Anaerobe xxx (2014) 1e7

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Clinical microbiology

Hydrogen sulfide production from subgingival plaque samples n A. Basic*, G. Dahle Oral Microbiology and Immunology, Sahlgrenska Academy, University of Gothenburg, Sweden

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

Periodontitis is a polymicrobial anaerobe infection. Little is known about the dysbiotic microbiota and the role of bacterial metabolites in the disease process. It is suggested that the production of certain waste products in the proteolytic metabolism may work as markers for disease severity. Hydrogen sulfide (H2S) is a gas produced by degradation of proteins in the subgingival pocket. It is highly toxic and believed to have pro-inflammatory properties. We aimed to study H2S production from subgingival plaque samples in relation to disease severity in subjects with natural development of the disease, using a colorimetric method based on bismuth precipitation. In remote areas of northern Thailand, adults with poor oral hygiene habits and a natural development of periodontal disease were examined for their oral health status. H2S production was measured with the bismuth method and subgingival plaque samples were analyzed for the presence of 20 bacterial species with the checkerboard DNAeDNA hybridization technique. In total, 43 subjects were examined (age 40e60 years, mean PI 95 ± 6.6%). Fifty-six percent had moderate periodontal breakdown (CAL > 3 < 7 mm) and 35% had severe periodontal breakdown (CAL > 7 mm) on at least one site. Parvimonas micra, Filifactor alocis, Porphyromonas endodontalis and Fusobacterium nucleatum were frequently detected. H2S production could not be correlated to periodontal disease severity (PPD or CAL at sampled sites) or to a specific bacterial composition. Site 21 had statistically lower production of H2S (p ¼ 0.02) compared to 16 and 46. Betel nut chewers had statistically significant lower H2S production (p ¼ 0.01) than non-chewers. Rapid detection and estimation of subgingival H2S production capacity was easily and reliably tested by the colorimetric bismuth sulfide precipitation method. H2S may be a valuable clinical marker for degradation of proteins in the subgingival pocket. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Karen Hill tribe Dental plaque Hydrogen sulfide Checkerboard DNAeDNA hybridization technique Betel chewing

1. Introduction Periodontitis is, along with caries, the most common oral disease worldwide. In severe cases it may lead to tooth loss and edentulousness. Under healthy conditions, the microorganisms in the richly colonized oral cavity live in balance (homeostasis) with the host, forming biofilms on mucosal surfaces and teeth [1]. When the bacterial load, by poor oral hygiene, is increased the balance can be disturbed and dysbiosis is formed [2]. The plaque (biofilm) on teeth surfaces accumulates and changes in composition over time, consequently leading to a soft tissue inflammation in the gingiva e gingivitis, diagnosed based on characteristic signs such as swelling

* Corresponding author. Department of Oral Microbiology and Immunology , University of Gothenburg, Box 450, 405 30 Gothenburg, Sweden. Tel.: þ46 31 7863266, þ46 73 5075959. E-mail addresses: [email protected] (A. Basic), [email protected]. n). se (G. Dahle

and redness. Gingivitis may over time, further result in periodontitis where the host response degrades the periodontal ligament and supporting alveolar bone, clinically registered as loss of clinical attachment (CAL) and increasing probing pocket depth (PPD). The disease progression and severity is highly subject dependent and leads to tooth loss in some individuals while others do not experience periodontal destruction despite persistent gingival inflammation for many years (long-standing gingivitis). Gingivitis is a reversible process where the inflammatory host response can be eliminated if the bacterial overload is reduced [3,4]. The lost supporting alveolar bone in periodontitis is, however, irreversible. Periodontal disease is induced by dysbiosis in the plaque but the exact mechanisms and role of the microorganism are still unclear, however, it is likely that bacterial activity, growth and production of toxic metabolites trigger the inflammatory response. According to the ecological plaque hypothesis [5], there is a change in the environment of the subgingival pocket, implying increased flow of gingival crevicular fluid, providing serum constitutes such as proteins, hormones and vitamins, favoring

http://dx.doi.org/10.1016/j.anaerobe.2014.09.017 1075-9964/© 2014 Elsevier Ltd. All rights reserved.

