Streptococcus mutans and Streptococcus sanguinis colonization correlated with caries experience in children.

The aim of this study was to examine the colonization of Streptococcus mutans and Streptococcus sanguinis in the oral cavity and the association with severe early childhood caries (S-ECC). Saliva and plaque samples were collected from 14 S-ECC children and 8 caries-free (CF) children. All S-ECC children were S. mutans positive; 100% of CF children and 93% of S-ECC children were S. sanguinis positive. The children's caries severity was positively correlated with levels of S. mutans (p < 0.001), total oral streptococci (p < 0.01), total cultivable oral bacteria (p < 0.05), and children's age (p < 0.05). Logistic regression analysis showed that the interaction of S. sanguinis with S. mutans was a significant factor associated with the caries status in children, suggesting that the relative levels of these two microorganisms in the oral cavity play an important role in caries development.

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.

Mode of delivery and other maternal factors influence the acquisition of Streptococcus mutans in infants.

S. mutans plays a key role in dental caries. The extent to which perinatal events influence the acquisition of S. mutans is unclear. We hypothesized that several maternal factors, including the mode of delivery, influence the initial acquisition of S. mutans in infants. A prospective cohort study was conducted in 156 mother-infant pairs. The study found that maternal gestational age (p = 0.04), S. mutans level (p = 0.02), caries score (p = 0.02), sexually transmitted disease (STD) infection experience (p = 0.01), and family income (p = 0.03) had significant effects on the acquisition of S. mutans. Among infants who became infected, those delivered by Caesarean section acquired S. mutans 11.7 mos earlier than did vaginally delivered infants (p = 0.038). C-section infants harbored a single genotype of S. mutans that was identical to that of their mothers (100% fidelity). Analysis of the data demonstrated the possible perinatal influences on infants' acquisition of a member of the cariogenic microbiota, and its potential effect on caries outcome.

Oral Lactobacilli and Dental Caries: A Model for Niche Adaptation in Humans.

Lactobacilli have been associated with dental caries for over a century. Here, we review the pertinent literature along with findings from our own study to formulate a working hypothesis about the natural history and role of lactobacilli. Unlike most indigenous microbes that stably colonize a host, lactobacilli appear to be planktonic, opportunistic settlers that can gather and multiply only in certain restrictive niches of the host, at least within the oral cavity. We postulate that the following essential requirements are necessary for sustained colonization of lactobacilli in humans: 1) a stagnant, retentive niche that is mostly anaerobic; 2) a low pH milieu; and 3) ready access to carbohydrates. Three sites on the human body meet these specifications: caries lesions, the stomach, and the vagina. Only a handful of Lactobacillus species is found in caries lesions, but they are largely absent in caries-free children. Lactobacilli present in caries lesions represent both a major contributor to caries progression and a major reservoir to the gastrointestinal (GI) tract. We extend the assertion from other investigators that lactobacilli found in the GI tract originate in the oral cavity by proposing that lactobacilli in the oral cavity arise from caries lesions. This, in turn, leads us to reflect on the health implications of the lactobacilli in the mouth and downstream GI and to ponder whether these or any of the Lactobacillus species are truly indigenous to the human GI tract or the oral cavity.

Sequence analysis of mutA and mutM genes involved in the biosynthesis of the lantibiotic mutacin II in Streptococcus mutans.

