Efficacy of natural antimicrobials in toothpaste formulations against oral biofilms in vitro

AbstractObjectivesTo evaluate the antimicrobial efficacies of two toothpaste formulations containing natural antimicrobials (herbal extracts and chitosan) against oral biofilms of different composition and maturational status.MethodsBacteria from a buffer suspension or fresh saliva were adhered for 2h to a salivary conditioning film and subsequently grown for 16h. Dual-species biofilms were prepared from Actinomyces naeslundii T14V-J1 and Streptococcus oralis J22, whilst multi-species biofilms were grown from freshly collected human saliva. Biofilms were exposed to 25wt% toothpaste supernatants. A chlorhexidine-containing mouthrinse and a buffer were used as positive- and negative-controls, respectively. Antibacterial efficacy was concluded from acute killing, bacterial removal, prevention of bacterial re-deposition and continued killing during re-deposition.ResultsThe herbal- and chitosan-based supernatants showed immediate killing of oral biofilm bacteria, comparable with chlorhexidine. Moreover, exposure of a biofilm to these supernatants or chlorhexidine, yielded ongoing killing of biofilm bacteria after exposure during re-deposition of bacteria to a matured 16h biofilm, but not to a much thinner initial biofilm formed by 2h adhesion only. This suggests that thicker, more matured biofilms can absorb and release oral antimicrobials.ConclusionsSupernatants based on herbal- and chitosan-based toothpastes have comparable immediate and ongoing antibacterial efficacies as chlorhexidine. Natural antimicrobials and chlorhexidine absorb in oral biofilms which contributes to their substantive action

Quantification and Qualification of Bacteria Trapped in Chewed Gum

Chewing of gum contributes to the maintenance of oral health. Many oral diseases, including caries and periodontal disease, are caused by bacteria. However, it is unknown whether chewing of gum can remove bacteria from the oral cavity. Here, we hypothesize that chewing of gum can trap bacteria and remove them from the oral cavity. To test this hypothesis, we developed two methods to quantify numbers of bacteria trapped in chewed gum. In the first method, known numbers of bacteria were finger-chewed into gum and chewed gums were molded to standard dimensions, sonicated and plated to determine numbers of colony-forming-units incorporated, yielding calibration curves of colony-forming-units retrieved versus finger-chewed in. In a second method, calibration curves were created by finger-chewing known numbers of bacteria into gum and subsequently dissolving the gum in a mixture of chloroform and tris-ethylenediaminetetraacetic-acid (TE)-buffer. The TE-buffer was analyzed using quantitative Polymerase-Chain-Reaction (qPCR), yielding calibration curves of total numbers of bacteria versus finger-chewed in. Next, five volunteers were requested to chew gum up to 10 min after which numbers of colony-forming-units and total numbers of bacteria trapped in chewed gum were determined using the above methods. The qPCR method, involving both dead and live bacteria yielded higher numbers of retrieved bacteria than plating, involving only viable bacteria. Numbers of trapped bacteria were maximal during initial chewing after which a slow decrease over time up to 10 min was observed. Around 10(8) bacteria were detected per gum piece depending on the method and gum considered. The number of species trapped in chewed gum increased with chewing time. Trapped bacteria were clearly visualized in chewed gum using scanning-electron-microscopy. Summarizing, using novel methods to quantify and qualify oral bacteria trapped in chewed gum, the hypothesis is confirmed that chewing of gum can trap and remove bacteria from the oral cavity

Oral biofilm models for mechanical plaque removal

In vitro plaque removal studies require biofilm models that resemble in vivo dental plaque. Here, we compare contact and non-contact removal of single and dual-species biofilms as well as of biofilms grown from human whole saliva in vitro using different biofilm models. Bacteria were adhered to a salivary pellicle for 2 h or grown after adhesion for 16 h, after which, their removal was evaluated. In a contact mode, no differences were observed between the manual, rotating, or sonic brushing; and removal was on average 39%, 84%, and 95% for Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, respectively, and 90% and 54% for the dual- and multi-species biofilms, respectively. However, in a non-contact mode, rotating and sonic brushes still removed considerable numbers of bacteria (24–40%), while the manual brush as a control (5–11%) did not. Single A. naeslundii and dual-species (A. naeslundii and S. oralis) biofilms were more difficult to remove after 16 h growth than after 2 h adhesion (on average, 62% and 93% for 16- and 2-h-old biofilms, respectively), while in contrast, biofilms grown from whole saliva were easier to remove (97% after 16 h and 54% after 2 h of growth). Considering the strong adhesion of dual-species biofilms and their easier more reproducible growth compared with biofilms grown from whole saliva, dual-species biofilms of A. naeslundii and S. oralis are suggested to be preferred for use in mechanical plaque removal studies in vitro

Self-perceived mouthfeel and physico-chemical surface effects after chewing gums containing sorbitol and Magnolia bark extract