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fastidious proteolytic bacterial species. This results in a biofilm of a high diversity of mainly Gram negative and anaerobe bacteria [6,7]. The diseased subgingival pocket is slightly alkaline with a negative redox potential, as compared to positive mV recordings at healthy sites [8]. The dysbiotic bacterial activity results in an increased production of enzymes and a cascade of metabolic end products such as nitrogen compounds (e.g., ammonia), volatile sulfur compounds (e.g., hydrogen sulfide, methyl mercaptan, dimethyl disulfide) and short carboxylic acids (butyric acid, valeric acid, propionic acid, caproic acid, phenyl acetic acid etc.) in addition to other bacterial products such as endotoxins and phenyl compounds (e.g., indole) [9]. The presence and activity of microorganisms in the subgingival pocket can be studied at different levels depending on the question addressed. New techniques are constantly developing in the search to “map” the entire plaque and identify the genome of all the species present. Another approach of interest is the expression of proteins, proteomics, associated to mixed bacterial communities rather than single species alone. The enzymatic activity concerning the hydrolysis of benzoyl-DL-arginine-naphthylamide is an example of a protein studied and a test developed, the so-called BANA, identifying a specific activity only found for a small number of bacterial species [10,11]. Yet another level in studying the plaque is the net effect of the entire biofilm, the result of the proteins e the metabolites, metabolomics. Hydrogen sulfide (H2S) is an end product of the proteolytic activity, proposed to be of interest in gingivitis and periodontitis [12] but the impact in disease development is largely unknown. It is a volatile, foul smelling gas produced by degradation of cysteine in the subgingival pocket [13e15]. It is a gas known for its toxic properties [16] with pro-inflammatory elements [17], recently proposed as a gasotransmitter along with nitric oxide (NO) and carbon monoxide (CO) [18]. The bacterial production of H2S in vitro has previously been reported for many species frequently encountered in subjects with periodontitis e.g., Fusobacterium spp., Parvimonas micra, and Treponema denticola by degradation of cysteine or glutathione [19e22]. The gas is of major interest in the research of halitosis (bad breath) and is believed to be one of the key factors for oral malodor [23,24]. Measurement of H2S in gingival crevices has previously been conducted in several studies where the production has been measured with lead acetate impregnated paper strips [13,14], trapping device and gas chromatographic analyses [12] and a technologically advanced probe for sulfide detection [25]. All these studies have tried to elucidate the presence and role of H2S in periodontal disease. Nevertheless, the methods used have been either time consuming and insensitive, such as the lead acetate method, or too advanced and expensive for use in the clinics or field studies, as the latter two. Therefore, the existing methods have not been used and tested in a greater extent at clinical settings. The fact that the gas is volatile and easily converts to polysulfides has further hampered this task. Thus, comprehensive knowledge of the role of H2S in bacterial metabolism, the possible effect on local environment and the host, and its relation to clinical parameters in health and disease is still lacking. H2S may serve as a marker molecule for the increased proteolytic activity by the bacteria in the subgingival pocket in gingivitis and periodontitis. Thus, it is likely that deep pockets and severe periodontitis cases have higher H2S production than shallow pockets and cases with no or little periodontal breakdown. Recently, our group modified a colorimetric method for H2S estimation in vitro based on bismuth sulfide precipitation (unpublished data). The method was applied for subgingival ex vivo measurements and tested under field conditions among adults of the Karen Hill tribe of northern Thailand, a population with a natural