Streptococcus mutans, along with many other gram-positive bacteria produce small antibacterial peptides called bacteriocins. Bacteriocins elaborated by S. mutans, termed mutacins, may provide a selective force necessary for initial or sustained colonization in dental plaque by this major dental pathogen. Previously, we purified and characterized mutacin II, the first lantibiotic found in S. mutans. Specific oligonucleotides designed according to the N-terminal amino acid sequence permitted amplification of 0.7 kb upstream and 2.1 kb downstream of the N-terminus, using single-specific-primer PCR (SSP-PCR). The gene encoding the mutacin II prepeptide, mutA, was subsequently cloned and sequenced. The complete prepeptide consists of 53 amino acids, including the 26 amino acid amphipathic leader peptide with the Gly(-2)-Gly(-1) sequence at the processing site. The prepeptide showed similarity to the lantibiotics lacticin 481, variacin, salivaricin and streptococcin A-FF22. A 3 kb open reading frame immediately downstream of mutA, denoted mutM, showed sequence similarities to LCNDR2 from Lactococcus lactis. By analogy, mutM is probably involved in post-translational modification of the mutacin prepeptide. Gene disruption with an insertional vector pVA891 showed that intact copies of mutA and mutM are required for production of mutacin II.

Associations of MHC genes with levels of caries-inducing organisms and caries severity in African-American women.

The aim of this investigation was to evaluate the relationship between MHC alleles at the HLA-DRB1, DQB1 and TNFa microsatellite loci and levels of oral bacteria that play a role in the etiology of dental caries, and the DMFS index in 186 AA primparous women. The average age of the cohort was 20.8+/-3.7 years. The median DMFS index was 9 (range 0-68). High levels of S. mutans were positively associated with DRB1*3 and DRB1*4 presence (p < or = 0.005). DRB1*8 was positively associated with higher levels of S. mutans as a percentage of total Streptococci (p = 0.04). DRB1*1 was positively associated with high levels L. casei (p = 0.04). DQB1 alleles were not observed associated with oral bacterial levels. TNFa allele 103 was negatively associated (p = 0.04), and TNFa 117 was positively associated (p = 0.007), with high levels of L. acidophilus. No significant associations were observed between any DRB1, DQB1 or TNFa allele and the DMFS index. These results support an hypothesis of an association between host HLA class II and TNFa genetic profile and colonization of S. mutans, L. casei, and L. acidophilus thought to be pathogens involved in the etiology of dental caries.

Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II.

The lantibiotic mutacin II, produced by Streptococcus mutans T8, is a ribosomally synthesized peptide antibiotic that contains thioether amino acids such as lanthionine and methyllanthionine as a result of post-translational modifications. The mutacin II leader peptide sequence shares a number of identical amino acid residues with class AII lantibiotic leader peptides. To study the role of these conservative residues in the production of active antimicrobial mutacin, 15 mutations were generated by site-directed mutagenesis. The effects of these substitutions vary from no effect to complete block-out. Mutations G-1A, G-2A, I-4D, and L-7K completely blocked the production of mature mutacin. Other mutations (I-4V, L-7M, E-8D, S-11T/A, V-12I/A, and E-13D) had no detectable effect on mutacin production. The changes of Glu-8 to Lys, Val-12 to Leu, Glu-13 to Lys reduced the mutacin production level to about 75%, 50%, and 10% of the wild-type, respectively. Thus, our data indicated that some of these conserved residues are essential for the mutacin biosynthesis, whereas others are important for optimal biosynthesis rates.

Microbially-produced peptides having potential application to the prevention of dental caries.

Strategies advanced to decrease the occurrence of dental caries have in the past typically focussed upon attempting to reduce plaque accumulation by application of broad-spectrum antibacterial agents. In recent years however there has been growing interest in the application of a more targeted approach to the selective elimination from plaque of those bacterial species that are specifically implicated as the aetiological agents of this disease. This review focuses upon a number of the small bacterially-produced peptide antibiotics known as bacteriocins that are currently being explored for their potential role in the treatment and prevention of dental caries.

Suppression of Streptococcus mutans in the mouths of humans by a dental prophylaxis and topically-applied iodine.

A prophylaxis followed by three topical applications of an iodine-potassium iodide solution significantly reduced the levels of Streptococcus mutans in fissure and approximal plaques and in saliva. Reductions persisted 20--24 weeks after treatment in salivary and approximal samples. A prophylaxis alone exerted a small and temporary reduction of S. mutans in occlusal fissure plaque, but did not reduce the levels of this organism in approximal plaque or in saliva. A significant relationship existed between the levels of S. mutans in saliva and the proportions of this organism in plaque. The dorsum of the tongue does not appear to constitute a significant reservoir for S. mutans following disinfecting procedures.

