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Table of Contents
1. A quantitative approach to the effectiveness of ozone against microbiota organisms colonizing toothbrushes....................................................................................................................................................

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A quantitative approach to the effectiveness of ozone against microbiota organisms colonizing toothbrushes
Author: Bezirtzoglou, Eugenia; Cretoiu, Silvia-Mariana; Moldoveanu, Mirela; Alexopoulos, Athanasios; Lazar, Veronica; Nakou, Mela Publication info: Journal of Dentistry 36. 8 (Aug 2008): 600-605. ProQuest document link Abstract: Toothbrushes are rapidly contaminated with different microorganisms, which colonize the oral cavity and interdental spaces. This can represent a possible cause of infection or reinfection. In this study, the ozone experimental effect upon toothbrushes microflora was estimated microbiologically before and after saturation with ozone gas. Fifty used toothbrushes coming from children and adults were entered our study. Microorganisms were enumerated and identified. Bristles from each brush were soaked in ozone saturated PBS solution for 5, 10, 15, 20 and 30min and the total microbial population was reassessed. Counts of microorganisms isolated per brush varied between 102 and 107 CFU. Candida albicans was present in used toothbrushes. No obligate anaerobes were isolated. Members of Streptococcaceae family were regularly found (65.2%) belonging to the following species: Streptococcus pyogenes , S. mutans , S. mitis , S. oralis , S. sobrinus , S. viridans , S. salivarius , S. sanguis , Aerococcus viridans . A. viridans and S. mutans were more frequently isolated on children toothbrushes while Staphylococcus aureus and S. epidermidis were found on adults brushes. Escherichia coli, Pseudomonas sp. and Enterococcus sp., were also recovered. We found that the ozone treatment decreased gradually the microbial load. However, a bacterial re-growth was effective following short ozonation period. Decontamination was complete after an extended exposure to ozone for 30min. Ozone application was found to remove the toothbrushes bristles microbiota following conventional brushing. Maximum decontamination efficacy of ozone treatment was observed after 30min while exposure for short time periods seems to be inefficient which probably reflect the low dose of ozone used in this study. Full Text: Species Children group Streptococcus pyogenes 20 40 1.000 20 Streptococcus oralisa 40 56 0.007 68 Streptococcus salivarius Prevalence (%) Adult group 28 0.289 44 Streptococcus mitis 14 60 0.008 16 Streptococcus viridansa 52 20 Significance (p ) Total 12 Streptococcus mutans 42 8 0.417 20 Streptococcus sobrinusa 36 36 0.046 32

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26 8 0.037 24 E. faecalis, E. faecium 4 16 0.007 60 Lactobacillus sp. 42 8 0.417 28 Candida albicans

0.520 36 Aerococcus viridansa 46 0 0.489 56 Staphylococcus epidermidisa 42 40 1.000 20 Pseudomonas aeruginosaa 16 52

Streptococcus sanguisa 22 68 0.004 8 Staphylococcus aureusa 36 24 0.020 44 Esherichia coli 14 4 0.048 36

