Biofilm reduction and staining potential of a 0.05% chlorhexidine rinse containing essential oils.

OBJECTIVES: To assess the biofilm reduction and discolouration potential of a new 0.05% chlorhexidine (CHX) digluconate solution, containing additional essential oil and alcohol components, compared with that of standard control CHX solutions (0.05% and 0.2% CHX). METHODS: The potential to reduce total viable counts of growing mixed microbial populations was examined using the Zurich biofilm model. Biofilms were created on sterile pellicle-coated hydroxyapatite discs and exposed to test substances at different time points. After 64.5 h, mean colony-forming units and SDs were determined. Colour change measurements using light reflection analysis were carried out on saliva preconditioned bovine dentin and enamel samples, as well as on composite and glass ceramic restorative materials, after successive immersions in a standardized tea brew and the CHX solutions. RESULTS: The test solution was able to reduce biofilm formation by 3 log steps compared with a negative (water) control. This was significantly less effective than the standard control CHX solutions, which reduced viable counts by 6 log steps. Both the test and control solutions exhibited staining on all surfaces. Staining was most pronounced on dentin, followed by enamel and to a significantly lesser degree on the restorative materials. Furthermore, the staining caused by the test solution on these restorative materials was generally lower than that caused by the control solutions. CONCLUSIONS: The test solution exhibited an antimicrobial activity. The composition, however, seems to hamper its effectiveness. Accordingly, it produced statistically significant, although by trend less, staining on restorative materials.

An in vitro oral biofilm model for comparing the efficacy of antimicrobial mouthrinses.

The ability of commercial mouthrinses to reduce total viable counts of mixed microbial populations was examined using a previously developed in vitro model of supragingival plaque. Exploratory experiments aimed at fine-tuning the model indicated that optimal correspondence between in vitro and clinical results for chlorhexidine-containing formulations were obtained at a saliva:medium ratio of 70:30 (v/v); moreover, expanding the microbial population from 5 bacterial species to 5 bacterial species + Candida albicans had no noticeable impact on overall results. The efficacies of 12 different mouthrinse proprietary products containing chlorhexidine, hexetidine, octenidine, Triclosan, plant extracts, or aminefluoride/stannous fluoride vis-à-vis biofilm clearance were compared. All mouthrinses promoted a statistically significant reduction in microbial load compared to distilled water. The herbal- and phenolic-based products were substantially less effective than most chlorhexidine-containing mouthrinses, or mouthrinses containing hexetidine or octenidine. No significant difference between the plaque-clearing plaque-clearing abilities of Listerine and Meridol was observed. This polyspecies biofilm model can be a valuable tool for preclinical testing of antiplaque formulations, particularly during the product development stage.

Treponema lecithinolyticum sp. nov., a small saccharolytic spirochaete with phospholipase A and C activities associated with periodontal diseases.

Strong phospholipase A (PLA) and phospholipase C (PLC) activities as potential virulence factors are the outstanding characteristics of eight strains of small oral spirochaetes isolated from deep periodontal lesions. By qualitative dot-blot DNA-DNA hybridization and 16S rDNA sequence comparison, these spirochaetes form a distinct phylogenetic group, with Treponema maltophilum as its closest cultivable relative. Growth of these treponemes, cells of which contain two endoflagella, one at each pole, was autoinhibited by the PLA-mediated production of lysolecithin unless medium OMIZ-Pat was prepared without lecithin. N-Acetylglucosamine was essential and D-ribose was stimulatory for growth. All isolates were growth-inhibited when 1% foetal calf serum was added to the medium. Growth on agar plates supplemented with human erythrocytes produced haemolysis. In addition to PLA and PLC, the new isolates displayed strong activities of alkaline and acid phosphatases, beta-galactosidase, beta-glucuronidase, N-acetyl-beta-glucosaminidase and sialidase, intermediate activities of C4- and C8-esterases, naphthol phosphohydrolase and alpha-fucosidase and a distinctive 30 kDa antigen detectable on Western blots. This phenotypically and genotypically homogeneous group is proposed as a novel species, Treponema lecithinolyticum sp. nov., with isolate OMZ 684T designated as the type strain. A molecular epidemiological analysis using a T. lecithinolyticum-specific probe showed this organism to be associated with affected sites when compared with unaffected sites of periodontitis patients. This association was more pronounced in patients with rapidly progressive periodontitis than in those with adult periodontitis.

Powdered milk micellar casein prevents oral colonization by Streptococcus sobrinus and dental caries in rats: a basis for the caries-protective effect of dairy products.