The fidelity of initial acquisition of mutans streptococci by infants from their mothers.

Previous cross-sectional studies using bacteriocin profiles, serotyping, or genotyping suggest that mothers are the principle source of mutans streptococci (MS) to their infants. This study determined the commonality of MS genotypes between mothers and their infants at the time of initial acquisition. Oral bacteria of mothers and their infants were monitored from birth for approximately 3 years at three-month intervals. Genotypes of MS in infants appeared identical to those present in mothers in approximately 71% of 34 mother-infant pairs studied. Interestingly, female infants acquired MS genotypes identical to their mothers' with significantly greater fidelity than male infants (88% vs. 53%). Homology of genotypes between mothers and their infants at initial acquisition strongly suggests that MS strains were transmitted from mother to infant and that this transfer exhibited gender specificity. In no instance did we observe homology of genotypes between fathers and infants or fathers and mothers, further supporting the notion that acquisition of MS in humans follows maternal lines. Although the prevalence of dental caries was low in this young child population (11/34; 32%), we observed that male children who harbored the same genotype as their mothers had a 13 times greater likelihood of having caries than female children who acquired their mothers' strain; this difference was statistically significant (p < 0.01). Although we do not know the biological mechanisms governing fidelity of acquisition between a mother and her infant, our data suggest that caries outcome may be, in part, determined by both the source of MS and the presence of a specific genotype of MS.(ABSTRACT TRUNCATED AT 250 WORDS)

Root surface caries subsequent to gingivectomy in rats inoculated with Streptococcus sobrinus (mutans) and Actinomyces viscosus.

We studied the effect of oral infection with cariogenic micro-organisms on alveolar bone loss and root surface caries subsequent to gingivectomy in rats. Thirty-six rats were fed diet MIT 200 (67% sucrose); one-half, the controls, had antibiotics added to the diet. At ages 18, 19, and 21 days, the experimental group was orally infected with streptomycin-resistant Streptococcus sobrinus (mutans) 6715 and Actinomyces viscosus M-100. At age 30 days, all animals were subjected to a gingivectomy on maxillary and mandibular left quadrants. The experimental group was re-inoculated at age 36 days. Animals were killed seven weeks after surgery. Jaws were stained, and planimetric measurements of exposed root surface area and caries on lingual/palatal surfaces of 1st and 2nd molars were made from video images with a computer/digitizer. Compared with the non-operated contralateral quadrants, gingivectomy significantly increased exposed root surface area in the maxilla and in the mandible in both the control and experimental groups. Oral inoculation significantly increased exposed root surface in mandibular (but not maxillary) quadrants which had received a gingivectomy, but had no effect on non-gingivectomized quadrants. There was no caries in the control group, whereas ten rats in the experimental group had root surface caries lesions, all in quadrants which had received a gingivectomy. This rat model should prove useful in further studies of root surface caries.

Effect of topically-applied solutions of iodine, sodium fluoride, or chlorhexidine on oral bacteria and caries in rats.

Antibacterial levels of iodine, chlorhexidine or sodium fluoride were topically applied to the molar teeth of rats. The effect of these agents on buccal and sulcal caries, as well as on certain oral bacterial populations was assayed. All three agents exerted a significant cariostatic effect which was site specific. Buccal caries incidence and reductions appeared correlated to S. mutans counts, while sulcal caries was better correlated to total cultivable counts. These studies suggested that the simultaneous application of iodine and NaF might exert an additive cariostatic effect.

Initial acquisition of mutans streptococci by infants: evidence for a discrete window of infectivity.

