabolome in IOH (Figure 2). We also hypothesized that removal of TC would improve the taste perception.
Tongue coating
TC, a thin “white coat” present in healthy people, is relatively thick in people with IOH. TC might be the predominant cause of IOH in individuals with healthy periodontium. In traditional Chinese and Ayurvedic medicine, diagnosis of gastrointestinal and other organ disorders is based on the presence and certain characteristics of the TC (color, moisture, and texture). In these medical traditions, the
tongue is the mirror of the body, reflecting changes in other organs and aiding in disease diagnosis. Advances in medical instrumentation for diagnosing systemic disease have superseded TC-based diagnosis in general. Recently, an epidemiological study on TC showed that TC is more prevalent (28%) among conditions such as geographic tongue (16.4%) and fissured tongue (14.9%) [1]. Renewed interest in the meaning of TC in systemic disease diagnosis may arise because some aspects of TC recently have been linked to diabetes mellitus [2].
Fig. 2. Overview of this study
Methods for evaluating tongue coating
IOH is associated with increased TC. Therefore, evaluating the quantity of TC is useful in developing a treatment strategy and can serve as a biomarker for systemic diseases. Evaluation of the TC based on quantity is mainly done by visual indices and criteria involving thickness, covered area, and discoloration of the coating. An alternative method includes collection of a tongue scraping and determining its wet weight. Of these methods, the visual index is a simple, quick, and reliable approach. Several visual index options are the Kojima, Miyazaki, Gomez, and Winkel (WTCI: Winkel Tongue Coating Index). Among these, the WTCI is the most commonly used.
CHAPTER 6
The visual WTCI has a chance for inter- and intra-examiner bias, and therefore, standardizing this method to minimize bias is important for reproducibility. A potential option is the digital tongue coating index [3], but this method has practical difficulties because of restricted mouth opening of some individuals. Other biomarkers must be developed to allow for better description and characterization of the TC. Understanding the TC microbiome and its metabolites could help lead to specific biomarkers.
Tongue coating microbiome
The papillary structure of the tongue provides an ecological niche for growth of the tongue microbiome [4]. The tongue microbiome metabolizes the organic material of saliva, food residues, plaque, and epithelial cells, producing volatile sulfur compounds (VSCs), the key elements of bad breath [5]. Understanding the composition of the tongue microbiome in health and IOH may assist in the development of new treatment strategies for IOH. In vitro studies have shown that oral bacteria can produce VSCs, and studies of IOH thus have focused on characterization of the tongue microbiome. We aimed to characterize the TC microbiome in IOH using 16S amplicon sequencing of the V3-V4 hypervariable region, with an Illumina MiSeq platform. Sequence analysis of the 16S ribosomal RNA (rRNA) gene has been widely used in different approaches to identify bacterial species and perform taxonomic studies. The bacterial 16S rRNA gene contains nine hypervariable regions (V1–V9) that represent sequence diversity among different bacterial species. V2 and V3 are the most used for distinguishing bacterial species to the genus level, except for closely related species. The current trend in the study of the human microbiome is the exploration of bacterial communities particularly in health and disease. Illumina MiSeq is an advanced technology with greater sequence coverage and depth of the target genes. 16S rRNA amplicon sequencing can be targeted specifically against bacteria and does not require the availability of reference genome sequences. This technique had a significant influence on the outcome of this study because previous studies on IOH were based on culture technique and cloning-based Sanger sequencing. Sanger sequencing, a classical approach, can address only a single gene, and the sequence depth is comparatively less than that of the Illumina MiSeq system. Depth of sequencing is important in microbiome analysis at the species level. Moreover, the previous studies reported species based on their prevalence in an individual with halitosis, and because the sample sizes were small, no statistical analysis was performed.