Table 1 - Prevalence of the normal microbiota organisms found in used toothbrushes originated from children (n : 25, mean age: 8 years) and adults (n : 25, mean age: 40 years) 1 Introduction Toothbrushing is the most common method of personal hygiene for removal of dental plaque and promoting oral health. The prevention of caries and the control of the oral hygiene, focus mainly on the mechanical and/or chemical biofilm removal.1 Toothbrushes upon use, rapidly become contaminated by a plethora of oral microorganisms including bacteria, viruses and fungi.1,2 Bacteria isolated from conventional brushes showed increasing rates when compared to antibacterial brushes used by the same individuals. However, not statistically significant difference was demonstrated.1-3 The majority of general dental practitioners together with manufacturing companies recommend to their patients to replace their toothbrushes every 2-3 months. The inference of these recommendations is that a toothbrush used for such a period, may be less effective in plaque removal than a brand new brush. 4 The toothbrushes are considered to be potential sources of oral, blood and systemic infection or reinfection.1,5,6 They may act as a reservoir of opportunistic pathogens including Staphylococcus and Pseudomonas -like organisms.1 Staphylococci, among other potentially oral pathogens, may be transient members of the oral microbiota and the importance of toothbrushes as a potential source of staphylococcal infection has not been extensively explored.6,7 Staphylococci were isolated from the floor of the palate, tongue and crevicular fluid of patients with periodontal disease as well as from healthy controls.6-8 Their numbers are usually low in healthy individuals oral microbiota.6,7 Staphylococci found on toothbrushes, may originate not only from the oral cavity but also from the environment where brushes are stored.8 Indeed, toothbrushes may harbour pathogens from patients having infections which may therefore serve as vehicles for reinfection by these organisms. Moreover, toothbrushes may harbour pathogens from the external environment which may cause infection. This contamination presents the possibility of reinfection of a patient by used toothbrushes harbouring the pathogenic microorganisms. Aware of the possible hazards for a mouth infection with bacteria originated from a toothbrush, dentists often recommend various methods for sanitizing brushes. Soaking in alcohol was the first recommended procedure for toothbrush disinfection in 1920s.10 Other methods for sanitation were proposed such as exposing toothbrushes to sunlight, salt, formaldehyde gas, ultraviolet light, spraying with antimicrobial solutions, use of a microwave oven and washing of the toothbrush in a dishwater. 23 February 2013 Page 2 of 9 ProQuest

1,9,10 Some authors investigated the impact of the accumulated toothpaste debris on toothbrush's microbial contamination, while the efficacy of the incorporation of an antimicrobial substance in the toothbrush to avoid bacterial contamination was assessed in other studies. 11,12 Bacteria isolated from conventional brushes showed increasing colonization rates when compared to antibacterial brushes used by the same individuals. However, non-statistically significant difference was demonstrated.1-3 Ozone (O3) on microorganisms acts against different cellular substances including proteins lipids, peptidoglycans, enzymes and virus capsids,13 but effective microbial inactivation seems to be dependent on several environmental factors.14,15 The oxidizing mechanisms of ozone may involve direct reactions of molecular ozone and free radical-mediated destruction. The main advantage of ozone usage consists of its superiority compared to other disinfectants like chlorine, for three main reasons; firstly, is reporting to be 1.5 times stronger than chlorine and acting 3000 times faster without producing harmful decomposition products. 