Faculty Bibliography

Several methods for determining the diversity of Lactobacillus spp were evaluated with the purpose of developing a realistic approach for further studies. The patient population was comprised of young children with an oral disease called severe early childhood caries. The ultimate goal of these studies was to ascertain the role of lactobacilli in the caries process. To accomplish that goal, we evaluated several methods and approaches for determining diversity including AP-PCR, chromosomal DNA fingerprinting, denaturing gradient gel electrophoresis, and 16S rRNA gene sequencing. Central to these methods was the gathering and screening of isolates from cultivation medium. Using various estimates of diversity, we addressed the question as to how many isolates represent the overall diversity and how cultivation compares to non-cultivation techniques. Finally, we proposed a working approach for achieving the goals outlined framed by both practical constraints in terms of time, effort and efficacy while yielding a reliable outcome

The oral biofilm community consists of >800 microbial species, of which Streptococcus mutans is considered a primary pathogen for dental caries. The genomic island, TnSmu2, of S. mutans comprises >2% of the genome. In this study, we demonstrate that TnSmu2 harbors a gene cluster encoding nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), as well as accessory proteins and regulators involved in non-ribosomal peptide (NRP) and polyketide (PK) biosynthesis. Interestingly, the sequences of these genes and their genomic organizations and locations are highly divergent among different S. mutans strains, yet each TnSmu2 region encodes NRPS/PKS and accessory proteins. Mutagenesis of the structural genes and putative regulatory genes in strain UA159, UA140, and MT4653 resulted in colonies that were devoid of their yellow pigmentation (for UA140 and MT4653). In addition, these mutant strains also displayed retarded growth under aerobic conditions and in the presence of H2O2. HPLC profiling of cell surface extracts identified unique peaks that were missing in the mutant strains, and partial characterization of the purified product from UA159 demonstrated that it is indeed a hybrid NRP/PK, as predicted. A genomic survey of 94 clinical S. mutans isolates suggests that TnSm2 gene cluster may be more prevalent than previously recognized

Human saliva can be separated by centrifugation into cell pellet and cell-free supernatant, which are called cellular phase and liquid phase in this study. While it is well documented that the cellular phase of saliva contains hundreds of oral bacteria species, little is known whether the liquid phase of saliva contains any information related to oral microbiota. In this study, we analyzed the bacterial nucleic acid contents of the liquid phase of saliva. Using primers universal to most eubacterial 16S rDNA, we detected large amounts of bacterial 16S rRNA and rDNA in the cell-free phase of saliva. Random sequencing analysis of forty PCR amplicons from the cell-free phase of saliva led to 15 operational taxonomic unit (OTU) groups. Furthermore, using denaturing gradient gel electrophoresis (DGGE), we compared 16S rRNA/rDNA profiles derived from liquid phases and cellular phases of saliva samples, and found positive correlations (Pearson Correlation=0.822, P<0.001) between these sample groups. These findings indicate that the liquid phase of saliva contains numerous bacterial 16S rRNA/rDNA molecules that have correlations with bacteria existing in the cellular phase.

Streptococcus mutans is one of several members of the oral indigenous biota linked with severe early childhood caries (S-ECC). Because most humans harbor S. mutans, but not all manifest disease, it has been proposed that the strains of S. mutans associated with S-ECC are genetically distinct from those found in caries-free (CF) children. The objective of this study was to identify common DNA fragments from S. mutans present in S-ECC but not in CF children. Using suppressive subtractive hybridization, we found a number of DNA fragments (biomarkers) present in 88 to 95% of the S-ECC S. mutans strains but not in CF S. mutans strains. We then applied machine learning techniques including support vector machines and neural networks to identify the biomarkers with the most predictive power for disease status, achieving a 92% accurate classification of the strains as either S-ECC or CF associated. The presence of these gene fragments in 90 to 100% of the 26 S-ECC isolates tested suggested their possible functional role in the pathogenesis of S. mutans associated with dental caries.