progression of periodontal disease without disturbing intervention on the subgingival microbiota by regular dental treatment. The aim of this study was to investigate the H2S producing ability of subgingival samples in relation to clinical and microbiological parameters on site and individual level. 2. Materials and methods 2.1. Subjects The subjects participating in the study were members of the Karen Hill tribe selected from five villages in Omgoi district, Chiang Mai province in northern Thailand. During a week our group accompanied a mobile dental team organized by The Princess Mother Medical Voluntary Foundation, Bangkok, Thailand, that also ethically approved to the study. Inclusion criteria were subjects between 40 and 60 years of age. The participants were selected randomly, informed of the study and voluntarily consented to participate. The subjects were interviewed for their age, smoking habits, betel chewing habits, oral hygiene habits and sugar consumption. This population is living in a remote area with underdeveloped infrastructure, and has no access to dental care and therefore represent an inviolate population for a cross-sectional study on H2S production in subgingival plaque in relation to clinical parameters and the composition of the microorganisms inhabiting the biofilm. 2.2. Clinical examination The clinical examination was performed in day light with the help of a mouth mirror and a dental and periodontal probe prior to the examination and potential treatment performed by the mobile dental team. Following parameters were registered: Decayed Missed Filled Teeth (DMFT), Probing Pocket Depth (PPD)  4 mm at four sites per tooth, Bleeding on Probing (BoP), Clinical Attachment Level (CAL)  3 mm and Plaque Index (PI). The periodontal status of the participants was divided into three groups; gingivitis (BoP but 3 mm CAL), mild periodontitis (CAL > 3 mm but 0.05), while betel chewing was more common among women (p ¼ 0.002, phi ¼ 0.53). More than 40% of the subjects never brushed their teeth and a

Fig. 1. Bismuth test scores (bars) in relation to plaque sampling sites. The hydrogen sulfide (H2S) producing capacity from dental plaque was measured ex vivo at four mesial sites (16, 21, 31, 46) in 43 subjects with the bismuth test. The test ranged from no H2S production, registered visually by no color production (score 0), to maximum H2S production showing black color production (score 3).

Fig. 2. Frequency (percent) of sites with bacterial species in one of the sampling sites (46) in 43 subjects, detected with the checkerboard DNAeDNA hybridization technique.

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Table 1 Characteristics of the 43 examined adults of the Karen population. Variable

Women (%) Betel chewing (%) Tobacco smoking (%) Both betel and smoking (%) Oral hygiene habits (%)b Never Sometimes Every day Remaining teeth (mean ± SDc) d DMFT (mean ± SD) PIe (mean ± SD) BoPf (mean ± SD) >4 < 7 mm Subjects with PPD (%)g 7 mm Subjects with CAL (%)h >3 < 7 mm 7 mm Sampled sites with PPD (%) >4 < 7 mm 7 mm Sampled sites with CAL (%) >3 < 7 mm 7 mm Localized chronic periodontitis (%)i Generalized chronic periodontitis (%)j Bismuth test score median (range)k

Total (na ¼ 43)

Age group 40e49 (n ¼ 20)

24 (56) 28 (65) 27 (63) 18 (42) 18 (44) 13 (32) 10 (24) 27 ± 1.8 1.1 ± 1.9 95 ± 6.6 92 ± 16 17 (40) 12 (28) 24 (56) 15 (35) 12 (7.0) 2 (1.2) 15 (8.7) 2 (1.2) 25 (58) 14 (33) 1.5 (0e3)

13 (65) 13 (65) 11 (55) 8 (40) 6 (30) 9 (45) 5 (25) 28 ± 0.81 1.0 ± 1.4 95 ± 8.1 91 ± 17 10 (50) 4 (20) 12 (60) 6 (30) 5 (6.3) 1 (1.3) 6 (7.5) 1 (1.3) 11 (55) 7 (35) 1.75 (0e3)

Table 2 The pattern/structure matrix from factor analyses.a Speciesb

Pattern/structure coefficients

50e60 (n ¼ 23)

Factor analyses (variance)

Factor 1 (28.5%)

Factor 2 (10.9%)

Factor 3 (7.7%)

Factor 4 (7.0%)

Factor 5 (5.8%)

Factor 6 (5.4%)

11 (48) 15 (65) 16 (70) 10 (43) 12 (52) 4 (17) 5 (22) 27 ± 2.3 1.3 ± 2.3 96 ± 5.2 92 ± 16 7 (30) 8 (35) 12 (52) 9 (39) 7 (7.6) 1 (1.1) 9 (9.8) 1 (1.1) 14 (61) 7 (30) 1.5 (0.5e3)

