Anaerobe 27 (2014) 64e70
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pH and bacterial proﬁle of dental plaque in children and adults of a low caries population Elisabeth Raner a, Lina Lindqvist a, Soﬁa Johansson a, Haidar Hassan a, Anette Carlén a, Narong Suksu-art b, Gunnar Dahlén a, * a
Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Box 450, 40530 Göteborg, Sweden Princess Mother Medical Voluntary Foundation, Bangkok, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2013 Received in revised form 8 January 2014 Accepted 21 March 2014 Available online 28 March 2014
Objective: This study compares pH and microbiological proﬁle of dental plaque in children and adults of a low caries population. Material and methods: Thirty-nine children, 12e14 years of age and 45 adults between 20 and 39 years of age in 5 Karen villages of the Tak province, Northern Thailand were examined for plaque, calculus, caries (DMFT) and pH measurements in resting plaque and after a sucrose rinse. Information on dietary and oral hygiene habits was obtained through interviews using a ﬁxed questionnaire. Microbiological proﬁle of plaque samples was analyzed with DNAeDNA checkerboard technique. Results: Mean DMFT was 0.77 1.56 and 87% of the adults and 67% of the children were caries free (p < 0.05). The mean resting pH was for both age groups in the range of 7.0e7.1 and signiﬁcantly higher than a Swedish caries free reference group. Karen adult men had signiﬁcantly lower pH minimum than females and children (p < 0.05). Supragingival plaque samples showed high levels of low acidogenic and anaerobic species, which dominated over strong acid producers such as streptococci. Conclusion: The study indicates that the Karen children and adults has a plaque physiology and microbiology predominating by low acidogenic anaerobes, which in addition to the low sucrose intake explains the low caries prevalence in this population. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Dental plaque Supragingival microbiota Caries pH DNAeDNA hybridization Acidogenicity
1. Introduction Caries is a worldwide dental disease, which has been more prevalent and severe in industrial than in developing countries . The caries prevalence among children has declined in the industrial countries and mainly due to increased ﬂuoride prophylaxis. The caries condition of children in the developing countries is, however, more uncertain. The major etiological factors for caries are considered to be high consumption of reﬁned sugars (sucrose) and presence of an acidogenic ﬂora in the dental plaque . Earlier, the speciﬁc plaque hypothesis constituted the basis for the caries etiology, and mutans streptococci were considered the arch-villain for caries initiation and progression [3,4]. The close relation between mutans streptococci and caries was explained by sucrose fermentation by which the bacteria produce lactic acid and insoluble
* Corresponding author. Tel.: þ46 31 7863262; fax: þ46 31 825733. E-mail address: [email protected]
(G. Dahlén). http://dx.doi.org/10.1016/j.anaerobe.2014.03.010 1075-9964/Ó 2014 Elsevier Ltd. All rights reserved.
polyglucans . Later studies have suggested the caries development to be much more complex  and the focus on mutans streptococci in caries development as well as in caries risk assessment has been reevaluated . In view of the ecological plaque hypothesis, the total acidity in plaque is essential and many bacterial species in the dental plaque are capable of producing acids from various sugars . An increased plaque acidity will favor genuine acid tolerant bacteria e.g. lactobacilli. Others such as streptococci may adapt to a lower pH, while those that are acid sensitive will be diminished . Populations such as the Karen Hill tribes living in remote villages of Northern Thailand seem to have low caries prevalence [9,10]. This was primarily explained by little access to reﬁned sugars in the diet. On the other hand, these populations have other risk factors such as poor oral hygiene and low ﬂuoride exposure. We know little about the cariogenic ﬂora in these populations, and studies performed so far have focused only on Streptococcus mutans [11,12], while little is known about the frequency and amounts of other bacterial species in the dental plaque in this population.