The European Food Safety Authority recognizes the contribution of sugar-free chewing gum to oral health through increased salivation, clearance of food debris, and neutralization of biofilm pH. Magnolia bark extract is a gum additive shown to reduce the prevalence of bad-breath bacteria but its effects on self-perceived mouthfeel are unknown. This paper aims to relate the effects of sorbitol-containing chewing gum, with and without Magnolia bark extract, on tooth-surface hydrophobicity and salivary-film composition with self-perceived mouthfeel. In a crossover clinical trial, volunteers chewed sorbitol-containing gum, with or without Magnolia bark extract added, three times daily during a 4-wk time period. A subset of volunteers also chewed Parafilm as a mastication control. Oral moistness and tooth smoothness were assessed using questionnaires, and intra-oral water-contact angles were measured before, immediately after, and 60min after, chewing. Simultaneously, saliva samples were collected, placed on glass slides, and the compositions of the adsorbed film were measured using X-ray photoelectron spectroscopy. Chewing of gum, regardless of whether or not it contained Magnolia bark extract, improved self-perceived mouthfeel up to 60min, concurrent with a more hydrophilic tooth surface and an increased amount of O-1s electrons bound at 532.6 eV in salivary films. Chewing of Parafilm affected neither tooth-surface hydrophobicity nor salivary-film composition. Accordingly, adsorption of sorbitol, rather than the presence of Magnolia bark extract or increased salivation, is responsible for improved self-perceived mouthfeel

Magnolia bark extract increases oral bacterial cell surface hydrophobicity and improves self-perceived breath freshness when added to chewing gum

Magnolia bark extract (MBE) is a natural product used as an anti-inflammatory, anti platelet, and chemo-preventive agent. Here, we investigate the effects of MBE on the self perceived freshness of breath evaluated in ten human volunteers, who chewed gum with and without MBE added, as a functional food. Furthermore, the effects of exposing oral bacteria to MBE on cell-surface hydrophobicity were determined and bactericidal effects of MBE in human saliva were assessed. Volunteers perceived freshness of breath similar directly after chewing a gum with or without MBE added, but 60 min after chewing MBE gum, freshness was perceived significantly better. Also, cell-surface hydrophobicity of Gram-negative, but not of Gram-positiye bacteria, increased dose-responsively after exposure to MBE, while spiking of saliva with MBE did not affect bacterial viability. Taken together, MBE gum improves self-perceived freshness of breath likely due to an increase in cell-surface hydrophobicity of Gram-negative bacteria, instead of a bactericidal mechanism. (C) 2016 Elsevier Ltd. All rights reserved

Bacteria trapped in two different types of spearmint gums chewed by human volunteers as function of time.

<p>The number of bacteria trapped in chewed gums for two types of spearmint gums as a function of the chewing time. Error bars denote the standard deviation over a group of five volunteers, with each volunteer having chewed the same gum twice for all time points. A. CFUs trapped per gum piece obtained after molding, sonication and agar-plating. B. Total number of bacteria trapped per gum piece obtained after dissolving the gum and performing qPCR.</p

Diversity of bacterial strains and species trapped in chewed gum in comparison with the bacterial diversity in the salivary microbiome and the micobiome adhering to tooth surfaces.

<p>A. The number of bands in DGGE gels in bacterial DNA obtained from pieces of chewed gum as a function of the chewing time. Error bars denote the standard deviation over a group of five volunteers. No statistically significant differences were observed. B. Percentage of species detected in the microbiome adhering to tooth surfaces or in the salivary microbiome relative to the number of species found in chewed gum (10 min of chewing) set at 100%. Error bars denote the standard deviation over a group of five volunteers. No statistically significant differences were observed. C. Percentage of species found in chewed gum based on origin, i.e. found in chewed gum and the adhering microbiome, chewed gum and the salivary microbiome and found in gum and both microbiomes. The category “other origin” indicates species that were solely found in chewed gum and below detection in the salivary and in the adhering microbiome.</p

Calibration curves for bacterial trapping in finger-chewed gums.

<p>Calibration curves for bacterial trapping after finger-chewing known numbers of bacteria into gum. Results are obtained from three independent experiments with separately cultured bacteria. Data are corrected for losses of bacteria due to adhesion to the glove-finger and during water rinsing. Error bars denote the standard deviation over triplicate experiments and linear relations are presented by the equations with their corresponding correlation coefficients. A. The number of CFUs retrieved as a function of the numbers of CFUs finger-chewed in a gum piece for the four different bacterial strains, obtained by sonication of chewed pieces of gum, molded into a standard dimension and followed by sonication and agar-plating. B. The number of threshold cycles (Ct) at fixed relative fluorescence units as a function of the total number of bacteria finger-chewed in a gum piece for the four different bacterial strains, obtained after dissolving the gum in chloroform and TE buffer and performing qPCR.</p