Oral bacterial levels of 46 mother-child pairs were monitored from infant birth up to five years of age so that the acquisition of mutans streptococci (MS) by children could be studied. The initial acquisition of MS occurred in 38 children at the median age of 26 months during a discrete period we designated as the "window of infectivity". MS remained undetected in eight children (17%) until the end of the study period (median age of 56 mo). The levels of both MS and lactobacilli in saliva of mothers of children with and without MS were not significantly different. Comparisons between a caries-active cohort colonized by MS (nine of 38) and children without detectable MS revealed similar histories in terms of antibiotic usage, gestational age, and birth weight. Interestingly, half of the children between the ages of one and two years who were not colonized by MS were attended by caretakers other than the mother, while all of the caries-active children during this same time period were cared for by their mothers; the difference was statistically significant. Here we report for the first time that MS is acquired by infants during a defined period in the ontogeny of a child. Support for the notion of a discrete window of infectivity comes from other sources, including animal models.

Dental caries. An infectious and transmissible disease.

Of the infectious diseases that affect humans, dental caries may be the most prevalent. Pediatric primary medical care providers are usually the first health care providers to examine the oral cavity of a child and so need to be able to recognize suspicious dental lesions. This article provides information on recognizing caries and reviews the epidemiology, pathogenesis, and treatment of caries and odontogenic infections.

Colonization by mutans streptococci in the mouths of 3- and 4-year-old Chinese children with or without enamel hypoplasia.

This case-control study compared the prevalence and concentration of mutans streptococci (MS) in saliva between children with and without enamel hypoplasia (EHP). A total of 486 3- or 4-year-old Chinese children were initially screened for EHP, then distributed into two groups: 234 children diagnosed as having EHP were assigned to the case group; 252 who were free of EHP were included in the control group. The concentration of MS in saliva was assayed for each child. Nutritional status was deduced from body height and weight. Birth weight, prematurity, and nursing history were also determined. MS were found in 94.7% of the study population. The differences in MS concentrations were not associated with low birth weight, prematurity, length of breast feeding, or body height and weight. A statistically significant association existed between the presence of EHP and high counts of MS (p < 0.001). High MS counts were correlated with severity of enamel defects (p < 0.001). When the caries status of the children was controlled as the confounding factor in statistical analyses, the association between EHP and MS decreased but still remained significant (p = 0.025). This study shows that high MS counts are correlated with EHP, suggesting that irregularities in enamel surfaces could be a contributing factor that fosters the increased colonization of MS in the mouths of children.

Genetic characterization of the oral Actinomyces.

Actinomyces are difficult to identify using serological and biochemical methods but genotyping is an efficient and reliable means of bacterial characterization and can be used to determine clonal identity. The purpose here was to genotype 13 American type culture collection (ATCC) reference strains representing six different oral Actinomyces spp. by using chromosomal DNA fingerprinting (CDF), arbitrarily primed-polymerase chain reaction (AP-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In CDF analysis, BamHI, BstEII and SmaI yielded digestion patterns revealing characteristic differences among the known Actinomyces spp., with SmaI demonstrating optimal resolution. Amplicons generated by AP-PCR with primer OPB-07 displayed banding patterns that permitted discrimination of all Actinomyces strains tested. PCR-RFLP with MnlI digests generated fragment patterns that also characterized the reference strains. Collectively, genotypic profiles generated by CDF, AP-PCR and PCR-RFLP permitted differentiation of all 13 ATCC Actinomyces strains. SmaI CDF analysis of 18 clinical isolates of catalase-positive A. naeslundii genospecies 2 revealed extensive genetic diversity among these strains. These molecular approaches may be useful in determining genetic diversity within oral Actinomyces populations and fidelity of Actinomyces transmission between mother and child.

Genetic diversity of Actinomyces naeslundii genospecies 2 in mother-child pairs.

Actinomyces naeslundii genospecies 2 (gsp-2) are members of the autochthonous oral flora. Chromosomal DNA fingerprinting (CDF) with SmaI revealed extensive genetic diversity among A. naeslundii gsp-2 strains within individual mothers and children. There was a low prevalence of genotype match among A. naeslundii gsp-2 strains between all mother and child pairs.