A. actinomycetemcomitans A. odontolyticus A. oris C. gracilis C. rectus C. ureolyticus E. corrodens F. alocis F. nucleatum L. fermentum P. endodontalis P. gingivalis P. intermedia P. micra P. tannerae S. anginosus S. mutans S. salivarius T. denticola T. forsythia

0.060

0.005

0.120

0.028

0.357

0.651

0.715 0.619 0.363 0.172 0.010 0.750 0.200 0.107 0.739 0.137 0.009 0.010 0.230 0.140 0.739 0.019 0.261 0.023 0.001

0.081 0.294 0.326 0.254 0.024 0.021 0.366 0.191 0.014 0.831 0.127 0.481 0.813 0.645 0.221 0.035 0.132 0.240 0.008

0.100 0.078 0.102 0.679 0.185 0.076 0.094 0.071 0.112 0.187 0.810 0.268 0.111 0.170 0.057 0.045 0.404 0.548 0.714

0.492 0.131 0.007 0.182 0.150 0.401 0.762 0.819 0.122 0.152 0.114 0.310 0.138 0.421 0.303 0.089 0.040 0.065 0.242

0.022 0.084 0.599 0.108 0.670 0.016 0.031 0.125 0.150 0.044 0.065 0.281 0.009 0.252 0.040 0.227 0.429 0.467 0.121

0.060 0.076 0.027 0.132 0.007 0.071 0.057 0.063 0.083 0.027 0.097 0.145 0.059 0.125 0.036 0.802 0.415 0.030 0.102

a

Number of subjects examined. N ¼ 41. c Standard deviation. d Decayed missed filled teeth. e Plaque index. f Bleeding on probing. g At least one pocket with PPD (probing pocket depth) >4 < 7 mm or 7 mm. h At least one pocket with CAL (clinical attachment level) >3 < 7 mm or 7 mm. i Localized 30% of sites with CAL. j Localized >30% of sites with CAL. k Median within the age group, calculated from mean of four measuring sites per subject. b

generally high PI and BoP was recorded in both age groups. Severe periodontitis and tooth loss was only sporadically seen and few of the sampled sites had high PPD and/or CAL. Almost all subjects were positive for H2S production and only one individual was negative on all four sites. The sites 16 and 46 had significantly higher bismuth test scores than 21 (p ¼ 0.02) (Fig. 1). The bismuth test in relation to PPD and CAL showed no statistical significance with the Chi-square test for independence for any of the sites (p > 0.05, phi < 0.14). The checkerboard analyses detected Streptococcus anginosus, Actinomyces oris and Eikenella corrodens in all subjects (Table 3). Aggregatibacter actinomycetemcomitans and Campylobacter ureolyticus were detected in 7 subjects (16%). Fig. 2 illustrates the percent sites colonized by different bacterial species at site 46 for the 43 subjects tested. The results from site 16, 21 and 31 showed a similar pattern, and no statistical difference of colonizing species between different sites was found. To reduce the relatively high number of bacterial species, related to each other in the subgingival plaque, a factor analyses was conducted resulting in six factors (KMO ¼ 0.819, Bartlett's test p ¼ 0.000), explaining a total of 65.25% of the variance (Table 2). Four factors explained 54% of the variance. The first factor included Actinomyces spp., E. corrodens, Lactobacillus fermentum and S. anginosus and was negatively correlated to sulfide production, although, not statistically significant. The second factor included Porphyromonas endodontalis, Parvimonas micra and Prevotella tannerae and the third Porphyromonas gingivalis, Tannerella forsythia, Campylobacter rectus and Treponema denticola. The third factor included the bacteria of Socransky's red complex, proposed to be associated with periodontal disease [28]. Fusobacterium

