Caries-active Individuals Research Articles

Genotypic diversity and virulence factors of Streptococcus mutans in caries-free and caries-active individuals

The objective of this study was to analyze the genotypic diversity, frequency of serotypes and the detection of mutacins from Streptococcus mutans isolates in caries-free and caries-active individuals.A total of 260 S. mutans isolated from 28 individuals with and without dental caries were subjected to AP-PCR and PCR screening of glucosyltransferase B, mutacin and serotype genes, which showed the presence of. 70 different genotypes. There was no statistically significant association between the presence of genes for serotypes and mutacins with dental caries. However, there was a statistically significant and a strong association between the higher genotypic diversity in the subjects with caries (r = 0.72, p = 0.001). There was an increase in the number of genotypes with increasing age (p <0.01).

Genetic diversity of ATP synthase cab subunits amplified from Streptococcus mutans clinical isolates from Uyghur children with different caries susceptibility

To investigate the aciduricity and genetic diversity of ATP synthase subunit gene uncEBF derived from Uyghur children Streptococcus mutans (Sm) clinical isolates and the relationship between the genetic diversity of ATP synthase and Sm aciduric ability and caries susceptibility. Forty-one Sm strains derived from 24 caries-active individuals and 17 caries-free individuals, including 16 strains displaying high acid tolerance and 17 strains displaying low acid tolerance. Solutions of all isolated Sm with same density were made and cultured at pH 4.0 to 7.0 brain heart infusion (BHI) liquid. Terminal growth situation was compared. Gene uncEBF of these isolates were amplified with specific primers from Sm genomic DNA, and the polymerase chain reaction (PCR) products were analyzed by PCR-restriction fragment length polymorphism (RFLP) and sequenced. Aciduric ability of Sm isolated from the high caries-susceptible children were higher than that isolated from caries-free group (P = 0.023). Alu I digested fragments of uncEBF displayed two different patterns A and B. The distributions of A and B genotype strains with different acidurance were different (P = 0.039). A genotype included 7 strains displaying high acid tolerance and 2 strains displaying low acid tolerance;B genotype included 9 strains displaying high acid tolerance and 15 strains displaying low acid tolerance. The distributions of A and B genotype strains in different caries-sensitivity groups were different (P = 0.009). A genotype included 7 high caries-susceptible strains and 12 caries-free strains; B genotype included 17 high caries-susceptible strains and 5 caries-free strain. Some of these amplified uncEBF genes from different genotype were sequenced and testified that there existed variation of Alu I recognized sites. The high cariogenecity of Sm strains isolated from caries-active children shows a close relationship with the high aciduric ability of the isolated Sm strains. uncEBF gene of Sm F-ATPase obviously exhibits genetic diversity.

Microbial profiles in saliva from children with and without caries in mixed dentition

The purpose of this study was to determine the bacterial profiles in saliva of the isolated children for studying caries etiology. Samples were collected from isolated children from 6 to 8years old including 20 caries-free (dmfs=0) (healthy) and 30 caries-active individuals (dmfs>8) (patients). 16S rRNA genes were amplified by PCR from bacterial DNA of saliva sample and labeled via incorporation of Cy3-dCTP in second nested PCR. After hybridization of labeled amplicons on HOMIM, the microarray slides were scanned and original data acquired from professional software. Collectively, 94 bacterial species or clusters representing six bacterial phyla and 30 genera were detected. A higher bacterial diversity was observed in patients than in healthy samples. Statistical analyses revealed eight species or clusters were detected more frequently in diseased patients than in healthy samples, while six different species were detected more frequently in healthy as compared to diseased patients. The diversity of microbe within saliva derived from isolated population increased in caries-active status, and there are some bacteria in salivary flora can be as candidate biomarkers for caries prognosis in mixed dentition. The imbalances in the resident microflora may be the ultimate mechanism of dental caries.

AP-PCR detection of Streptococcus mutans and Streptococcus sobrinus in caries-free and caries-active subjects