PURPOSE: This two armed, self-controlled, investigator blinded, clinical study tested the efficacy of an ultraviolet (UV) light toothbrush holder (Violight) to decrease toothbrush bacterial contamination. METHODS: 25 subjects were randomly assigned to control or experimental groups and received two toothbrushes for home use on either even or odd days. The control group rinsed both toothbrushes after use in cold tap water with no mechanical manipulation. The experimental group rinsed one toothbrush in cold running water while storing the other toothbrush in the Violight toothbrush holder after use. The toothbrushes were returned after 2 weeks use in sealed plastic bags and were analyzed for the number of colony forming units (CFU) of S. mutans, S. salivarius, lactobacilli, E. coli, and other coliforms, and total bacterial counts by culture. An additional analysis of the total bacterial profile was performed using denaturing gradient gel electrophoresis (DGGE). RESULTS: The Violight toothbrush holder reduced total CFU by an average of 86% (ANCOVA, P = 0.037). In addition, a tendency was noted for a reduction in total bacterial population as detected by DGGE.

Early childhood caries (ECC) is the single most common chronic childhood disease in the United States. Associated with a characteristic clinical presentation, ECC results from a culmination of risk factors, including cariogenic bacteria, inappropriate feeding practices, and social variables. It is essential that nurses become knowledgeable about ECC health promotion and prevention interventions to facilitate their efforts in everyday patient encounters and improve the overall oral health status of infants and children. This article provides an overview of ECC along with suggestions for actions that nurses can take to improve the oral health of 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

The purposes of this literature review were to: (1) review the sources of mutans streptococci (MS) colonization in children and the effect of MS levels of primary caregivers on children's MS colonization; and (2) evaluate studies examining interventions to reduce transmission of MS from caregivers to their children. Forty-six studies were reviewed. Strong evidence demonstrated that mothers are a primary source of MS colonization of their children. A few investigations showed other potential sources of children's MS colonization, notably fathers. The role of other factors influencing transmission, such as socioeconomic status (SES) and specific cultural or behavioral practices, are unclear. There were at least 12 reports of microbiological interventions to reduce transmission of MS from caregivers to their children. Even though most studies found a reduction of MS in the children and 2 showed significant caries reduction, these studies generally lock consistent findings regarding caries reduction, hove a small sample size and inadequate control groups, and lock blindness of investigators and subjects. The efficacy of microbiological approaches on the caregivers to reduce caries risk in children still needs to be established through more rigorously designed clinical trials

There are suggestions that the phylogeny of Streptococcus mutans, a member of the human indigenous biota that is transmitted mostly mother to child, might parallel the evolutionary history of its human host. The relatedness and phylogeny of plasmid-containing strains of S. mutans were examined based on chromosomal DNA fingerprints (CDF), a hypervariable region (HVR) of a 5.6-kb plasmid, the rRNA gene intergenic spacer region (IGSR), serotypes, and the genotypes of mutacin I and II. Plasmid-containing strains were studied because their genetic diversity was twice as great as that of plasmid-free strains. The CDF of S. mutans from unrelated human hosts were unique, except those from Caucasians, which were essentially identical. The evolutionary history of the IGSR, with or without the serotype and mutacin characters, clearly delineated an Asian clade. Also, a continuous association with mutacin II could be reconstructed through an evolutionary lineage with the IGSR, but not for serotype e. DNA sequences from the HVR of the plasmid produced a well-resolved phylogeny that differed from the chromosomal phylogeny, indicating that the horizontal transfer of the plasmid may have occurred multiple times. The plasmid phylogeny was more congruent with serotype e than with mutacin II evolution, suggesting a possible functional correlation. Thus, the history of this three-tiered relationship between human, bacterium, and plasmid supported both coevolution and independent evolution.

For nearly a century, lactobacilli (LB) in the oral cavity have been generally associated with dental caries. Here, we characterized the LB isolated from the saliva of 6 women with active caries using genetic-based taxonomical identification methods. From each subject, 30 isolates growing on Rogosa medium and presumed to be LB were analyzed. Of the 180 isolates, 176 were further characterized by biotyping, DNA melting points, DNA chromosomal fingerprinting, genotyping, and phylogenetic cluster assessment. We found a total of 30 unique genotypes of LB in the saliva of caries-active women, with each woman harboring between 2 and 8 distinct genotypes. Although Lactobacillus vaginalis, L. fermentum, and L. salivarius were found in 4 of 6 of the subjects, results from other studies using comparable methods show an entirely different array of LB associated with caries. These collective observations lead us to surmise that LB associated with dental caries are likely exogenous and opportunistic colonizers, arising from food or other reservoirs outside the oral cavity.