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These remote populations might have other caries preventing habits such as betel chewing and also saliva and plaque factors that are different from western countries, e.g. pH, buffer capacity, oral clearance, saliva secretion rate etc. The overall aim of a project in the Karen population in Northern Thailand was to describe the caries conditions and caries risk factors among children compared to adults. The focus for the present study was to determine caries risk factors in the two age groups by mapping parts of the oral microbiota and measure the dental plaque pH before and after a sugar rinse. The hypothesis was that the Karen Hill tribe children would be more susceptible to caries than the adults due to the inﬂuence of a suspected higher intake of sucrose in the diet. It was further presumed that there would be less acid-generating bacterial species in caries free compared to caries active subjects. 2. Materials and methods 2.1. Study design The present study had a cross sectional, descriptive design. It was performed in 5 different remote villages of the Karen Hill tribe in the Tak province of Northern Thailand in cooperation with the Princess Mother Medical Voluntary Foundation (PMMVF) and their mobile dental clinics. The study was organized and ethically approved by the PMMVF. All subjects participated voluntarily and were informed about the study and agreed to participate. All subjects were afterwards offered dental treatment according to their needs. The caries prevalence was recorded as DMFT. The dietary and oral hygiene habits were described through a questionnaire and through measurements of the amount of plaque and calculus. The plaque microbiota was analyzed with DNA checkerboard technique. The ability of the plaque bacteria to produce acid after a 1% sucrose rinse was estimated during 30 min. The sucrose rinse was compared with a reference group consisting of 36 (21 females and 15 males) caries free Swedish voluntary dental students (22e 28 yrs). 2.2. Subjects The subjects were recruited among children (12e14 yrs) and adults (30e50 yrs). The study proceeded during a total period of 5 days during which 98 subjects were screened by the staff of the mobile dental team. They were selected as randomly and with an equal gender distribution, as possible. Each day the ﬁrst 15e20 subjects in the 5 villages meeting our criteria in age and gender were selected to participate in the study. Ten of these were not fully examined due to lack of cooperation and were excluded from the study. Another 4 were excluded due to misunderstandings regarding age. In total, 39 children (22 girls, 17 boys) and 45 adults (19 women, 26 men) were included. 2.3. Questionnaire Before the oral examination, the subjects were interviewed concerning their dietary and oral hygiene habits. Two assistants from the PMMVF, who translated the questions from questionnaire in English into the local spoken language, conducted interviews. Betel chewing and tobacco smoking were especially recorded. 2.4. Clinical examinations The clinical examinations were performed by three examiners in the open air and in daylight using instruments such as mouth
mirror, probe, diode lamp, and pH strips. The three examiners (ER, LL, SJ) were calibrated for the clinical examinations and pH measurements. During the examinations a fourth dentist was consulted in case of any uncertainty regarding caries registration or other measurements. 2.5. Plaque and calculus registration Plaque and calculus were recorded and indexed in six dental regions as described below; right and left premolar/molar region and anterior regions of the maxilla and mandible (17e14, 13e23, 24e27, 47e44, 43e33, 34e37). The amount of plaque and calculus was graded separately on a scale of 0e2. When denoted abundant the index was given 2, 1 denoted sparse amount of plaque/calculus and 0 denoted zero or no visible plaque/calculus. Clearly visible continuous bands of plaque/calculus on several teeth in an area were deﬁned as abundant. A thin band of plaque/calculus on one tooth in an area alternatively non-continuous plaque/calculus on one or several teeth was deﬁned as a sparse amount. Plaque was always recorded on sites in presence of calculus. 2.6. Caries registration Dental caries was recorded clinically in all teeth except for the third molar and was expressed as DMFT (Decayed, Missing, and Filled Teeth index) and DT (Decayed Teeth index). 