Our study using the MiSeq platform showed a clear taxonomic and phylogenic identification. We observed nearly 7 genera, 27 phyla, and 825 species. The main finding of our study is that the microbial community composition in IOH is
GENERAL DISCUSSION highly similar compared to controls, with a similarity of 97%. Minor differences were related to OTUs (operational taxonomic units) in the IOH group and include
Clostridiales, SRI, TM7, Campylobacter, Dialister, Leptotrichia, Peptostreptococcus, Prevotella, Selenomonas, Peptococuus, Aggregatibacter, Capnocytophaga, Parvimonas, Treponema, and Tannerella. A previous study using anaerobic culture
identified a few of the above-mentioned species such as Campylobacter, Dialister,
Leptotrichia, Peptostreptococcus, Prevotella, and Selenomonas [6]. Another study
based on cloning and 16S sequencing identified TM7 and Dialister as markers for IOH [7]. Earlier, S. moorei was reported as the prevailing species in an IOH group and was identified only in IOH samples [8]. In contrast, another study found S.
moorei in both IOH and control samples [9]. In the present work, no significant
difference in S. moorei counts could be established between IOH and controls [10]. Of interest, in our study, we identified Clostridiales, SRI, and Peptococuus as markers for IOH, which has not been reported in previous studies.
Based on these observations, there seems to be no rationale for microbial diagnosis in IOH. In the future, a possible area of research is evaluating the tongue microbiome before and after treatment, considering environmental factors (i.e., bacterial adaptability, oral cavity pH, food debris, dead epithelial cells) that might reveal more significant candidates for microbial therapy. Based on the conclusions of our study (Chapter 3), we speculate that changes in the physiological adaptability of the tongue microbiome in response to unknown environmental stress might be responsible for IOH. Also, we further hypothesize that microbial-derived metabolites play a role in IOH.
Metabolomics of the tongue coating
Metabolomics provides a real picture of the dynamic processes in the cell and is more closely related to the phenotype of the disease. Metabolomics provides information about the presence and amounts of small molecules of enzymatic intermediates and metabolic end-products such as fatty acids, sugars, lipids, amino acids, pigments, nucleotides, organic acids, and vitamins under certain physiological conditions over time. The amount of metabolites may reflect disease progression influenced by environmental factors and genetic modifications, changes in microbiome, and altered kinetic activity of enzymes. A recent study on the metabolomics of dental caries has revealed a direct link between the presence of Streptococcus mutans and its metabolite lactic acid, which initiates dental caries. Here, we profiled the TC metabolites in participants with and without IOH using the LC-MS/MS approach. As far as we are aware, our study is the first to investigate the microbial metabolic profile of TC.
The visual WTCI has a chance for inter- and intra-examiner bias, and therefore, standardizing this method to minimize bias is important for reproducibility. A potential option is the digital tongue coating index [3], but this method has practical difficulties because of restricted mouth opening of some individuals. Other biomarkers must be developed to allow for better description and characterization of the TC. Understanding the TC microbiome and its metabolites could help lead to specific biomarkers.
Tongue coating microbiome
The papillary structure of the tongue provides an ecological niche for growth of the tongue microbiome [4]. The tongue microbiome metabolizes the organic material of saliva, food residues, plaque, and epithelial cells, producing volatile sulfur compounds (VSCs), the key elements of bad breath [5]. Understanding the composition of the tongue microbiome in health and IOH may assist in the development of new treatment strategies for IOH. In vitro studies have shown that oral bacteria can produce VSCs, and studies of IOH thus have focused on characterization of the tongue microbiome. We aimed to characterize the TC microbiome in IOH using 16S amplicon sequencing of the V3-V4 hypervariable region, with an Illumina MiSeq platform. Sequence analysis of the 16S ribosomal RNA (rRNA) gene has been widely used in different approaches to identify bacterial species and perform taxonomic studies. The bacterial 16S rRNA gene contains nine hypervariable regions (V1–V9) that represent sequence diversity among different bacterial species. V2 and V3 are the most used for distinguishing bacterial species to the genus level, except for closely related species. The current trend in the study of the human microbiome is the exploration of bacterial communities particularly in health and disease. Illumina MiSeq is an advanced technology with greater sequence coverage and depth of the target genes. 16S rRNA amplicon sequencing can be targeted specifically against bacteria and does not require the availability of reference genome sequences. This technique had a significant influence on the outcome of this study because previous studies on IOH were based on culture technique and cloning-based Sanger sequencing. Sanger sequencing, a classical approach, can address only a single gene, and the sequence depth is comparatively less than that of the Illumina MiSeq system. Depth of sequencing is important in microbiome analysis at the species level. Moreover, the previous studies reported species based on their prevalence in an individual with halitosis, and because the sample sizes were small, no statistical analysis was performed.