The aim of the study was to identify the number and distribution of genotypes of Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) isolated from caries-free and caries-active subjects. Non-stimulated saliva, buccal smooth surface of the right upper teeth, fissures of sound occlusal surface, and carious surface were sampled from 7 caries-free and 7 caries-active (DMFT ≥ 6) students aged 22-24 years. S. mutans and S. sobrinus were isolated using Chelex-100 and primarily identified by colony morphology and biochemical characteristics. The isolates of S. mutans were genotyped using arbitrarily primed polymerase chain reaction. A total of 516 isolates of S. mutans were genotyped from 47 sites in 14 students, and 44 different genotypes were determined. All of the caries-free individuals harbored S. mutans but not S. sobrinus, although individuals 3 and 7 had no S. mutans in their saliva. The CFU value of S. mutans on carious surfaces was the highest, and values in saliva, fissures, and occlusal surfaces were higher in caries-active individuals than in caries-free individuals. We detected 28 genotypes of S. mutans in caries-free individuals, each of who carried more than 3 genotypes. However, we found only 16 genotypes of S. mutans in caries-active individuals, each of who carried no more than 3 genotypes. More genotypes are harbored in the saliva, fissures, and smooth surfaces of caries-free individuals than of caries-active individuals. The proportion of samples positive for S. mutans and S. sobrinus was significantly higher in caries-active individuals than in caries-free individuals, and the presence of these species is a risk factor for high DMFT in dental caries. Isolates of S. mutans exist that have apparent genetic diversity. The genotypes of isolates might relate to differences in caries susceptibility.

Differential adhesion of Streptococcus mutans to metallic brackets induced by saliva from caries-free and caries-active individuals.

The purpose of this study was to evaluate the influence of saliva obtained from caries-free and caries-active individuals on the adhesion rates of Streptococcus mutans to metallic brackets. The unstimulated whole saliva of four caries-free (decayed, missing, or filled surfaces = 0) volunteers and four caries-active (decayed, missing, or filled surfaces >12) patients were collected. The saliva samples from each group were mixed and clarified. Acquired pellicles were formed onto 30 metallic edgewise brackets for premolars for each saliva group. The brackets were put in contact with planktonic cells of Streptococcus mutans ATCC 25175. Adhesion rates were assessed by crystal violet retention technique. A higher streptococcal adhesion pattern (P < 0.00001) was observed on acquired pellicles formed by saliva from caries-active donors. The results showed that saliva from caries-active patients tends to increase the mutans adhesion to surfaces, which is a point of concern for orthodontists.

Caries Experience and Quantification of Streptococcus mutans and Streptococcus sobrinus in Saliva of Sudanese Schoolchildren

Streptococcus mutans and Streptococcus sobrinus are among the most commonly isolated bacterial species implicated as etiological agents of dental caries. Details of the composition of the oral microflora related to dental caries should aid in assessing the prevalence and risk of disease at an individual level. The aim of the present study was to determine the presence and relative amounts of S. mutans and S. sobrinus in the saliva samples obtained from schoolchildren in Khartoum State, the Sudan, and to study the association of the amounts of S. mutans and S. sobrinus with caries experience, socioeconomic status and sugar-sweetened snacks in this population. 140 samples, 30 of which were from individuals with caries experience, were examined using quantitative real-time polymerase chain reaction (qRT-PCR) with specific oligonucleotide primers. The mean ratio of fold differences of S. mutans to S. sobrinus was 0.77 (SD 5.4) and 2.29 (SD 6.0) for samples obtained from caries-free and caries-active individuals, respectively. This suggested that the proportion of S. sobrinus was higher than S.mutans in the caries-active group when compared to the caries-free group. An association was found between children with caries-active lesions and the frequent consumption of sticky desserts and higher socioeconomic status. S. sobrinus seems to be associated with caries experience in the studied population. A proposal of caries screening programs designed to test for S. sobrinus in this population may be developed.

Health promotion and dental caries

The central idea of the Brazilian health system is to prevent the establishment of disease or detect it as early as possible. Prevention and treatment of dental caries are related to behavioral factors, including dietary and oral hygiene habits, which are related to many chronic diseases. Dental health promotion therefore should be fully integrated into broadly based health-promoting strategies and actions such as food and health policies, and general hygiene (including oral hygiene), among others. For decades, a linear relationship between sugar consumption and caries has been observed. Recent data has indicated that this relationship is not as strong as it used to be before the widespread use of fluoride. However, diet is still a key factor acting in the carious process. Oral hygiene is a major aspect when it comes to caries, since dental biofilm is its etiological factor. Oral hygiene procedures are effective in controlling dental caries, especially if plaque removal is performed adequately and associated with fluoride. An alternative to a more efficient biofilm control in occlusal areas is the use of dental sealants, which are only indicated for caries-active individuals. If a cavity is formed as a consequence of the metabolic activity of the biofilm, a restorative material or a sealant can be placed to block access of the biofilm to the oral environment in order to prevent caries progress. The prevention of dental caries based on common risk-factor strategies (diet and hygiene) should be supplemented by more disease-specific policies such as rational use of fluoride, and evidence-based dental health care.