Three animal studies were performed to investigate the influence of the macromolecular structure of milk casein on caries incidence and the possible ecological changes of the oral microbiota by such casein fractions. Towards this end, rats were infected with mixed bacterial suspensions of Streptococcus sobrinus OMZ 176 and Actinomyces viscosus Ny1. Various milk protein fractions were incorporated into carefully balanced powdered cariogenic diets to constitute the sole major protein component. Diets containing micellar casein had a pronounced and highly significant effect on almost all clinical and microbiological parameters examined. Both the formation of advanced dentinal fissure (B) and smooth surface (E) caries lesions was inhibited by diets containing micellar casein; this caries-inhibiting effect appeared to be due mainly to modifications within the plaque microbiota. The proportion of S. sobrinus in the oral cavity of rats was reduced (73-80%) by micellar casein-containing preparations, whereas the A. viscosus population was increased. Both these microbiological parameters were always negatively correlated. This appears to be the first example of a food component other than dietary sugars, selectively modifying the composition of the dental plaque microbiota of rats in such a way as to reduce its pathogenic potential. It also demonstrates the importance of establishing a molecular basis for the role of food components, which prove to be beneficial to oral health.

Treponema amylovorum sp. nov., a saccharolytic spirochete of medium size isolated from an advanced human periodontal lesion.

A highly motile, medium-size, saccharolytic spirochete was isolated from an advanced human periodontal lesion in medium OMIZ-Pat supplemented with 1% human serum. The growth of this organism is dependent on either glucose, maltose, starch, or glycogen. The cells contain six endoflagella, three per pole, which overlap in the central region of the cell body. On the basis of its cell morphology and enzyme activities, as well as its sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein and antigen profiles, this organism is clearly distinct from all previously cultured spirochetes. The presence of a novel species is supported by the 16S rRNA sequence of this organism, which places it in phylotype 19 of Choi et al. (B. K. Choi, B. J. Paster, F. E. Dewhirst, and U. B. Göbel, Infect. Immun. 62:1889-1895, 1994). The only isolate, strain HA2P, is designated the type strain of a novel species, for which we propose the name Treponema amylovorum.

Caries and plaque inhibition in rats by five topically applied dentifrices.

The effectiveness of 5 different brands of dentifrices on caries and plaque were tested in rats in 2 experiments. 10 litters of rats each were infected with Streptococcus sobrinus OMZ-176 and Actinomyces viscosus Ny1. The rats were offered the diet 2000a containing either 40% of sucrose (study I) or 10% of sucrose (study II). The following products were tested topically: (1) H2O, (negative control); (2) chlorhexidine diacetate, (positive control); (3) Parodontax NF; (4) Meridol; (5) Colgate Total; (6) Mentadent C; (7) Dent Xpress. Comparisons with the water control in study I showed that plaque extent was decreased by chlorhexidine diacetate, Dent Xpress and by Mentadent C only. All fluoride-containing dentifrices tested reduced caries. In study II, comparisons with the water control showed that plaque extent was reduced by chlorhexidine diacetate, Parodontax, Meridol, Dent Xpress and by Mentadent C. All dentifrices reduced caries, however, the incidence was low. These 2 studies confirm that fluorides incorporated in dentifrices decrease dental caries in the rat. Differences in the effectiveness of caries inhibition between the 5 dentifrices could not be explained either by the form of fluoride utilized or by the antimicrobials added. Rather, the performance of a given dentifrice depended upon the concerted action of the particular ingredients. It appears that the effect of antiplaque agents should be evaluated in the rat model using a diet containing sucrose at a low level.

Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci.

The protective effects of milk and milk products against dental caries have been demonstrated in many animal studies. We have shown that this effect was mediated by micellar casein or caseinopeptide derivatives. A reduction in the Streptococcus sobrinus population in the oral microbiota of animals fed diets supplemented with these milk components was consistently observed. A possible explanation for these findings is that milk components are incorporated into the salivary pellicle, thereby reducing the adherence of S. sobrinus. This hypothesis was tested in vitro by the incubation of bovine enamel discs with unstimulated saliva. The resulting pellicle was washed and incubated with caseinoglycomacropeptide (CGMP) and/or caseinophosphopeptide (CPP) labeled with 17- and 12-nm gold particles. All samples were prepared for electron microscopy by high-pressure freezing followed by freeze-substitution. It was demonstrated by high-resolution scanning electron microscopy with back-scattered electron imaging, as well as by transmission electron microscopy, that both peptides were incorporated into the pellicle in exchange for albumin, confirming previous findings. This protein was identified with a mouse anti-human serum albumin followed by goat anti-mouse IgG labeled with 25-nm gold particles. Incorporation of CGMP and/or CPP into salivary pellicles reduced the adherence of both S. sobrinus and S. mutans significantly. It is suggested that the calcium and phosphate-rich micellar casein or caseinopeptides are incorporated into the pellicle. The resulting ecological shifts, together with the increased remineralization potential of this biofilm, may explain its modified cariogenic potential.