Our study using the MiSeq platform showed a clear taxonomic and phylogenic identification. We observed nearly 7 genera, 27 phyla, and 825 species. The main finding of our study is that the microbial community composition in IOH is
highly similar compared to controls, with a similarity of 97%. Minor differences were related to OTUs (operational taxonomic units) in the IOH group and include
Clostridiales, SRI, TM7, Campylobacter, Dialister, Leptotrichia, Peptostreptococcus, Prevotella, Selenomonas, Peptococuus, Aggregatibacter, Capnocytophaga, Parvimonas, Treponema, and Tannerella. A previous study using anaerobic culture
identified a few of the above-mentioned species such as Campylobacter, Dialister,
Leptotrichia, Peptostreptococcus, Prevotella, and Selenomonas [6]. Another study
based on cloning and 16S sequencing identified TM7 and Dialister as markers for IOH [7]. Earlier, S. moorei was reported as the prevailing species in an IOH group and was identified only in IOH samples [8]. In contrast, another study found S.
moorei in both IOH and control samples [9]. In the present work, no significant
difference in S. moorei counts could be established between IOH and controls [10]. Of interest, in our study, we identified Clostridiales, SRI, and Peptococuus as markers for IOH, which has not been reported in previous studies.
Based on these observations, there seems to be no rationale for microbial diagnosis in IOH. In the future, a possible area of research is evaluating the tongue microbiome before and after treatment, considering environmental factors (i.e., bacterial adaptability, oral cavity pH, food debris, dead epithelial cells) that might reveal more significant candidates for microbial therapy. Based on the conclusions of our study (Chapter 3), we speculate that changes in the physiological adaptability of the tongue microbiome in response to unknown environmental stress might be responsible for IOH. Also, we further hypothesize that microbial-derived metabolites play a role in IOH.
Metabolomics of the tongue coating
Metabolomics provides a real picture of the dynamic processes in the cell and is more closely related to the phenotype of the disease. Metabolomics provides information about the presence and amounts of small molecules of enzymatic intermediates and metabolic end-products such as fatty acids, sugars, lipids, amino acids, pigments, nucleotides, organic acids, and vitamins under certain physiological conditions over time. The amount of metabolites may reflect disease progression influenced by environmental factors and genetic modifications, changes in microbiome, and altered kinetic activity of enzymes. A recent study on the metabolomics of dental caries has revealed a direct link between the presence of Streptococcus mutans and its metabolite lactic acid, which initiates dental caries. Here, we profiled the TC metabolites in participants with and without IOH using the LC-MS/MS approach. As far as we are aware, our study is the first to investigate the microbial metabolic profile of TC.
CHAPTER 6
The striking result of our study was the finding that the tongue metabolomic profiles were significantly different in IOH (TC-IOH) compared to the healthy control group (TC-Control). These novel findings revealed the mechanism of TC formation and the VSC production pathway, including production of H2S, one of the primary causes of IOH. Most important, in the TC-IOH, we observed enrichment of the metabolite branched-chain fatty acids (BCFAs) produced during degradation of branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine in an anaerobic environment. To support this finding, we observed a significant amount of BCAA in our TC-IOH samples compared to the TC-Controls. Clostridia species have been reported to produce BCFA from BCAA by the so-called “Stickland reaction” [11]. Of interest, in our microbiome study, we found that Clostridiales was significantly elevated in those with TC-IOH compared to those without [10]. BCFA can form a cheesy wax-like coating in newborn babies [12], suggesting that it might be responsible for TC formation in the oral cavity. BCFA-fermented food products like natto and dairy products significantly increase bad odor [13]. We speculate that the BCFAs are tied to the fermentation of TC debris (most likely food debris), thus producing bad breath.