2.7. Recording of pH The subjects were asked to rinse with 10 ml of a 1% sucrose solution for 1 min. Plaque pH was measured with a pH strip according to Carlén et al. . Brieﬂy, the test is based on plastic pHstrips (Spezialindikator, Merck, Darmstadt, Germany) cut into thinner strips that easily can be applied approximally between the selected teeth for 10 s. The color change is then compared to a reference according to the manufacturer. Primarily we used the pH strip with the range 4.0e7.0 or alternatively 6.5e10.0 if necessary. Determination of pH was performed on the distal surface of 15. In case of lacking teeth or heavy bleeding, pH was measured on the distal surface of 25. The pH was measured prior to the rinse (baseline or resting pH) and at 1, 2, 3, 5, 10, 20 and 30 min after the rinse. The examiners were trained in pH measurements prior to the commencement of the study using pH strips and the examiners were then calibrated through cross-examining of 7 subjects. There was an inter-examiner agreement in 80e90% of the cases. For comparison, pH was similarly measured in a reference group of Swedish students after 1 min rinsing with 1% sucrose. They were told to abstain from oral hygiene procedures during 24 h prior to the test. 2.8. Checkerboard DNAeDNA hybridization Supragingival plaque samples were collected from 4 approximal sites, 31/41, 13/14, 16/17 and 46/47, using a curette. The samples were transferred to separate Eppendorf tubes and immediately mixed with 100 ml 0.1 M NaOH. The samples were kept cold on ice or refrigerator until analysis. The analysis of the samples was made in the department of Oral Microbiology at the Institute of Odontology of Gothenburg University, 6 months after sampling. The presence and level of 19 species associated to the caries process (Figs. 4 and 5) were determined with the aid of the checkerboard DNAeDNA hybridization methodology according to WalleManning et al. . In brief, whole genomic DNA probes were prepared from the panel of bacterial type strains. DNA was extracted with
E. Raner et al. / Anaerobe 27 (2014) 64e70
Fig. 1. Plaque index of dental sites in girls/boys and women/men of a Karen hill tribe population.
mutanolysin and lysozyme as previously described  and the DNA quality was evaluated from the UV spectrum between 200 and 300 nm using a Gene Quant spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). Probe DNA (1 mg) was labeled with deoxygenin using the DIG High Prime kit according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). A volume of 100 ml of each plaque sample was boiled for 5 min, neutralized with 800 ml 5 M ammonium acetate, transferred onto nylon membranes (Minislot device, Immunetics, Cambridge, MA) and ﬁxed by ultraviolet light (UV Stratalinker 1800, Stratagene, La Jolla, CA). After 2 h of pre-hybridization, the DNA probes (1e10 ng) were allowed to hybridize over night in lanes vertically to the plaque samples using a Miniblotter device (Immunetics) at 42 C. Buffer-set 2  was used as buffers for pre-hybridization and hybridization. After a series of stringency washes at 70 C, hybrids were detected using phosphatase-conjugated anti-digoxygenin
Fig. 2. Calculus index of dental sites in girls/boys and women/men of a Karen Hill tribe population.
Fig. 3. Dental plaque pH of Karen adults and children compared with Swedish dental students (control) after 1 min rinsing with 1% sucrose.
antibodies and the signals were visualized with a chemiluminescent substrate (CDP Star, Roche Diagnostics). Evaluation of the number of bacteria in the samples was performed by comparing visually the obtained signals with the ones generated by two standard samples containing respectively 106 and 105 cells of each of the 19 type strains. The signals were categorized and coded on a scale 0e5 where 0 indicated no signal, 1 indicated a low intensity corresponding to 105 but < 106 cells, score 4 indicates 106 cells and score indicates 5 > 106 cells.
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(DMFT ¼ 0), a statistically signiﬁcant difference (p < 0.05). Fiftyeight (including some children) of the 84 subjects chewed betel and in this group 79% were caries free, while 65% among the nonbetel chewers were caries free. DMFT was 0.7 1.6 for betel chewers and 0.9 1.5 for non-betel chewers (Data not shown in table). It should be noticed that existing cavitated lesions appeared as arrested with a leathered texture and dark brown staining indicating a low caries activity even in those individuals with open carious lesions. 3.4. pH registration
Fig. 5. Frequency distribution (score 3e5) of 19 microbial species determined with the DNAeDNA hybridization techniques in men and women of the Karen Hill population. Score 3 indicates >105 but < 106 cells, score 4 indicates 106 cells and score indicates 5 > 106 cells.