16 In 1995, ozone was declared as Generally Recognized as Safe (GRAS) by the FDA for treatment of bottled water. Finally, its application was extended as GRAS to food processing a few years later.17 In dentistry, the efficacy of ozone on oral sanitation procedures or as a given therapy on a multispecies oral biofilm has been tested in vitro , with promising results.13,18 The aim of this study was to evaluate the microbial contamination of used toothbrushes and the susceptibility of the detached microbial load harboured on these brushes to ozone. 2 Materials and methods 2.1 Sampling preparation Fifty manual toothbrushes were investigated in this study. Toothbrushes were used by the owners for a period of 1 to over 3 months and none of them were sanitized in any way or had any antibacterial properties. Brushes were divided in two major groups: those used by children (7-10 years of age) and those by adults (20-70 years of age). Volunteers were asked to bring their brushes to the laboratory in plastic sterilized bags and they had their teeth brushed by means of the classic mechanical technique. After use, each toothbrush was rinsed under running tap water and processed immediately. The bristles from each brush were aseptically trimmed off and transferred into a tube with 9ml sterile Phosphate Buffered Saline (PBS) solution. The content was vigorously vortexed for 3min in order for the non-strongly adherent cells to be detached. Afterwards, this same content was vigorously shaken in a cold bath for 10min,in order for the sessille cells to be detached.19 Moreover, serial tenfold dilutions in PBS were prepared for microbiological evaluation. 2.2 Microbiological analysis One hundred microliters from each dilution were plated onto different selective and non-selective growth media as follows: Mannitol salt agar (Oxoid Ltd., Basingstoke, Hampshire, UK) was used to quantitatively detect Staphylococcus sp. Representative colonies were picked and subjected to Gram-stain, catalase and coagulase tests (Staphylex, Oxoid Ltd.). MacConkey agar (Oxoid Ltd.) was used to enumerate presumptively Enterobacteriaceae. Characteristic colonies appeared within 24h were confirmed by the IMVC test (Indole, Methyl Red, Voges-Proskauer, Citrate). Moreover, the following media were used: MRS agar (Oxoid Ltd.) for the recovery of Lactobacillus, Mitis salivarius agar (Becton Dickinson Co., USA) for total Streptococci and Columbia agar supplemented with blood (5%) for the aerobes and facultative anaerobes. Finally, Sabouraud Dextrose agar (Oxoid Ltd.) was used for cultivation of yeasts and moulds. Incubation of the plates was performed accordingly to the involved medium aerobically for 24h at 37C or anaerobically for at least 5 days at 37C. Identification of the strains was based upon their colony and cell morphology, respiration requirements and biochemical characteristics. These included microscopic examination of Gram-stained cells; motility, catalase and oxidase reaction and growth in anaerobic conditions. Finally, biochemical tests by the aid of commercial kits (API from BioMrieux S.A., Marcy l'Etoile, France and Microgen ID from Microgen Bioproducts Ltd., UK), were performed for further identification of the isolates to the species level. 2.3 Ozone sanitation trials In the second phase of the study, the decimal dilutions of our samples (T : 20C) were ozone saturated by using a commercial ozone gas generator (Air &Water System PC1325, USA) through a sterilized microdiffuser and samples for microbiological analysis were drawn after 5, 10, 15, 20 and 30min. 100l from the ozonated suspensions were plated on all previous growth media and incubated accordingly. A quantitative evaluation of the microbial growth achieved by inoculation of 10 -1 to 10-7 serial dilutions from each sample 23 February 2013 Page 3 of 9 ProQuest