Factor loadings > 0.5 are given in bold. a The results from factor analyses, with Varimax rotation, for log counts of identified bacterial species. The six factors illustrated explain 65.25% of the variance. b Abbreviations: P. gingivalis, Porphyromonas gingivalis; P. intermedia, Prevotella intermedia; T. forsythia, Tannerella forsythia; A. actinomycetemcomitans, Aggregatibacter actinomycetemcomitans; F. nucleatum, Fusobacterium nucleatum; T. denticola, Treponema denticola; P. micra, Parvimonas micra; P. endodontalis, Porphyromonas endodontalis; F. alocis, Filifactor alocis; P. tannerae, Prevotella tannerae; S. anginosus, Streptococcus anginosus; A. oris, Actinomyces oris; A. odontolyticus, Actinomyces odontolyticus; E. corrodens, Eikenella corrodens; C. ureolyticus, Campylobacter ureolyticus; L. fermentum, Lactobacillus fermentum; S. mutans, Streptococcus mutans; C. gracilis, Campylobacter gracilis; C. rectus, Campylobacter rectus; S. salivarius, Streptococcus salivarius.

nucleatum was in the forth factor along with Filifactor alocis. The number of the four sites per subject investigated, colonized by the different factors for each subject is illustrated in Table 3. The investigated sites were frequently colonized by species from factors 1, 2 and 4. No clear pattern was seen for detected bacterial species and median bismuth score. Multilevel regression analyses were performed on clinical parameters (Tables 4 and 5) and on factors from factor analyses (Table 6) in relation to low/high H2S production. None of the sitespecific clinical parameters (BoP, PPD, CAL) were statistically significant with regard to H2S production, nor any of the factors from factor analyses. Only betel chewing showed statistical significance where subjects chewing betel had lower sulfide producing levels (p ¼ 0.10). 4. Discussion This study explored H2S production in relation to periodontal status using a colorimetric method for H2S estimation and checkerboard DNAeDNA hybridization technology for bacterial detection and quantification. H2S production was recorded generally on both site and subject levels with the ex vivo bismuth test. An in vivo methylene blue test was also tested and was generally negative (unpublished observation), probably due to a high detection limit along with the volatile property of the gas and the fast conversion to polysulfides. The production of the gas was found in a higher amount in the molar regions compared to site 21. Variations at different sites have previously been reported for pH [29] and urea and ammonia [30]. Since the upper front region showed less CAL,

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Table 3 Number of colonized sites out of four tested per individual for 20 bacterial species.a

a Plaque samples from four sites (16, 21, 31, 46) per subject, analyzed with checkerboard DNAeDNA hybridization technique. The bacterial species are divided in groups from the factor analyses. The table cell was colored black if all 4 sites were colonized and white if no bacteria were detected at any of the four sites. The bismuth test scores are median values from the four investigated sites.

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6 Table 4 Multilevel regression analyses for clinical parameters. H2S production low/high

Coefficient

p-Value

Table 6 Multilevel regression analyses for factors from factor analyses. 95% Confidence interval Lower