2.9. Statistical analysis One sample conﬁdence interval was used and calculated to be DMFT 0.9 0.4 for children and 0.7 0.5 for adults. Two samples Ttest with unequal variances were used for calculating statistical signiﬁcance. Level of statistical signiﬁcance was set to p < 0.05. 3. Results 3.1. Questionnaire All 84 subjects included in the study answered the questionnaire. 90% of the subjects in both age groups had an intake of sugar 0) (data not shown in table). 3.5. Microbiological analysis DNAeDNA checkerboard analysis showed a predominance (prevalence of the bacteria at score 3e5) of the anaerobic species Prevotella intermedia and Fusobacterium nucleatum, together with Actinomyces species e.g. A. oris and A. odontolyticus detected in >105 cells per sample (Fig 4) in both children and adults. The microbial composition of the dental plaque did not differ signiﬁcantly between men and women (Fig 5), although men showed generally higher frequencies of most bacterial species (including streptococci) analyzed. Streptococci were even lower than some Lactobacillus species (e.g. L. salivarius and L. casei). The mutans streptococci (S. mutans and Streptococcus sobrinus) showed a low prevalence but higher in adults compared to children (not statistically different). The bacteriological data did not differ signiﬁcantly between betel chewers and non-betel chewers or between those with caries (DT > 0) and those with no caries (DT ¼ 0) individuals (data not shown in ﬁgure). 4. Discussion
Plaque and calculus indexes are illustrated as mean values for different age and gender in Figs. 1 and 2. Less plaque was generally registered in the upper anterior region and most calculus in the lower anterior region. The children had a lower amount of plaque (not statistically signiﬁcant) and a statistically signiﬁcant (p < 0.05) lower amount of calculus than the adults. The females and girls had less plaque (not statistical signiﬁcant) and calculus (statistically signiﬁcant children vs adults) than the males and boys (Table 1). 3.3. Caries registration The caries prevalence (DMFT) among all the 84 subjects was 0.8 1.6. It was 0.7 1.7 in the adult group and 0.9 1.8 among the children (Table 1). DT was 0.5 1.6 in the adult group and 0.7 1.3 for the children. Men had signiﬁcantly (p < 0.05) less caries than women. 87% of the adults and 67% of the children were caries free
This study conﬁrmed the low caries experience in the Karen Hill tribe population and found proportionally and signiﬁcantly more caries free adults than children. While the oral hygiene levels was poor and the amount of calculus high, the sugar intake was low in both groups. All Karen groups showed a higher baseline pH (resting plaque pH) than Swedish dental students. Female adults and the children showed a similar pH response on a sucrose rinse as seen for the Swedish control subjects, while the male adults had a greater pH drop. Prevotella intermedia, F. nucleatum and Actinomyces species predominated over streptococci in the supragingival plaque of both children and adults. The low caries prevalence among Karen Hill tribe children and adults conﬁrmed earlier studies [10e12]. The ﬂuoride content in the drinking water of the study area was reported to be 0.02e
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Table 1 Clinical recordings of children and adults of a Karen Hill tribe population. Children
Age (Mean SD) PlI (Mean SD) Calculus (Mean SD) Number of Betel chewers (%) DMFT (Mean SD) DT (Mean SD) Number of Caries free (%)
Girls (n ¼ 22)
Boys (n ¼ 17)
All (n ¼ 39)
Women (n ¼ 19)
Men (n ¼ 26)
All (n ¼ 45)
13.3 1.1 0.9 0.7 0.4 0.6 11 (50) 0.9 1.4 0.7 1.6 15 (68)
12.9 0.9 1.4 0.6 0.6 0.7 8 (47) 0.9 1.3 0.8 1.3 11 (65)
13.1 1.0 1.2 0.7 0.5 0.7* 19 (49) 0.9 1.8 0.7 1.3 26 (67)
35.6 4.9 1.2 0.7 1.0 0.7 16 (84) 1.7 2.5 1.1 2.4 13 (68)
37.0 5.0 1.6 0.6 1.3 0.7 23 (88) 0.2 0.4 0.1 0.3** 24 (92)
37.71 5.3 1.4 0.7 1.2 0.7* 39 (87) 0.7 1.7 0.5 1.6 39 (87)***
*Adults had signiﬁcantly (p < 0.05) more calculus than children. **Men had signiﬁcantly (p < 0.05) less caries than women. ***Adults were signiﬁcantly (p < 0.05) often caries free than children.