Correlations of oral bacterial arginine and urea catabolism with caries experience

Alkali generation by oral bacteria plays a key role in plaque pH homeostasis and may be a major impediment to the development of dental caries. To determine if the capacity of oral samples to produce ammonia from arginine or urea was related to caries experience, the arginine deiminase system (ADS) and urease activity in saliva and dental plaque samples were measured in 45 adult subjects. The subjects were divided into three groups according to caries status; 13 caries-free (CF) individuals (decayed, missing, and filled teeth = 0); 21 caries-active (CA) individuals (decayed teeth >or= 4); and 11 caries-experienced (CE) individuals (decayed teeth = 0; missing and filled teeth > 0). Real-time polymerase chain reaction was used to quantify the proportion of certain acid- or alkali-producing organisms in the samples. The amount of ammonia generated from the test substrates by plaque samples was generally higher than that produced by salivary samples in all groups. Significantly higher levels of salivary ADS activity and plaque urease activity were observed in CF subjects compared to CA subjects (P = 0.0004 and P = 0.014, respectively). The proportions of Streptococcus mutans from saliva and dental plaque of CA subjects were significantly higher than those from the CF group (P = 0.0153 and P = 0.0009, respectively). In the CA group, there was an inverse relationship between urease activity and the levels of S. mutans (P < 0.0001). This study supports the theory that increased caries risk is associated with reduced alkali-generating capacity of the bacteria colonizing the oral cavity; providing compelling evidence to further our understanding of oral alkali-generation in health and disease.

Heritability of Oral Microbial Species in Caries-Active and Caries-Free Twins

Oral microbes that colonize in the mouths of humans contribute to disease susceptibility, but it is unclear if host genetic factors mediate colonization. We therefore tested the hypothesis that the levels at which oral microbes colonize in the mouth are heritable. Dental plaque biofilms were sampled from intact tooth surfaces of 118 caries-free twins. An additional 86 caries-active twins were sampled for plaque from carious lesions and intact tooth surfaces. Using a reverse capture checkerboard assay the relative abundance of 82 bacterial species was determined. An integrative computational predictive model determined microbial abundance patterns of microbial species in caries-free twins as compared to caries-active twins. Heritability estimates were calculated for the relative microbial abundance levels of the microbial species in both groups. The levels of 10 species were significantly different in healthy individuals than in caries-active individuals, including, A. defectiva, S. parasanguinis, S. mitis/oralis, S. sanguinis, S. cristatus, S. salivarius, Streptococcus sp. clone CH016, G. morbillorum and G. haemolysans. Moderate to high heritability estimates were found for these species (h(2) = 56%-80%, p < .0001). Similarity of the overall oral microbial flora was also evident in caries-free twins from multivariate distance matrix regression analysis. It appears that genetic and/or familial factors significantly contribute to the colonization of oral beneficial species in twins.

Genetic diversity of F-ATPase subunits gene uncA amplified from Streptococcus mutans clinical isolates

To study the genetic diversity of F-ATPase alpha subunit gene uncA derived from Streptococcus mutans (S. mutans) clinical isolates and to investigate the relationship between the genetic diversity of acidurance factor and S. mutans aciduric ability, also and the cariogenicity. Sixty-four S. mutans strains derived from 34 caries-active individuals and 30 caries-free individuals, including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncA was amplified with specific primers from S. mutans genomic DNA, then the PCR products were analyzed by RFLP and sequenced. Two genotypes A and B of PCR-RFLP were revealed when digested with Hph I. Mbo II also produced two different pattern C and D. The distributions of A and B genotype strains with different caries-sensitivity groups were different (P < 0.05), and the proportion of A genotype strains from caries-activity group was higher than that from caries-free one. The distributions of C and D genotype strains with different acidurance strains were different (P < 0.05), and the proportion of C genotype strains from high acid tolerance group was higher than that from low acid tolerance group. These amplified uncA genes from different group were sequenced and there existed variation of Hph I and Mbo II recognized sites. This study indicates that uncA gene of S. mutans F-ATPase obviously displayed genetic diversity. The different Hph I-RFLP and Mbo II-RFLP genotypes could be related to the cariogenicity and acid tolerance of S. mutans strains.

Survey of Oral Microbial Diversity using PCR-based Denaturing Gradient Gel Electrophoresis

Polymicrobial biofilms in the human oral cavity exhibit marked diversity. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) surveys microbial diversity by displaying PCR-generated 16S rDNA fragments that migrate at different distances, reflecting the differences in the base-pair (i.e., % G+C) composition of the fragment. This study examined DGGE-generated diversity profiles of cultivable bacteria from individuals with different caries status. Initially, we developed a set of PCR-DGGE running conditions appropriate to oral bacteria. Next, we assessed migration standards from known oral bacterial reference strains. To test the methods, we profiled 20 bacterial saliva samples cultivated from young adults. The study produced a battery of species-specific 16S rDNA amplicons that could be used as a migration distance standard necessary for computer-assisted profile analysis. From the clinical samples, we found a significantly greater diversity of oral microbes in caries-free individuals compared with caries-active individuals (P = 0.01). These findings suggest thtat a portion of oral microbiota of caries-active individuals may be absent, suppressed, or replaced.

