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Research Studies - Indiana University Microbiological Testing
"Inhibition of growth active would reduce the ability of any of these microbes to adhere to salivary pellicle-coated tooth surfaces. Overall the data provided indications that the extract was more effective at inhibition of S. sanguis and A. viscosus growth than the other bacteria indicating that there was no significant affect on overall total bacteria numbers. This provides evidence of anti-plaque activity of the chlorophyll test product and may suggest that is has potent effects on oral hygiene in addition to its well demonstrated anti-malodor activity."
Aim
This study was conducted to determine if the provided anti-microbial tablets have growth inhibitory activity against six representative normal oral bacteria.
Introduction
The normal human oral cavity contains at least 300 different bacterial species and many of these species are also present in the oral flora of companion animals. The great majority of these microorganisms are found in dental plaque. Among these plaque microorganisms are streptococci (early and late plaque formers and major causative agents of dental caries), actinomyces (involved in plaque formation and may have a role in caries formation), lactobacilli (late plaque colonizers and play a role in synthesizing organic acids for demineralization of enamel), staphylococci (components of plaque) and black-pigmented bacteria that produce volatile sulfur compounds (VSC; hydrogen sulfide, methyl mercaptan and dimethyl sulfide) as a result of sulfur-containing amino acid (cysteine and cystine) metabolism and are the primary causes of malodor.
Experimental Design
Inhibition of bacterial growth. The bacteria were cultured in Mueller-Hinton (MH) broth in a 5% CO2 incubator at 37oC for 24 h. 105 cells/ml were inoculated into 1 ml of MH broth and dispensed into 96 well microtiter plates (180 ul in each well). The tablet material was dissolved in deionized H2O at the stated concentration and added to the wells (20 ul); the plates were then incubated for 18 h. The minimum inhibitory concentration (MIC) was determined by serial 2-fold dilutions of the product in the presence and in the absence of the material. MIC is defined as the concentration of the product that completely inhibits growth during the 18 h of incubation. Growth was assayed with a microplate reader (Molecular Devices, Menlo Park, CA) by following absorbance at 540 nm.
Test Specimens: Tablet powder product.
Control Specimens: MH broth not containing antimicrobial powder (negative control); and CHX at 0.12% (as a positive control).
Results
Inhibition of bacterial growth: The ability of antimicrobial agents to inhibit S. mutans growth is critical to preventing caries activity. Inhibition of S. mutans bacterial growth is indicative of a strong ability to provide anti-caries activity. In addition, it is important to ascertain the ability of an antimicrobial agent to inhibit plaque formation particularly by early plaque colonizing organisms such as S. sanguis and A. viscosus. However, it is also important that antimicrobial agents do not provide anti-bacterial effects against other normal oral biofilm agents. Other important oral biofilm species examined in this study included Lactobacci caseii, S. salivarius and P. nigrescens. P. nigrescens was used in place of P. intermedius as it grew better and is also a strong VSC and odor-producing oral microbe. S. salivarius was used in place of S. aureus as it is a much more common oral bacterium. Inhibition of growth activity would reduce the ability of any of these microbes to adhere to salivary pellicle-coated tooth surfaces.
The chlorophyll extract (test product) was assessed for growth inhibition of 6 different oral bacteria using a spectrophotometric assay. As noted above, the assay consisted of incubation of cultures of S. mutans, S. sanguis, A. viscosus, L. caseii, S. salivarius or P. nigrescens with the extract for 24 hours and the measurement of growth using spectrophotometry at 540 nm. Controls consisted of bacteria with PBS or media only with the product. The following figure is representative of a typical microtiter plate used in this study.
The results are shown in the following tables each of which contains a graphical presentation of the results observed with that particular microorganism. Briefly, the extract inhibited S. sanguis and A. viscosus growth (seeTables 1 and 2). The diluted extract (as low as 10 ug/ml) significantly inhibited growth of both S. sanguis and A. viscosus.
However, the extract did not inhibit Lactobacci caseii, S. salivarius, S. mutans and P. nigrescens at any concentration (see Tables 3-6). The CHX control at 0.12% totally inhibited the growth of all bacteria.
The data from the P. nigrescens cultures during the initial study (Table 5) was considered to be incomplete as the cells did not grow properly probably due to then use of a fewer number of hours of incubation than is required for this species as it is slower growing than the others. We repeated this experiment with a longer growing period and these data are included as Tables 6 and 7.
Conclusions
Overall, the data provided indications that the extract was more effective at inhibition of S. sanguis and A. viscosus growth than the other bacteria; whether or not this effect results in a significant reduction in the overall total bacterial numbers remains to be seen. These results provide evidence of anti-plaque activity of the chlorophyll test product and may suggest that it has potent effects on oral hygiene in addition to it's well-demonstrated anti-malodor activity.
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