onto Columbia blood agar. Ozone concentrations were estimated photometricaly with the indigo method by using a commercially available kit (Hach Co., Loveland Co., USA). Final ozone concentration succeeded is reported to 3-3.5ppm. A quantitative evaluation of the microbial growth was achieved after the end of the ozonation. The re-growth profile of bacteria was estimated by total counting of the recovered bacteria (CFU/ml). 2.4 Statistical analysis Differences in the occurrence of microbial species between each age group were examined by using Fisher's exact test. Differences in microbial counts before and after ozonation were estimated with Mann-Whitney z -test after logarithmic transformation of counts. Linear regression models for the inactivation of microbes over time were also estimated. All statistical analyses were performed with the SPSS v13 statistical software package (SPSS Inc., USA) at 95% level of significance. 3 Results The results of the microbiota's qualitative assessment from all samples are shown in Table 1. A total of 16 different species of bacteria were identified from adults and children toothbrushes. Overall, most abundant were the Streptococcus genus with an occurrence of 31.5%, (average count: 4.5log CFU) equally distributed among children (32%) and adults (31%) and with typical representatives Streptococcus pyogenes , S. mutans , S. mitis , S. oralis , S. sobrinus , S. viridans , S. salivarius and S. sanguis . From the above strains, S. oralis and S. viridans were those mostly isolated (60% and 68%, respectively) from our samples. In children, except Streptococcus sp., Aerococcus viridans (68%), Candida albicans (52%) and Lactobacillus sp. (40%) were also recovered, while Staphylococci (S. aureus and S. epidermidis ) were dominant over the adult group. Less abundant species were Escherichia coli , S. faecalis , S. faecium and Pseudomonas aeruginosa (average count less than 3logs) and particularly in children's brushes. The quantitative evaluation of the microbiota revealed that the total number of microorganisms ranged between 102 and 106 CFU per toothbrush. The most evident bacterial growth was observed on Columbia blood agar with the maximum growth estimated on 6.9107 for the children and 6.7107 for adults, indicating no actual differences. Total counts for C. albicans on Sabouraud Dextrose media was 1.87102 for the children and 1.12102 for adults. A 48% from children brushes and 64% from adults was free from C. albicans colonization. Fisher's exact test revealed statistical significant differences regarding the occurrence of the various species between the two groups in almost half of the verified strains. As can be seen from Table 1, three of those species (S. oralis , S. sobrinus and A. viridans ) were more frequent in children while, five species (S. viridans , S. sanguis , S. aureus , S. epidermidis and P. aeruginosa ) were more frequent in adults, indicating a variable pattern of microflora between the two groups. Concerning the ozone experiments, in both age groups, only after 10min of ozonation (and in the consequent exposures) statistical significant differences with the initial microbial concentrations were observed (Mann-Whitney z : -4.77 and -4.96, p <0.05). After 5min of exposure a slightly and insignificant decrease of 0.5logs was recorded. A full sanitation efficiency was observed after 30min of ozonation, since no viable counts were observed for those samples. Linear fitted models for both groups indicated a decrease in total counts by -0.17 to -0.18logs for every 5min of ozonation (Fig. 1). This similarity of slopes did not disrupted when controlling for the age of the subjects, or the period the toothbrushes were used. 4 Discussion Based on our results, used toothbrushes harbouring an increased number of aerobic and facultative anaerobic bacterial species. This finding is in accordance with the results of previous studies1,3,6-8 and indicates that an actual risk of recolonization exists after each brushing. That risk is of particular importance for periodontal patients under therapy.3 The profile of total microbiota isolated and identified in our samples included the species: Streptococcus sp., Aerococcus sp., Enterococcus sp., Staphylococcus sp., Lactobacillus sp., Pseudomonas sp., E. coli , and Candida . It was observed that the most abundant strains were those of the Streptococcus genre like S. pyogenes , S. mutans , S. mitis , S. oralis , S. sobrinus , S. viridans , S. salivarius , S. sanguis and A. viridans . A part of those species isolated both from children and adults and their occurrence probably resulted by disruption of bacterial plaque after tooth brushing. From Micrococacceae family, two species were identified: S. aureus and S. epidermidis usual commensals on skin, known as potential pathogens and capable of biofilm formation.12,13 Both isolated species of E. faecium and E. faecalis were recovered from adult's brushes and thus, indicating a dental superinfection, especially 23 February 2013 Page 4 of 9 ProQuest