Frequency of four different mutacin genes in Streptococcus mutans genotypes isolated from caries-free and caries-active individuals

The ability of Streptococcus mutans to produce mutacins, combined with the production of other virulence factors such as lactic acid, may contribute to the pathogenesis of this bacterium. In the present study, the detection of genes encoding mutacin types I/III, II and IV was performed by PCR with specific primers to each type in a total of 63 S. mutans genotypes isolated from caries-active and caries-free individuals. In the caries-free group, PCR screening for mutacin IV revealed that 31.8% of strains were positive for this mutacin. PCR for the other three mutacins tested (I/III and II) did not yield amplicons in any S. mutans strains in this group. The PCR with primers of mutacin IV showed 68.3% positive genotypes in the caries-active group, on the other hand, the amplicons of mutacins I/III revealed 41.5% positive strains that carried these genes. The chi square test showed significant differences in the number of positive strains to mutacin IV when comparing the caries-free and caries-active genotypes of S. mutans (P = 0.01). All tested S. mutans strains were negative by PCR for mutacin II. The low frequencies of detection of some mutacin genes suggest the existence of high diversity and polymorphism in the production of genetic determinants of mutacin-like substances. In addition, the production of a wide spectrum of mutacins can play an important biological role in colonization by S. mutans strains, mainly in the niche of high-complexity microbial communities.

Genotypic diversity and virulence traits of Streptococcus mutans in caries-free and caries-active individuals

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Genotypic diversity and virulence traits of Streptococcus mutans in caries-free and caries-active individuals.

The present study evaluated the relationship between clonal diversity and some virulence traits of Streptococcus mutans isolated from eight caries-free and eight caries-active subjects. A total of 155 S. mutans isolates from caries-free subjects and 144 isolates from caries-active subjects were obtained from samples of saliva, dental plaque and tongue surface and identified by PCR. The isolates were submitted to arbitrarily primed (AP)-PCR (OPA-2 and OPA-13) and multilocus enzyme electrophoresis (MLEE) to establish the genotypic diversity. Production of water-insoluble glucan (WIG) (monitored by SDS-PAGE), final pH of cultures and the ability of bacterial cells to adhere to smooth glass in the presence of sucrose were measured. High and comparable abilities of MLEE and AP-PCR were found to distinguish S. mutans genotypes, using Simpson's index of discrimination (0.971 and 0.968, respectively). The results showed a significant difference (P < 0.01) in the number of genotypes when caries-free and caries-active groups were compared by both fingerprinting methods used. Final pH (P = 0.32) and the percentage of adherence to a glass surface (P = 0.62) did not show differences between the two groups; however, the intensities of WIG bands from the caries-active group were greater than those from the caries-free group (P < 0.01). In addition, WIG was positively correlated with the ability of S. mutans to adhere to a glass surface (r = 0.34, P = 0.02) from caries-active subjects. These data showed that AP-PCR analysis and MLEE are both effective methods for assessing the genetic relatedness of S. mutans. Using these techniques, it was found that there is a larger number of genotypes of S. mutans with increased ability to synthesize WIG in caries-active individuals.

Factors associated with caries incidence in an elderly population.

The purpose of this paper was to identify baseline factors associated with future caries development in older adults (age 60+) during a 3-year study period. Poisson regression analysis was used to determine the association between potential risk factors and disease incidence. The significant factors associated with high coronal caries incidence rates were high baseline root DMFS (P<0.001), high counts of mutans streptococci and lactobacilli (P=0.036), male gender (P=0.007), and Asian ethnicity (P=0.002). These factors had small to moderate effects on incidence rates, with relative risk values of approximately 1.2 to 2. The significant factors associated with higher disease incidence on root surfaces were baseline coronal DMFS (marginally significant, P=0.078), high bacterial counts (P=0.002), and Asian ethnicity (P=0.009). The predictive value of the models was low for both coronal and root caries. This result may be because this population had a higher than usual caries incidence rate, making discrimination among these caries-active individuals difficult. The current study affirmed the value of baseline DMFS and salivary variables to modeling caries incidence and introduced ethnicity as a variable useful for the study of dental caries in older adults.