Differences in the detection and enumeration of mutans streptococci due to differences in methods.

Different methods reported for assessing mutans streptococci (MS) make the direct comparisons of results across studies difficult. To quantitate the variations of MS estimates attributable to differences in method, stimulated and unstimulated saliva samples and oral swab samples were compared with pooled dental-plaque samples. Detection of MS in stimulated saliva samples was in excellent agreement with the presence of MS in pooled plaque samples. MS detection in unstimulated saliva samples, however, was significantly discordant with that in either pooled plaque or oral swab samples. When caries status was used as the criterion of validity of MS estimates, stimulated saliva samples demonstrated a sensitivity (94%) similar to that of pooled plaque samples, but exhibited a lower specificity (11%) than that of pooled plaque samples (17%). As a result, the measure of association between MS and caries was biased (towards null) when MS status was based on stimulated saliva samples. Interestingly though, in enumerating MS, stimulated saliva samples yielded significantly higher levels of MS (about 1.5 log10 increase) with a lower variability compared to unstimulated saliva samples. The use of different culture media for detection of MS gave different results as well. MS detection was poor (kappa = 0.31) and MS levels were lower (p = 0.0001) when samples were grown on glucose-sucrose-potassium tellurite-bacitracin agar compared to mitis-salivarius-bacitracin agar. Together with the relative ease of sampling and processing, these findings collectively justify the use of plaque samples for the qualitative assessment of MS and stimulated saliva samples for the quantitative assessment of MS, while providing a basis for adjustment of estimates when comparing results across studies.

Effect of conventional dental restorative treatment on bacteria in saliva.

Dental caries results from the dissolution of mineralized dental tissues by the metabolic by-products of oral bacteria colonizing the surface of teeth. The principal modality for dealing with this infectious process is through restorative treatment which removes the pathologic tissue and replaces it with a variety of inert materials. The purpose of this study was to evaluate the effect of traditional restorative treatment on select oral bacterial populations. Fifty-two females demonstrating high levels of mutans streptococci (greater than or equal to 2.5 x 10(4) colony forming units (cfu) per ml saliva) with no more than four missing posterior teeth were recruited for this study. Salivary levels of mutans streptococci, lactobacilli, total streptococci, and total cultivable bacteria were evaluated before, during, and after restorative treatment. Initial DMFS scores were established by two standardized examiners using bitewing radiographs and clinical examination, which was conducted under optimal conditions. All restorative treatment was completed by faculty members of the University of Alabama School of Dentistry using treatment plans developed by the DMFS examiners. The participants received a mean of 16.4 restored surfaces, which resulted in significant reductions of all the bacterial populations tested. All microbial populations monitored were predicted to return to their baseline levels within 151 days after restorative treatment in 50% of the participants. This study shows that conventional restorative treatment results in a significant reduction of bacterial populations including those associated with the dental caries process, i.e., mutans streptococci and lactobacilli.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmission of mutans streptococci to infants following short term application of an iodine-NaF solution to mothers' dentition.

In a randomized clinical trial, six applications of an I2-NaF or a placebo solution were administered to the mothers' dentition at the time of tooth emergence of her infant, to evaluate its effect on transmission of mutans streptococci (MS) to the infant. MS and other bacterial levels were periodically monitored in 48 mother-infant pairs until the child's third birthday. There were significant reductions in maternal salivary MS (P = 0.04), lactobacilli (P = 0.04), total streptococci (P = 0.002), and total cultivable organisms (P = 0.004) immediately following treatment. In children, 3-yr incidence of MS colonization and the time of acquisition of MS or the caries experience did not differ significantly between the two groups. We conclude that short-term application of I2-NaF to the maternal dentition of predentate infants significantly lowers MS, lactobacilli, total streptococci, and total cultivable bacteria in mothers' saliva, but does not influence the incidence and the time of acquisition of MS or the caries experience in children.