associated with elderly people. A. viridans and some of the Streptococcus sp., were more frequently isolated from children brushes in contrast to S. aureus and S. epidermidis originating from adult's brushes. The contamination with those non-fastidious bacteria has its' origin in the oral cavity as S. aureus is frequently isolated from periodontal patients but also from manual handling of the toothbrushes. In general, the oral Streptococci group was significantly present in the microbiota isolated from all toothbrushes. P. aeruginosa and E. coli were present on both groups, probably as the result of the storage conditions (bathrooms with increased humidity, increased temperature and/or untreated tap water). S. mutans and species from Lactobacillus genre were present on different frequencies in children brushes and in adults; which indicates somewhat different dental conditions. No periodontopathogens like A. actinomycetemcomitans , P. gingivalis or F. nucleatum were recovered, meaning that among our subjects none with apparent oral infection occurred. However, bacterial variability of potential pathogens recovered on toothbrushes was considerable. Results from ozone sanitation experiments showed that exposure of the brushes bristles to ozonated Phosphate Buffered Saline solution for 5, 10, 15 and 20min did not result in a radical effect, as slight microbial re-growth afterwards was evident. Only after 30min of exposure a complete sanitation was observed. A possible explanation is the effect of ozone upon formed microbial assemblage on brushes. It is known the fact that many bacterial species are responsible of biofilm formation on different surfaces like toothbrushes and ozone can disrupt the biofilm structure. 16,17,20 Ozone has been used extensively to in vitro experiments for the deactivation of oral microorganisms and dental plaque treatment17 with remarkable results. However, those investigators reported a complete bactericidal activity after 10s of exposure to 4mg/l concentrations, which differs substantially from our results of 30min even thought, in our experiments ozone concentration was slightly lower (3-3.5mg/l). Delayed sanitation times for toothbrushes, have been reported by other investigators, which experimented with different agents. Caudry et al., 11 reported a complete disinfection after 20min of soaking in antiseptic, while Nelson et al.,20 after 20h. It is assumed that this delayed effect on toothbrushes decontamination could be the result of a stiffer biofilm formed on the bristles by the microbes, in combination with a protective coating offered to them by the accumulated toothpaste debris. Furthermore, our results also showed a quantitative stimulation (rebound) effect of bacterial growth after exposure to the ozone for short time periods (i.e. after 5min) and only after the biofilm is already detached and viability of the cells is affected (i.e. after 30min exposure) no microbial growth was observed. Another possible explanation should be the early bacteriostatic effect of ozone upon microorganisms growth. Extensive ozonation for more than 30min results at a relevant bactericidal effect. Comparing to our original baseline population, we evaluate the qualitative bacterial profile involved in this rebound effect (re-growth), which collects aerobic or facultative anaerobic bacteria. No strict anaerobic bacteria were found. The direct oxidative effect of ozone upon the anaerobic microflora is obvious. However, ozone seems like to act as an oxygen donator for the aerobic and facultative anaerobic bacteria which use it for their re-growth. Extensive exposure to ozone showed a complete bactericidal effect upon all microorganisms involved. It is of importance to state here the reductant capacity of the chosen media (as Sodium chloride, Potassium chloride, Disodium hydrogen phosphate, Potassium dihydrogen phosphate, contained in PBS) in our experimentation to produce an early redox reaction which will reinforce the global ozone effect upon the microbiota, as we applied only a low ozone concentration (3-3.5ppm). The redox reaction will occur then in an early stage, before the ozone itself has time to react directly upon microorganisms. Specifically, ozone diffuses quickly in water and exerts its bactericidal effect by rupturing microorganisms membranes. 16-21 As toothbrushes are a possible source of microbial contamination, we must advice sanitation of toothbrushes by soaking them in large volumes of ozonated water which actually could showed a sterilizing effect even with lower ozone concentrations. Usually the soaking in the ozonated water will be for many hours and even considering the half-life of ozone in water there could still be some ozone remaining even after 8h. Therefore, the fact that we achieved an excellent result only after 30min by using a very low ozone concentration could hypothetize that a future study assessing the ability of much greater ozone concentrations in water should sterilize efficiently toothbrushes for people. The 23 February 2013 Page 5 of 9 ProQuest