The Stickland reaction enables bacteria to grow on amino acids and use them as a carbon and nitrogen source for growth, relying on coupled amino acid oxidation and reduction to obtain energy [14]. Also, in this reaction, ammonia and organic acids are produced that alter the pH of the ecosystem. Another important aspect is that this reaction proceeds via acetyl phosphate formation. We found significantly higher acetyl phosphate metabolite levels in TC-IOH compared to TC-Control participants (Chapter 4). Overall, the above novel findings suggest a correlation among the tongue microbiome, metabolites, and bad breath.
TC and taste
In ancient medicine traditions such as Ayurveda, tongue cleaning was advised as a part of oral hygienic measures to improve oral health and digestion. Currently, tongue cleaning is not a part of standard oral health hygiene procedures. TC covers the taste papillae on the dorsal tongue surface and may block taste molecules from reaching taste cells, thereby decreasing taste sensitivity. The taste perception system gives us information on food quality. Stimulation of taste receptors, oral mechano- and temperature receptors, and olfactory receptors produces particular taste sensations. These taste sensations can be expressed as the intensity, palatability, or hedonic values of the tasted substances. Of the five basic taste capacities of humans, salt has more influence on general health. Increased salt intake is a risk factor for hypertension and its associated diseases like cardiac disease, stroke, and obesity. Several strategies have been used to reduce salt intake as a matter of public health,
GENERAL DISCUSSION and individual behavioral changes play an important role in this reduction. The aim of the study was to investigate the effects of mechanical TC removal on the intensity of salt taste perception. If simple tongue cleaning could change the perceived intensity of salt, individuals might better adapt to low sodium concentrations without changing food palatability. Our study is the first to report a significant increase in the intensity of salt perception after tongue cleaning (Chapter 5). Thus, tongue cleaning may help individuals reduce excess salt intake, in turn influencing general health. In conclusion, tongue cleaning should become a part of routine oral hygiene practice.
Conclusions
1. TC is associated with IOH, but little is known about the formation of this complex biofilm.
2. The compositions of the tongue microbiomes in healthy and IOH samples are very similar.
3. The small differences found at the OTU level between IOH and controls may explain the bacterial adaptability (physiological changes) in the IOH condition. 4. The physiological changes in bacteria may induce production of different IOH-related metabolites such as BCFA, which forms a white coating on the tongue. Clostridia species may play an important role in this process.
5. TC can serve as trap and a reservoir for food particles and cellular debris. 6. The present findings show a link between the oral microbiome and
metabolome in the formation of TC and bad odor.
7. TC covers the taste papillae and may block the taste buds; thus, mechanical removal helps to improve taste perception.
Future perspectives
1. Targeted metabolomics and lipidomics can help quantify BCFAs, which might facilitate understanding of the mechanism of TC formation.
2. Enzymes play a critical role in maintaining the redox state, and enzymological studies will be supportive in identifying the cause of IOH as it relates to TC. 3. Halitosis is a condition that might reflect diseases such as gastrointestinaldisorders. The tongue is the first organ of the digestive system, and
gastrointestinal disorders mostly manifest in the TC. In recent years, several studies have revealed changes in the gut microbiome in various gastrointestinal disorders. It will be of interest to study the changes in both TC and the gut microbiome. The TC microbiome may be used as a biomarker for gastrointestinal and other medical disorders.
The striking result of our study was the finding that the tongue metabolomic profiles were significantly different in IOH (TC-IOH) compared to the healthy control group (TC-Control). These novel findings revealed the mechanism of TC formation and the VSC production pathway, including production of H2S, one of the primary causes of IOH. Most important, in the TC-IOH, we observed enrichment of the metabolite branched-chain fatty acids (BCFAs) produced during degradation of branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine in an anaerobic environment. To support this finding, we observed a significant amount of BCAA in our TC-IOH samples compared to the TC-Controls. Clostridia species have been reported to produce BCFA from BCAA by the so-called “Stickland reaction” [11]. Of interest, in our microbiome study, we found that Clostridiales was significantly elevated in those with TC-IOH compared to those without [10]. BCFA can form a cheesy wax-like coating in newborn babies [12], suggesting that it might be responsible for TC formation in the oral cavity. BCFA-fermented food products like natto and dairy products significantly increase bad odor [13]. We speculate that the BCFAs are tied to the fermentation of TC debris (most likely food debris), thus producing bad breath.
