Faculty Bibliography

Dental caries is an infectious disease of bacterial origin. The use of antimicrobial agents to reduce or eliminate the bacteria associated with caries follows the approach used to combat other infectious diseases of humans. Unfortunately, only a few dozen studies have sufficient resolving power to make inferences as to the anticaries efficacy of the antimicrobial approach to caries management. Here, we comment on the findings of the RTI/UNC review concerning antimicrobials, discuss additional findings not covered in that review, and make recommendations based upon both the available literature and from our own experience. Even though the studies published thus far are inconclusive or lack sufficient demonstration of efficacy to recommend a specific approach involving antimicrobial agents in routine clinical practice, several pieces of information from these studies suggest future avenues of investigation.

Approximately 5% of strains of Streptococcus mutans contain plasmid DNA. Strain UA140 harbors a 5.6-kb cryptic plasmid, pUA140, with an overall G+C content of 32.7%. Five open reading frames (ORF), encoding peptides of larger than 100 amino acid residues, were initially designated as ORF1 to ORF5. These five ORFs were located on the same strand of pUA140. ORF1 (258 amino acids) resembled a replication protein, Rep. Upstream of the putative Rep gene, a double-stranded origin for plasmid replication that showed strong similarity to those of a number of plasmids in the pT181 family was identified. Further upstream was a region constituting the single-stranded origin of replication. A single-stranded DNA intermediate was detected during plasmid replication. Taken together, these results suggest that pUA140 replicated by the rolling circle replication mechanism but exhibited several characteristics that differ from those of other members of the pT181 plasmid family.

Streptococcus sanguis comprises a heterogeneous group of oral streptococci indigenous to the oral cavity of humans. A total of 289 isolates from an infant population (n=37) were tentatively identified as S. sanguis on the basis of the distinctive colony morphology as shown on MM10-sucrose non-selective medium. These isolates were divided into four biovars of S. sanguis as determined by an extended panel of biochemical attributes. Chromosomal DNA was extracted from each isolate, and an AP-PCR fingerprint profile was obtained to allow study of the diversity within and among the infants. In this study, all four biovars of S. sanguis were detected in the infants. A wide genotypic diversity of S. sanguis was observed among these isolates; on average, each infant harbored 2.7 unique amplitypes as shown by the AP-PCR fingerprints. To explore the phylogenic relationship among these S. sanguis isolates, 20 strains representing the four biovars were selected at random for sequencing of their 16S rDNA and 16S-23S rDNA intergenic spacer chromosomal loci. Two major sequence patterns were identified within the 16S rDNA sequences. A phylogenic analysis showed that members from each of the four biovars of S. sanguis bore close relationship with the type-strain ATCC 10556 sequence, and that all of the isolates representing the four biovars could be clustered into two main phylotypes. The biovars were distributed throughout the phylotypes, indicating no correlation between the genetic and phenotypic groupings.

Streptococcus mutans strains were isolated from cohorts of Brazilian nursery school children and genotyped by arbitrarily primed PCR and restriction fragment length polymorphism analysis. Of 24 children with two to five S. mutans isolates, 29% carried two or more genotypes. The presence of matching genotypes of S. mutans among children attending one nursery suggests horizontal transmission.