strategy of managing human infections by ozone application has been used since 1950s.16-22 Indeed, many scientists have discussed about its role as an alternative agent to chlorine for desinfection of the different water supplies.15,16 Adverse reactions of ozone were associated with high ozone concentrations.15,16 This is not the case for ozone used in watery solutions, where the ozone showed high solubility because it dissipates very quickly in water, especially in elevated concentrations. In this term, the use of ozone in watery ecosystems is considered quiet safe and a potential toxicity effect should not exclude its use. Last years, ozone seems to possess an important place as therapeutical agent in dentistry. Ozonated water has been found to act upon accelerating healing of the oral mucosa following tooth extraction processes or after surgical interventions as it associate a haemostatic action together with a bactericidal effect. Moreover, ozone was used in dental unit waterlines 22 and in denture cleaning bubbled solutions.22 Recently, ozone was proposed for the management of primary dental root caries.21,23 For this purpose, ozone was used as a gas directly into the middle of the lesion21,23 Elevated ozone concentrations, as high as 2.100ppm21 and for extremely short periods of times (10-20s) are necessary in gas form in order to act directly upon the injured site.24,25 Antimicrobial effect has been shown especially against cariogenic bacterial species, i.e. S. mutans , Lactobacillus sp. and others.25-27 Hence, some authors by employing high resolution proton nuclear magnetic resonance (NMR) describe that ozone has the capacity to clinically alleviate oral malodour via the direct oxidative inactivation of VSCs and their amino acid precurcors.28 In our study, as discussed already limited ozone levels gave an important disinfenctant effect. However, we must point out here that the exposition to the watery ozone was longer. The threshold of the ozone efficacy was studied by comparing the initial microbial contamination with that estimated after every ozone exposure time (Fig. 1). It was observed29 an uncommonly resistance of the uni- and multispecies biofilms to ozone by comparison with the sensibility of the planktonic cells, where the sterilization effect was 100%. As our results indicate, an efficient ozone effect was observed after a progressive application of the ozone, with 5min being the minimum and the 30min the optimum value. This procedure is more efficient than a unique application at one time. 5 Conclusions Owing to the fact that toothbrushes always are a possible source of microbial infection and reinfection, the sanitation after each use is advised. Ozone is a strong oxidizer of cell walls and cytoplasmatic membranes of bacteria and so considered to be a promising bactericidal, antiviral and antifungal agents. A practical application of the ozone should be the possibility to use it for prevention from different oral pathogens. It is then conceivable that high dose ozonated water should be tested directly on contaminated toothbrushes. Acknowledgements The study was sustained by Democritus University of Thrace, Faculty of Agricultural Development, Department of Food Science and Technology, Laboratory of Microbiology, Biotechnology and Hygiene and a European ERASMUS Program. References Sammons RL, Kaur D, Neal P: Bacterial survival and biofilm formation on conventional and antibacterial toothbrushes . Biofilms 1 123-130, 2004. Haffajee AD, Smith C, Torresyap G, Thompson M, Guerrero D, Socransky SS: Efficacy of manual and powered toothbrushes II. Effect on microbiological parameters . Journal of Clinical Periodontology 28 947-954, 2001. Efstratiou M, Papaioannou W, Nakou M, Ktenas E, Vrotsos IA, Panis V: Contamination of a toothbrush with antibacterial properties by oral microorganisms . Journal of Dentistry 35 331-337, 2007. Daly C, Marshal R, Lazarus R: Australian dentists' views on toothbrush wear and renewal . Australian Dental Journal 45 254-256, 2000. Brandle CR, Menghini GD, Marthaler TM: The determination of caries risk in schoolchildren based on microbiological-chemical analysis of the saliva and on the clinical dental status . Schweiz Monatsschr Zahnmed 101 993-996, 1991. Quirynen M, De Soete M, Pauwels M, Goossens K, Teughels W, Van Eldera J: Bacterial survival rate on tooth- and interdental brushes in relation to the use of toothpaste . Journal of Clinical Periodontology 28 1106-1114, 2001. Taji SS, Rogers AH: The microbial contamination of toothbrushes. A pilot study . Australian Dental Journal 43 2-5, 1998. Murdoch FE, Sammons RL, Chapple ILC: Isolation and characterization of subgingival Staphylococci from periodontitis patients and controls . Oral Diseases 10 155-162, 2004. Wetzel W-E, Schaumburg C, Ansari F, Kroeger T, Sziegoleit A: Microbial contamination of toothbrushes with different principles of filament anchoring . Journal of the American 23 February 2013 Page 6 of 9 ProQuest