Age Gender Smoking Betel chewing a

0.00 0.21 0.10 0.36

0.744 0.101 0.358 0.010a

0.02 0.47 0.12 0.62

H2S production low/High

0.01 0.04 0.33 0.09

Factor Factor Factor Factor a b c

H2S production may to some extent reflect the periodontal disease severity. There was, however, no association between H2S production and the composition of the bacterial species tested or the disease severity (PPD or CAL). Thus, no statistically significant differences were recorded for H2S production in relation to deep pockets or gingivitis sites. This can partly be explained by the low prevalence of severely diseased sites and partly by the fact that the subjects examined had long-standing gingivitis. Further, H2S may reflect a general proteolytic activity of the subgingival microbiota and gingival inflammation rather than disease severity and periodontal progression. Since the population represents individuals with long-standing gingivitis their plaque may possibly not be in a growing phase with a high metabolic activity. The dysbiotic bacterial community may have found a new homeostasis, in balance with the host, with a low or moderate metabolic activity. Betel chewing was the only parameter statistically associated with lower H2S production and may represent an external factor with significant impact of slowing down the bacterial growth and activity. The checkerboard DNAeDNA hybridization technique detected S. anginosus, A. oris and A. odontolyticus in almost all subjects, in accordance with previous studies reporting these as main plaque constitutes [28,31,32]. Periodontitis associated species were also frequently detected illustrating the presence of periodontal disease in this population, not differentiating between the severity of the disease. This study could not detect any significant correlation between ex vivo H2S levels and any of the investigated bacterial species or the groups from the factor analyses. Since many of the species were found in almost all subjects a discriminant analyses with significant strength could not be performed. Despite this, the factor analyses identified one factor representing low proteolytic but saccharolytic bacterial species such as S. anginosus, L. fermentum and Actinomyces spp. explaining nearly 30% of the variance with a tendency to a negative correlation to H2S production. Interestingly, factor 2, 3 and 4 clustered highly proteolytic bacterial species such as P. endodontalis, P. tannerae, P. micra (factor 2), the red complex bacteria P. gingivalis, T. denticola and T. forsythia (all three included in factor 3), and F. alocis and F. nucleatum (factor 4) that altogether explained > 25% of the variance. F. nucleatum has shown to be a strong H2S producer in vitro, also P. gingivalis, P. endodontalis, T. denticola and P. micra produced H2S

Table 5 Multilevel regression analyses for site-specific parameters. H2S production low/high

Coefficient

p-Value

95% Confidence interval Lower

Upper

BoPa PPDb CALc

0.01 0.00 0.02

0.070 0.966 0.453

0.00 0.10 0.03

0.13 0.09 0.07

a

c

Bleeding on probing. Probing pocket depth. Clinical attachment level.

p-Value

Upper

p < 0.05.

b

Coefficient

d

a

1 2b 3c 4d

Factor Factor Factor Factor

1: 2: 3: 4:

0.04 0.03 0.04 0.04

0.369 0.468 0.299 0.417

95% Confidence interval Lower

Upper

0.11 0.05 0.04 0.13

0.04 0.12 0.12 0.05

E. corrodens, L. fermentum, S. anginosus, A. odontolyticus, A. oris. P. endodontalis, P. micra, P. tannerae. P. gingivalis, T. forsythia, C. rectus, T. denticola. F. nucleatum, F. alocis.

from cysteine but at a slower pace (unpublished observations). The result that none of the factors from the factor analyses was correlated to H2S production can be due to several reasons. Firstly, if the subgingival plaque in vivo, in this population studied, represents a plaque in microbial homeostasis, then specific genes encoding for H2S production may not be expressed in sites with long-standing gingivitis and a low metabolic activity. Secondly, degradation of cysteine and production of H2S is only one metabolic pathway of the proteolytic activity by the subgingival microorganisms e including not only amino acids but also peptides and proteins in the gingival crevicular fluid. Other metabolic pathways produce other metabolites such as ammonia and short carboxylic acids, having potential impact on inflammation and maintaining gingivitis. Thirdly, we tested only 20 bacterial species while the subgingival plaque is known for its high diversity. In accordance with the ecological plaque hypothesis the microorganisms express different phenotypes under different environmental conditions. Therefore, it is expected that species produce variable amounts of the gas depending on the environmental settings. Although, certain species have been shown to be strong H2S producers in vitro, the production measured from the plaque samples is the net effect of the entire plaque sample, not single species production. The population studied, members of the Karen Hill tribe in northern Thailand, had high PI and BoP scores, in accordance with a previous study [33]. Also, as previously reported among this population [33e35], caries was not frequently encountered. Almost all subjects that volunteered to participate had sites with periodontal breakdown, with 56% of the individuals displaying moderate periodontal breakdown sites and 35% at least one site with severe destruction. In this part of the world, chewing of the mild stimulating and addictive betel nut (areca nut) is common and socially accepted, often in combination with tobacco and lime [36]. In accordance with previous studies, betel chewing in our study was more common among women [33,37]. The higher consumption of tobacco smoking among men reported by Baelum et al. [37] was, however, not seen in this study. Notably, this study found an inverse correlation between betel chewing and H2S production, while age, gender and smoking was not statistically significant factors for periodontal breakdown. Earlier studies on betel chewing [38e40] have suggested betel consumption to be associated with periodontitis progression and tooth loss. The effect of betel on oral microorganisms and periodontal disease is unclear but shows that external factors may influence the production of H2S in vivo. Further evaluation on H2S production in relation to periodontal disease should therefore be performed in non-betel chewing individuals. The disease susceptibility of the host has been proven to be of importance for the progression from gingivitis to periodontitis. Internal host factors (susceptibility) have been discussed along with external risk factors, such as smoking and poor oral hygiene.