The Stickland reaction enables bacteria to grow on amino acids and use them as a carbon and nitrogen source for growth, relying on coupled amino acid oxidation and reduction to obtain energy [14]. Also, in this reaction, ammonia and organic acids are produced that alter the pH of the ecosystem. Another important aspect is that this reaction proceeds via acetyl phosphate formation. We found significantly higher acetyl phosphate metabolite levels in TC-IOH compared to TC-Control participants (Chapter 4). Overall, the above novel findings suggest a correlation among the tongue microbiome, metabolites, and bad breath.
TC and taste
In ancient medicine traditions such as Ayurveda, tongue cleaning was advised as a part of oral hygienic measures to improve oral health and digestion. Currently, tongue cleaning is not a part of standard oral health hygiene procedures. TC covers the taste papillae on the dorsal tongue surface and may block taste molecules from reaching taste cells, thereby decreasing taste sensitivity. The taste perception system gives us information on food quality. Stimulation of taste receptors, oral mechano- and temperature receptors, and olfactory receptors produces particular taste sensations. These taste sensations can be expressed as the intensity, palatability, or hedonic values of the tasted substances. Of the five basic taste capacities of humans, salt has more influence on general health. Increased salt intake is a risk factor for hypertension and its associated diseases like cardiac disease, stroke, and obesity. Several strategies have been used to reduce salt intake as a matter of public health,
and individual behavioral changes play an important role in this reduction. The aim of the study was to investigate the effects of mechanical TC removal on the intensity of salt taste perception. If simple tongue cleaning could change the perceived intensity of salt, individuals might better adapt to low sodium concentrations without changing food palatability. Our study is the first to report a significant increase in the intensity of salt perception after tongue cleaning (Chapter 5). Thus, tongue cleaning may help individuals reduce excess salt intake, in turn influencing general health. In conclusion, tongue cleaning should become a part of routine oral hygiene practice.
Conclusions
1. TC is associated with IOH, but little is known about the formation of this complex biofilm.
2. The compositions of the tongue microbiomes in healthy and IOH samples are very similar.
3. The small differences found at the OTU level between IOH and controls may explain the bacterial adaptability (physiological changes) in the IOH condition. 4. The physiological changes in bacteria may induce production of different IOH-related metabolites such as BCFA, which forms a white coating on the tongue. Clostridia species may play an important role in this process.
5. TC can serve as trap and a reservoir for food particles and cellular debris. 6. The present findings show a link between the oral microbiome and
metabolome in the formation of TC and bad odor.
7. TC covers the taste papillae and may block the taste buds; thus, mechanical removal helps to improve taste perception.
Future perspectives
1. Targeted metabolomics and lipidomics can help quantify BCFAs, which might facilitate understanding of the mechanism of TC formation.
2. Enzymes play a critical role in maintaining the redox state, and enzymological studies will be supportive in identifying the cause of IOH as it relates to TC. 3. Halitosis is a condition that might reflect diseases such as gastrointestinaldisorders. The tongue is the first organ of the digestive system, and
gastrointestinal disorders mostly manifest in the TC. In recent years, several studies have revealed changes in the gut microbiome in various gastrointestinal disorders. It will be of interest to study the changes in both TC and the gut microbiome. The TC microbiome may be used as a biomarker for gastrointestinal and other medical disorders.
CHAPTER 6 References
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2. Tomooka K, Saito I, Furukawa S, et al (2018) Yellow tongue coating is associated with diabetes mellitus among japanese non-smoking men and women: the Toon Health Study. J Epidemiol. 28:287-291
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13. Wang DH, Yang Y, Lawrence, Brenna TJ (2017) Branched chain fatty acids content of natto. FASEB J. 31
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