Lactobacilli, Mutans streptococci and Dental Caries: A Longitudinal Study in 2-Year-Old Children up to the Age of 5 Years

A 3-year cohort study was carried out in 252 pre-school children for early identification of caries-active individuals. During this period information was collected about the acquisition of mutants streptococci and lactobacilli from the age of 2 till 5 years old. At baseline mutants streptococci were detected in 43% of the children while the detection frequency of lactobacilli was low (11.5%). On an individual level, numbers of colony-forming units of mutans streptococci and lactobacilli in plaque and saliva varied largely during the study period. The correlations between the numbers of lactobacilli and mutants streptococci in the saliva of the mother and the saliva and plaque of the child were low and never exceeded r = 0.22. Very low correlations (< r = 0.22) were also found between the numbers of mutans streptococci or lactobacilli and the diet in terms of the number of sugar intakes. Nevertheless, in children older than 2.5 years correlations between the clinical caries score and lactobacilli in saliva (range 0.31-0.62) and mutans streptococci in plaque or saliva (range 0.24-0.46) were highly significant (p < 0.01).

Comparison of the Initial Streptococcal Microflora on Dental Enamel in Caries-Active and in Caries-Inactive Individuals

This study compared the initial (4 h) microflora on enamel in 7 caries-active and in 7 caries-inactive adolescents. In both groups the microflora was dominated by streptococci which comprised 61 and 78% (median values) of the total viable counts in caries-active and caries-inactive individuals, respectively (p less than 0.01). Identification of a total of 700 streptococcal isolates according to a recently revised classification showed that the predominant streptococci belonged to the species Streptococcus oralis, Streptococcus mitis biovar 1, and Streptococcus sanguis. Early plaque from caries-inactive individuals differed from that of caries-active individuals by significantly higher proportions of S. sanguis (p less than 0.05) and IgA1 protease producing streptococci (p less than 0.05). In caries-active individuals, there was a tendency to elevated levels of S. mitis biovar 1 (p less than 0.10). In addition, caries-active individuals were colonized by significantly higher numbers of mutans streptococci on the enamel surfaces (p less than 0.01). However, in both groups Streptococcus mutans (serotype c) comprised less than or equal to 2% of the early streptococcal flora. Streptococcus gordonii, S. mitis biovar 2, and Streptococcus salivarius were present in low proportions and did not show differences in distribution that could be related to caries activity. The observed differences in the composition of the early streptococcal microflora may be a factor that governs the eventual cariogenic potential of dental plaque.

Streptococcus mutans establishment and dental caries experience in children from 2 to 4 years old.

39 children were studied longitudinally at the age of 2, 3, and 4 yr for the colonization of S. mutans in plaque and saliva and for caries experience. S. mutans was found in 38% of the children, and the predominant serotype group was c/e/f. A total of 16 children got caries before the age of 4. Children who harbored S. mutans in their plaque at the age of 2, appeared to be the most caries-active individuals. Their caries index values (number of decayed, missed and filled surfaces, dmfs = 10.6 +/- 5.3) at the age of 4 differed significantly from the values of children who harbored S. mutans later (dmfs = 3.4 +/- 1.8, P less than 0.005) or remained free from S. mutans infection (dmfs = 0.3 +/- 1.1, P less than 0.0003). It was thus concluded that the early establishment of S. mutans in the plaque of primary incisors indicated early and extensive caries attack in young primary dentition.

Monitoring of caries progression in permanent and primary posterior approximal enamel by bitewing radiography.

The work which has monitored caries progression in posterior approximal enamel surfaces by reference to the size of radiolucency seen on bitewing radiographs is reviewed. There are wide variations in the degree of standardisation achieved, in the composition of the study groups and in the duration of the studies. There is a paucity of information relating to adults, the elderly, groups with low caries prevalence, and fluoridated communities. The results indicate that for the majority approximal caries progresses slowly, and large numbers of lesions remain unchanged for long periods. The available results are compared using the mathematical model of a negative exponential. The 'mean' time during which a lesion remains radiographically confined to the enamel is of the order of 3-4 yr, although in caries active individuals much shorter times are reported. Some clinical implications are suggested and the need for further research using comparable methods is stressed.

Differential Utilization of Proteins in Saliva from Caries-active and Caries-free Subjects as Growth Substrates by Plaque-forming Streptococci

Mixed or parotid saliva from caries-active individuals consistently supported better growth of Streptococcus mutans (type c) than that from caries-free individuals. Electrophoretic studies revealed that certain proteins in caries-active salivas were susceptible to microbial attack, but similar proteins in caries-free salivas were refractory.