Human root caries: microbiota in plaque covering sound, carious and arrested carious root surfaces.

The plaque microbiota covering sound or carious root surfaces were studied and compared with that covering arrested root caries lesions. From each of these categories five extracted teeth were examined. The experimental design of the study allowed us to relate the qualitative and quantitative microbial composition to the degree of integrity of the root surface. Plaque was sampled by a newly developed 'mowing' technique. Plaque samples were cultured anaerobically on nonselective Columbia blood agar plates supplemented with 5% hemolyzed human blood and on media selective for Lactobacillus spp. and streptococci of the mutans group. The cultivable microbiota were quantitatively speciated using Rapid ID 32A, Rapid ID 32 Strep, API 20 Strep, API ZYM, and API 50 CH tests and SDS-PAG electrophoresis. Regardless of the state of mineralization, the microbiota on all surfaces resembled marginal plaque associated with gingivitis. In addition to the gram-positive predominant facultative anaerobic genera Streptococcus, Staphylococcus, Lactobacillus and Actinomyces, gram-negative anaerobes, predominantly Bacteroides, Prevotella, Selenomonas, Fusobacterium, Leptotrichia, and Capnocytophaga, showed the highest isolation frequencies. On all surfaces Actinomyces spp. predominated, with streptococci and lactobacilli forming a minor part of the microbiota. With respect to the detected proportions of anaerobes, microaerophiles, Actinomyces naeslundii, Prevotella buccae and Selenomonas dianae, significant differences were observed between the three categories of root surfaces. The total CFU's on both caries-free and caries-active surfaces were significantly higher than on arrested lesions. In general, the results support a polymicrobial etiology for caries initiation on root surfaces, with A. naeslundii, Capnocytophaga spp., and Prevotella spp. making specific contributions to the processes of cementum and dentin breakdown.

The antimicrobial activity of essential oils and essential oil components towards oral bacteria.

A method for reproducibly determining minimal inhibitory concentrations and minimal bactericidal concentrations of plant extracts towards fastidiously and facultatively anaerobic oral bacteria, predicated upon measurements of optical densities in microtitre plate wells, was devised. The antimicrobial properties of some botanical oils were surveyed; of these, Australian tea tree oil, peppermint oil, and sage oil proved to be the most potent essential oils, whereas thymol and eugenol were potent essential oil components.

[Microbiologic in vitro comparison of plaque-inhibiting mouthwashes]. / Mikrobiologischer In-vitro-Vergleich Plaquehemmender Mundspülmittel.

The colonization on rat molars of S. sobrinus OMZ 176, S. mutans OMZ 376, or a combination S. sanguis OMZ 9 and S. sobrinus OMZ 176 after a short exposition to various fluoride solutions and disinfectants was tested in vitro. The test solutions included Act, Candida, Veadent, sodium fluoride, amine fluoride, stannous fluoride, zinc fluoride hexetidine, stannous fluoride/amine fluoride solutions, chlorhexidine and water. The sterilized rat molars were immersed in the above test solutions for 60 seconds, then incubated with streptococci in broth for 8 hours, again dipped into the same test solutions for 60 seconds and reincubated for an additional 30 hours. The streptococcal suspension contained 14C labelled sucrose solutions. The deposits on the molars were dissolved in 6N potassium hydroxide during 16 hours. Finally, the beta rays emitted by the dissolved radiolabelled suspension were counted using a scintillation counter. The sodium fluoride containing solutions exerted no or a very limited effect on the bacterial deposits. In contrast to the other test solutions, sanguinaria extract (Veadent mouth rinse) mildly inhibited the S. sobrinus and S. mutans deposits, but plaque formation by the combination of streptococci was not hampered. Zinc fluoride/hexetidine, amine fluoride and stannous fluoride/amine fluoride solutions had a distinct and significant inhibitory effect on S. sobrinus and S. mutans deposits, but only a weak effect when mixed cultures were used for plaque formation. Chlorhexidine significantly inhibited the deposits of the three bacterial strains used in these experiments.