0.29 ppm  and 0.01e0.2 ppm (Narong Suksuart, unpublished data). The low prevalence of open caries lesions (DT of children 0.9 and adults 0.5) and the high frequency of caries free (e.g. DMFT ¼ 0) individuals might be surprising in view of the poor dental hygiene among the participants as indicated by high plaque scores (PlI > 1, which means visible plaque in all dental regions). Studies of a rural population with poor dental hygiene in the Suratthani and Songkla Provinces of Southern Thailand, the DMFT were 2.4 for 12-year olds and 6.0 for 30e39 year old adults [16,17] and only 30% of the children and 9% of the adults were caries free. The main explanation for the low caries experience in the Karen population is suggested to be the low intake of sucrose. This was conﬁrmed by the interview and also by the fact that it was practically no access to sucrose containing food (including snacks, candies and soft drinks) in the village “stores” available. The villages were all situated in very remote areas and were reached only after 4e5 h drive with 4 wheel-drive vehicles. Earlier studies on the Karen Hill tribes have shown a tendency for the caries disease to be more spread amongst children than the adults, which indicates that the caries prevalence might be changing [9,10]. This is in concordance with the present study where the percentage of caries free subjects was signiﬁcantly higher among the adults, (87%) than among the children (67%). These villages are developing increasing contacts with the outside world due to better infrastructure by which an increased exposure to sugar containing products follows. It can be speculated that the children and adolescents nowadays are more exposed to these products than the adults were during their youth despite the remote locations of the villages and with few sugar containing products. If this is true a higher caries rate would be expected in the future unless other caries preventive measures such as ﬂuoride containing tooth-paste is introduced in these villages. This will be an important future challenge for the mobile dental team and the PMMVF organization. Betel chewers had a slightly lower DMFT although not statistically signiﬁcant compared to non-betel chewers. This may, at least to some extent, explain the difference between children and adults. Even if half of the children already have been introduced to this habit, the adults have performed betel chewing more Table 2 Comparison between Karen children, adults and Swedish students in regard to dental plaque Baseline pH, Max pH drop and lowest pH (Min pH) (SD) after 1 min of sucrose rinse. Subject group Karen Karen Karen Karen
girls (n ¼ 22) boys (n ¼ 17) women (n ¼ 19) men (n ¼ 26)
Baseline pH 7.1 7.1 7.0 7.1
0.4 0.3 0.3 0.4
Max pH drop 0.9 1.0 0.9 1.4
0.3 0.7 0.4 0.5*
Min pH 6.3 6.1 6.1 5.7
* pH drop and min pH statistically signiﬁcant (p < 0.05) for men vs women.
0.3 0.7 0.4 0.6*
intensely and for a much longer time. Several studies have claimed that betel chewing might be cariostatic [18,19]. In a previous study  of the Karen Hill tribe population from the same region as in the present study and with the speciﬁc aim to study betel chewers versus non-chewers, both groups had similar low caries experience. Thus, the study could not demonstrate any statistical or clinical differences between chewers and non-chewers however it is still possible that betel chewing may contribute to the low caries activity and the arrested character the caries lesions had especially in adults. The pH was measured with the pH strip method  shown to be comparable with the micro-touch method. Most likely both methods measure pH on the plaque surface and in the interproximal dental space rather than in the depth of the dental plaque. The reliability of the strip method at repeated measurements of the same interproximal site is shown by the fact that the pH returns to baseline at prolonged measurements in both the study groups and in the reference group. The pH curve analysis after a rinse with 1% sucrose indicated a low acid producing ability of the dental plaque and the pH levels were similar to those obtained from a group of caries free Swedish students. The baseline pH level in both adults and children was even higher compared to the student group. In concordance with high pH levels, calculus was abundant in the adults. Also children more frequently had calculus compared to what is seen in western populations. It has been speculated if the South East Asian population is prone to form calculus in a higher degree than Caucasians [19-21]. In a previous study, 90% of the 13 year-old children in a school in Southern Thailand had calculus but only 36% were caries free . It was concluded in that study that no inverse correlation between the presence of caries and calculus occurred on a population level and that a high calculus score only marginally reduced the individual likelihood of having caries. Furthermore, no signiﬁcant difference was obtained for resting plaque pH in that study between those having the highest DMFT score in comparison with those having the highest calculus score. On the other hand, caries and calculus are usually differently distributed within the dentition. Calculus is most prevalent in the lower anterior region and in these populations (Thai as well as Karen) caries occurs most commonly in the lower ﬁrst molars. In the present study we speciﬁcally measured the pH mesially of the ﬁrst molars and the resting plaque pH levels were similar to or higher for both children and adults than the level seen for the Caucasian comparative group. Men had a lower caries experience than women as also seen among other population groups in rural areas of Thailand . This is plausibly due to frequent food intake by women during food cooking. The higher acid production from sucrose in the approximal plaque of men compared to women is explained by a poorer oral hygiene and thus more plaque and bacteria on the teeth.