Routine identification of Streptococcus mutans and Streptococcus sobrinus is generally based upon growth on various selective media, colony morphology and biochemical characteristics. We examined various approaches of differentiating these two species through a combination of the conventional phenotypic methodology with chromosomal DNA fingerprint (CDF) and arbitrarily primed polymerase chain reaction (AP-PCR) methods. Initially, ten ATCC type strains and 20 randomly selected clinical isolates of mutans streptococci (MS) were characterized and grouped into two major types based on patterns generated by the CDF using HaeIII digestion. The CDF's patterns with restriction fragments equal to or greater than 6.6 kb were defined as the CDF-1 group. The CDF's patterns with restriction fragments less than 6.6 kb were defined as the CDF-2 group. Both groups were then examined for biotype, serotype, and composition of DNA via thermal denaturation. AP-PCR was applied and evaluated for the capability of delineating S. mutans from S. sobrinus strains. Results of this study showed that all CDF-1 strains fit within a G+C range of 36.2% to 42.2%, whereas the CDF-2 strains had a G+C range of 45.8% to 47.0%. The serotyping assay exhibited 100% sensitivity, 90% specificity and 86.7% agreement with the CDF. The biotyping assay presented the poorest specificity (38.5%), indicating the highest variability. The capability of AP-PCR in differentiation of S. mutans from S. sobrinus was comparable to the CDF method, suggesting that either of these two approaches can and may serve as a viable alternative method to serotyping or biotyping of MS.

The heterogeneous group of oral bacteria within the sanguinis (sanguis) streptococci comprise members of the indigenous biota of the human oral cavity. While the association of Streptococcus sanguinis with bacterial endocarditis is well described in the literature, S. sanguinis is thought to play a benign, if not a beneficial, role in the oral cavity. Little is known, however, about the natural history of S. sanguinis and its specific relationship with other oral bacteria. As part of a longitudinal study concerning the transmission and acquisition of oral bacteria within mother-infant pairs, we examined the initial acquisition of S. sanguinis and described its colonization relative to tooth emergence and its proportions in plaque and saliva as a function of other biological events, including subsequent colonization with mutans streptococci. A second cohort of infants was recruited to define the taxonomic affiliation of S. sanguinis. We found that the colonization of the S. sanguinis occurs during a discrete "window of infectivity" at a median age of 9 months in the infants. Its colonization is tooth dependent and correlated to the time of tooth emergence; its proportions in saliva increase as new teeth emerge. In addition, early colonization of S. sanguinis and its elevated levels in the oral cavity were correlated to a significant delay in the colonization of mutans streptococci. Underpinning this apparent antagonism between S. sanguinis and mutans streptococci is the observation that after mutans streptococci colonize the infant, the levels of S. sanguinis decrease. Children who do not harbor detectable levels of mutans streptococci have significantly higher levels of S. sanguinis in their saliva than do children colonized with mutans streptococci. Collectively, these findings suggest that the colonization of S. sanguinis may influence the subsequent colonization of mutans streptococci, and this in turn may suggest several ecological approaches toward controlling dental caries.

Our previous study reported that the fidelity of mutans streptococci (MS) transmission from mother to infant was gender- and race-specific within a Birmingham, Ala., population. We hypothesized that fidelity might be a function of postnatal nurturing practices. The purpose of the present study was to investigate those factors that could be correlated with MS transmission among Chinese children whose nurturing histories were known. Forty-eight families with 2- to 3-year-old children were selected from two kindergartens of Beijing, China. A questionnaire concerning the childhood nursing practice was obtained from the parents. Dental caries status was examined, and bacterial samples were collected for all participants twice at 6-month intervals. An average of six isolates of MS was picked at random from selective medium from each individual at each visit. Chromosomal DNA fingerprints were performed for all MS isolates to determine the genomic similarity within each family and among individuals. The results showed that 66% of the children at 2-3 years of age harbored MS, and 46% were caries-active. Of those children, 70% were breast-fed. Among the children whose chromosomal DNA fingerprint genotypes of MS matched that of their mothers, 88% were breast-fed compared with only 12% who were not breast-fed (p = 0.03). We also found that children who were breast-fed for more than 9 months were likely to harbor strains of MS common to their mothers (p = 0.04) and experience more dental caries (dmft = 4.4) at 3 years of age compared with children who were breast-fed less than 9 months (dmft = 1.4, p = 0.04). The data suggest that breast-feeding, especially prolonged breast-feeding, may correlate with the fidelity of transmission and that prolonged breast-feeding may contribute to a higher caries rate.