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The role of the susceptibility of the host versus the microbiological biofilm formation is, however, still unclear. It may be argued that the metabolites of the proteolytic activity are of importance to maintain gingivitis and the host susceptibility of importance for the progression of the inflammatory response leading to the destruction of supporting tissue. Although, all subjects examined in this study had gingivitis and a bacterial profile associated with periodontal disease and H2S production in vitro, they exhibited little destruction of supporting tissue. Therefore, the role of the bacteria may be to induce the inflammation e gingivitis. In subjects with poor oral hygiene habits, such as the studied population, there may be a balance (homeostasis) between the bacterial load and host response resulting in long-standing chronic gingivitis never developing to periodontitis, or slowly developing and leading to low degree of destruction (chronic periodontitis). Here the activity of the biofilm may be somewhat suppressed, compared to new biofilm formation, with lower bacterial activity and thus smaller production of H2S. 5. Conclusion Hydrogen sulfide (H2S) production from subgingival plaque samples was easily detected and estimated with the bismuth sulfide test. No statistically significant correlation was seen, in the population studied, between periodontal disease severity or bacterial composition and H2S production. Subgingival plaque from the upper front teeth showed lower H2S production than plaque from molar sites. Betel chewers had a reduced H2S production. Since, H2S mainly is produced by bacterial degradation of proteins and amino acids in the subgingival pocket it may be used as a marker for protein degradation and further periodontal disease. Acknowledgments Special thanks to Dr Suksu-art, our contact person at The Princess Mother Medical Voluntary Foundation, Bangkok, Thailand and the entire group for their support during fieldwork. Further, we wish to thank Susanne Blomqvist for technical assistance with the checkerboard analyses. Statistical advice by Torgny Alstad, Kjell Pettersson and Vibeke Baelum is gratefully appreciated. The study was supported by TUA-Grant (TUAGBG-67191) and by Gothenburg Dental Society (GTS). References [1] Marsh PD. Dental plaque: biological significance of a biofilm and community life-style. J Clin Periodontol 2005;32:7e15. [2] Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011;55:16e35. [3] Loe H, Theilade E, Jensen SB. Experimental gingivitis in man. J Periodontol 1965;36:177e87. € e H. Experimental gingivitis in man. II. A [4] Theilade E, Wright WH, Jensen SB, Lo longitudinal clinical and bacteriological investigation. J Periodont Res 1966;1: 1e13. [5] Marsh PD. Are dental diseases examples of ecological catastrophes? Microbiology 2003;149:279e94. [6] Moore WE, Moore LV. The bacteria of periodontal diseases. Periodontol 2000 1994;5:66e77. [7] Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001;183:3770e83. [8] Kenney EB, Ash Jr MM. Oxidation reduction potential of developing plaque, periodontal pockets and gingival sulci. J Periodontol 1969;40:630e3. [9] Fine DH, Mandel ID. Indicators of periodontal disease activity: an evaluation. J Clin Periodontol 1986;13:533e46.

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n G, Hydrogen sulfide production from subgingival plaque samples, Anaerobe (2014), http:// Please cite this article in press as: Basic A, Dahle dx.doi.org/10.1016/j.anaerobe.2014.09.017

Hydrogen sulfide production from subgingival plaque samples.

Periodontitis is a polymicrobial anaerobe infection. Little is known about the dysbiotic microbiota and the role of bacterial metabolites in the disea...
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