Dental Association 136 758-765, 2005. Tan E, Daly C: Comparison of new and 3-month-old toothbrushes in plaque removal . Journal of Clinical Periodontology 29 645-650, 2002. Caudry SD, Klitorinos A, Chan ECS: Contaminated toothbrushes and their disinfection . Journal of the Canadian Dental Association 61 512-516, 1995. Sato S, Yoko I, Guimaraes Lara EH, Panzeri H, Ferreira de Albuquerque H, Pedrazzi V: Bacterial survival rate on toothbrushes and their decontamination with antimicrobial solutions . Journal of Applied Oral Science 12 99-103, 2004. Muller P, Guggenheim B, Schmidlin PR: Efficacy of gasiform ozone and photodynamic therapy on multispecies oral biofilm in vitro . European Journal of Oral Science 115 77-80, 2007. Weschler CJ: Ozone's impact on public health: contribution from indoor exposures to ozone and products of ozone-initiated chemistry . Environmental Health Perspectives 114 1489-1496, 2006. Voidarou C, Tzora A, Skoufos I, Vassos D, Galogiannis G, Alexopoulos A, Bezirtzoglou E: Experimental effect of ozone upon some indicator bacteria for preservation of an ecologically protected watery system . Water, Air, &Soil Pollution 181 161-171, 2007. Bocci V: Oxygen-ozone therapy: a critical evaluation . The Netherlands: Kluwer Academic Publishers, 2002. [ISBN 14020-0588-1] Graham DM: Use of ozone for food processing . Food Technology 51 72-75, 1997. Shu M, Wong L, Miller JH, Sissons CH: Development of multi-species consortia biofilms of oral bacteria as en enamel and root caries model system . Archives of Oral Biology 45 27-40, 2000. Nagayoshi M, Fukuizumi T, Kitamura C, Yano J, Terashita M, Nishihara T: Efficacy of ozone on survival and permeability of oral microorganisms . Oral Microbiology and Immunology 19 240-246, 2004. Nelson FP, Macari S, Faria G, Assed S, Ito IY: Microbial contamination of toothbrushes and their decontamination . Pediatric Dentistry 22 381-384, 2000. Baysan A, Lynch E: Effect of ozone on the oral microbiota and clinical severity of primary root caries . American Journal of Dentistry 17 56-60, 2004. Baysan A, Lynch E: The use of ozone in dentistry and medicine . Primary Dental Care 12 47-52, 2005. Baysan A, Lynch E: The use of ozone in dentistry and medicine Part 2: ozone and root caries . Primary Dental Care 13 37-41, 2006. Holmes J: Clinical reversal of root caries using ozone, double-blind, randomised, controlled 18-month trial . Gerontology 20 106-114, 2003. Baysan A, Lynch E: Clinical reversal of root caries using ozone: 6-month results . American Journal of Dentistry 20 202-208, 2007. Baysan A, Whiley RA, Lynch E: Antimicrobial effect of a novel ozone-generating device on microorganisms associated with primary root carious lesions in vitro . Caries Research 34 498-501, 2000. Montanaro L, Campoccia D, Rizzi S, Donati ME, Breschi L, Prati C: Evaluation of bacterial adhesion of Streptococcus mutans on dental restorative materials . Biomaterials 25 4457-4463, 2004. Grootvedld M, Silwood CJL, Lynch E: High resolution 1 H NMR investigations of the oxidative consumption of salivary biomolecules by ozone: relevance to the therapeutic applications of this agent in clinical dentistry . Biofactors 27 5-18, 2006. Loret JF, Roberts S, Thomas V, Cooper AJ, McCoy WF, Levi Y: Comparison of disinfectants for biofilm, protozoa and Legionella control . Journal of Water and Health 3 423-433, 2005. AuthorAffiliation Athanasios Alexopoulos, Eugenia Bezirtzoglou. Democritus University of Thrace, Faculty of Agricultural Development, Department of Food Science and Technology, Laboratory of Microbiology, Biotechnology and Hygiene, GR68200, Orestiada, Greece; Corresponding author . Tel.: +30 2552041149. Silvia-Mariana Cretoiu. National Institute for Research and Development in Microbiology and Immunology "Cantacuzino" - Molecular Microbiology Laboratory, 050096, Bucharest, Romania Mirela Moldoveanu. Biology Institute of Romanian Academy, 060031, Bucharest, Romania Veronica Lazar. University of Bucharest, Faculty of Biology, 76201, Bucharest, Romania Mela Nakou. University of Athens, Dental School, Oral Microbiological Laboratory, Department of Periodontology, GR11527, Athens, Greece Subject: Microbiology; Studies; Bacteria; Microorganisms; Free radicals; Drinking water; Disinfection&disinfectants; Nuclear magnetic resonance--NMR; Organisms; Hygiene; Methods Identifier / keyword: Oral hygiene, Toothbrush, Microbial contamination, Ozone, Disinfection, Microbiota Publication title: Journal of Dentistry Volume: 36

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Issue: 8 Pages: 600-605 Publication year: 2008 Publication date: Aug 2008 Year: 2008 Publisher: Elsevier Limited Place of publication: Oxford Country of publication: United States Journal subject: MEDICAL SCIENCES--DENTISTRY ISSN: 03005712 Source type: Scholarly Journals Language of publication: English Document type: EDB, Journal Article DOI: http://dx.doi.org/10.1016/j.jdent.2008.04.007 ProQuest document ID: 1030081579 Document URL: http://search.proquest.com/docview/1030081579?accountid=17242 Copyright: 2008 Elsevier Ltd Last updated: 2012-07-30 Database: ProQuest Nursing&Allied Health Source,ProQuest Research Library

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Bibliography
Citation style: APA 6th - American Psychological Association, 6th Edition Bezirtzoglou, E., Silvia-Mariana Cretoiu, Moldoveanu, M., Alexopoulos, A., Lazar, V., & Nakou, M. (2008). A quantitative approach to the effectiveness of ozone against microbiota organisms colonizing toothbrushes. Journal of Dentistry, 36(8), 600-605. doi: http://dx.doi.org/10.1016/j.jdent.2008.04.007

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