Improved method for rapid and accurate isolation and identification of Streptococcus mutans and Streptococcus sobrinus from human plaque samples.

Mutans streptococci (MS), specifically Streptococcus mutans (SM) and Streptococcus sobrinus (SS), are bacterial species frequently targeted for investigation due to their role in the etiology of dental caries. Differentiation of S. mutans and S. sobrinus is an essential part of exploring the role of these organisms in disease progression and the impact of the presence of either/both on a subject's caries experience. Of vital importance to the study of these organisms is an identification protocol that allows us to distinguish between the two species in an easy, accurate, and timely manner. While conducting a 5-year birth cohort study in a Northern Plains American Indian tribe, the need for a more rapid procedure for isolating and identifying high volumes of MS was recognized. We report here on the development of an accurate and rapid method for MS identification. Accuracy, ease of use, and material and time requirements for morphological differentiation on selective agar, biochemical tests, and various combinations of PCR primers were compared. The final protocol included preliminary identification based on colony morphology followed by PCR confirmation of species identification using primers targeting regions of the glucosyltransferase (gtf) genes of SM and SS. This method of isolation and identification was found to be highly accurate, more rapid than the previous methodology used, and easily learned. It resulted in more efficient use of both time and material resources.

Genotypic characterization and comparison of Streptococcus mutans in American Indian and Southeast Iowa children.

Early childhood caries is a complex health care issue that has a multifactorial etiology. One aspect of this etiology is the colonization and propagation of acidogenic bacteria at an early age. There have been several bacterial species associated with caries but 1 common species is Streptococcus mutans. Here, we describe genotypic diversity and commonality of Streptococcus mutans recovered from children representing 2 groups with similar socioeconomic demographics: a Northern Plains American Indian Tribe and a Southeast Iowa population. Forty 36-month-old American Indian children were selected from a cohort of 239 mothers and children, and forty 2- to 5-year-old children from Southeast Iowa were selected to compare the genotypic profiles of Streptococcus mutans recovered from each child's plaque. S. mutans isolates were selected from whole mouth plaque samples; DNA was extracted and amplified via AP-PCR to show specific genotype patterns. These patterns were compared with GelComparIIv6.5 gel analysis software. We found 18 distinct genotypes from 524 isolates; 13 of which were common between the 2 communities. Five genotypes were unique to only the American Indian children while the Southeast Iowa children harbored no unique genotypes. Although the American Indian children had some genotypes that were not present in the Southeast Iowa children, these were not widely distributed among the community. Furthermore, the levels of genotypic diversity and commonality were similar between the 2 populations. This study sets the groundwork for a comprehensive comparison of genotypes and caries among larger subsections of both populations.

Genotypic characterization of initial acquisition of Streptococcus mutans in American Indian children.

BACKGROUND: Severe-early childhood caries (S-ECC) is one of the most common infectious diseases in children and is prevalent in lower socio-economic populations. American Indian children suffer from the highest levels of S-ECC in the United States. Members of the mutans streptococci, Streptococcus mutans, in particular, are key etiologic agents in the development of caries. Children typically acquire S. mutans from their mothers and early acquisition is often associated with higher levels of tooth decay. METHODS: We have conducted a 5-year birth cohort study with a Northern Plains Tribe to determine the temporality and fidelity of S. mutans transmission from mother to child in addition to the genotypic diversity of S. mutans in this community. Plaque samples were collected from 239 mother/child dyads at regular intervals from birth to 36 months and S. mutans were isolated and genotyped by arbitrarily primed-polymerase chain reaction (AP-PCR). RESULTS: Here we present preliminary findings from a subset of the cohort. The focus for this paper is on initial acquisition events in the children. We identified 17 unique genotypes in 711 S. mutans isolates in our subset of 40 children, 40 mothers and 14 primary caregivers. Twelve of these genotypes were identified in more than one individual. S. mutans colonization occurred by 16 months in 57.5% of the children and early colonization was associated with higher decayed, missing and filled surface (DMFS) scores (p=0.0007). Children colonized by S. mutans shared a common genotype with their mothers 47.8% of the time. While multiple genotypes were common in adults, only 10% of children harbored multiple genotypes. CONCLUSION: These children acquire S. mutans at an earlier age than the originally described 'window of infectivity' and often, but not exclusively, from their mothers. Early acquisition is associated with both the caries status of the children and the mothers.

An in vitro comparative study determining bactericidal activity of stabilized chlorine dioxide and other oral rinses.

OBJECTIVE: The study was conducted to determine the bactericidal activity of a stabilized chlorine dioxide oral rinse (CloSYS Oral Rinse) compared to products currently available on the market. METHODS: Oral bacteria associated with gingivitis and periodontitis were exposed to rinses for one minute and five minutes. The numbers of colony forming units per milliliter (CFU/ml) were measured prior to and following exposure to determine the bactericidal activity. RESULTS: As expected, Listerine and Crest Pro-Health demonstrated complete kill on all bacteria exposed within one minute. Breath Rx exhibited the weakest levels of bactericidal effects overall. CloSYS and chlorhexidine rinses proved identical 100% kills against the periodontal pathogens at five minutes; in some cases, CloSYS oral rinse achieved a higher kill at the one-minute mark over the chlorhexidine rinse. CONCLUSION: The results demonstrated that CloSYS Oral Rinse has potential for providing a therapeutic benefit, making it an attractive option to induce compliance in patients concerned about taste and tooth discoloration during oral health therapy.

Bactericidal activity of stabilized chlorine dioxide as an endodontic irrigant in a polymicrobial biofilm tooth model system.

INTRODUCTION: The purpose of this study was to determine bactericidal efficacy of 0.04% stabilized chlorine dioxide, 3% sodium hypochlorite, 2% chlorhexidine gluconate, and sterile distilled water in a polymicrobial biofilm model. METHODS: Roots of 35 permanent bovine incisors had pulps extirpated, and their apical size and root length were standardized. Teeth were coated with mucin, inoculated with standardized suspensions of Streptococcus sanguinis, Actinomyces viscosus, Fusobacterium nucleatum, Peptostreptococcus micros, and Prevotella nigrescens and incubated anaerobically. Teeth were randomly divided into four groups and rinsed for 3 minutes with 15 mL of irrigant. Biofilms were harvested and spiral-plated on selective media. Numbers of bacteria in the harvested biofilms was determined via the standard spiral-plating methodology. Treatment groups were evaluated using the nonparametric Kruskal-Wallis procedure. Pair-wise comparisons among the four groups and five organisms were made using the Wilcoxon-Mann-Whitney procedure. Adjustments for multiple comparisons were made using the Holm method with p < 0.05. RESULTS: Results provide strong evidence of a significant difference in levels of bactericidal activity associated with the type of irrigant for all five bacterial species tested. Levels of bactericidal activity were significantly higher for the NaOCl group than for the stabilized chlorine dioxide (ClO2) group for S. sanguinis, A. viscosus, and P. nigrescens. Results for F. nucleatum and P. micros were not significant after the adjustment for multiple comparisons. CONCLUSIONS: The triple-inoculation bovine tooth model system is a robust, consistent, and reproducible model system to study polymicrobial biofilms. It should be used with the knowledge expansion of biofilm structure and function as well as the development of antimicrobial protocols.