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A pH drop below 5.0e5.5, which is the critical pH for enamel net loss demineralization, generally occurs at sucrose rinse. The dental plaque in both Karen children and adults responded to a sucrose rinse but to a much lower extent than seen in Caucasians [13,22,23]. There are several possible explanations for this. First, the higher resting pH at baseline points on the possibility that alkali (e.g. ammonia from ureolysis) is formed in a higher extent in the Karen population than in Caucasians. In a study of the Karen population in the same area showed a strong ureolytic capacity of the dental plaque . A second explanation could be that the plaque of the Karen individuals has a comparatively low level of strong sucrose fermenting bacteria outnumbered by less sugar dependent microorganisms. Both this study and the study by Appelgren et al.  indicate that the Karen population has a higher resting plaque pH and supragingival plaque microbiota that are distinctly different from those of caucasians. The speciﬁc plaque hypothesis has constituted for decades the basis to explain the caries development and progression e.g. by mutans streptococci and lactobacilli [3,4]. The importance of speciﬁc microorganisms has been reevaluated and the role of microorganisms in the dental plaque in the caries process is complex  and an alternative hypothesis has been proposed [7,24,25]. According to the ecological plaque hypothesis the caries process is due to an ecological imbalance in the dental plaque as a result of frequent sugar intake, frequent pH drops and an increasing acidurity of the plaque. Most streptococci and lactobacilli are strong acid producers and both genera show a high variation between species and strains [26,27]. The production is strong not only at pH 7 but also at lower pH (acidurity) due to inherent acid tolerance and to adaptation [5,8,28]. The streptococci is therefore claimed to be “products of their environment”  and explains why caries associated plaque is predominated by streptococci including the mutans streptococci. In the dental plaque of caries free or low-caries population as the one investigated in this study, a predominance of streptococci would therefore not be expected. In this population both children and adults seemed to have a predominance of anaerobic species such as P. intermedia and F nucleatum as well as Actinomyces species e.g. A. oris and A. odontolyticus. Due to the poor oral hygiene the plaque matures over a long time under anaerobic conditions and low fermentation of non-sucrose carbohydrates prevailing. Streptococci are not favored in such an environment. This explanation to a signiﬁcantly different plaque ﬂora, low in streptococci and comparably rich in anaerobic species, which was obtained from the Karen population needs to be conﬁrmed in other villages, with other sampling approaches and other analytical methods before more conclusive statements can be made. However, our results show that studies of the dental plaque ﬂora should not be restricted to evaluating the microbial composition but should also include measurements of physiological characteristics such as pH, to better evaluate the nature and ecology of the dental plaque. 5. Conclusions The study population of 13e15 years old children and young adults of the Karen Hill tribes living in remote villages of Northern Thailand had a very low caries experience despite poor oral hygiene and no ﬂuoride exposure. However, they also had a very low intake of reﬁned sugars, and moderately acidogenic and anaerobic bacteria predominated over the streptococci in their supragingival plaque. The pH levels at baseline were slightly higher while the pH drop after a rinse with sucrose corresponded to the level seen for Swedish caries free students. Little difference in plaque pH was found between those with caries lesions compared to those with no
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