Inhibition of biofilms associated with dentures and toothbrushes by tetrasodium EDTA
2007; Oxford University Press; Volume: 103; Issue: 6 Linguagem: Inglês
10.1111/j.1365-2672.2007.03491.x
ISSN1365-2672
AutoresDeirdre DeVine, Rimondia S. Percival, David J. Wood, Tobias J. Tuthill, P. Kite, R. A. Killington, Philip D. Marsh,
Tópico(s)HIV/AIDS oral health manifestations
ResumoJournal of Applied MicrobiologyVolume 103, Issue 6 p. 2516-2524 Free Access Inhibition of biofilms associated with dentures and toothbrushes by tetrasodium EDTA D.A. Devine, D.A. Devine Department of Oral Biology, Leeds Dental Institute, University of Leeds, UKSearch for more papers by this authorR.S. Percival, R.S. Percival Department of Oral Biology, Leeds Dental Institute, University of Leeds, UKSearch for more papers by this authorD.J. Wood, D.J. Wood Department of Oral Biology, Leeds Dental Institute, University of Leeds, UKSearch for more papers by this authorT.J. Tuthill, T.J. Tuthill Biochemistry and Molecular Biology, University of Leeds, UKSearch for more papers by this authorP. Kite, P. Kite Aseptica Ltd, Seattle, WA, USASearch for more papers by this authorR.A. Killington, R.A. Killington Biochemistry and Molecular Biology, University of Leeds, UKSearch for more papers by this authorP.D. Marsh, P.D. Marsh Department of Oral Biology, Leeds Dental Institute, University of Leeds, UKSearch for more papers by this author D.A. Devine, D.A. Devine Department of Oral Biology, Leeds Dental Institute, University of Leeds, UKSearch for more papers by this authorR.S. Percival, R.S. Percival Department of Oral Biology, Leeds Dental Institute, University of Leeds, UKSearch for more papers by this authorD.J. Wood, D.J. Wood Department of Oral Biology, Leeds Dental Institute, University of Leeds, UKSearch for more papers by this authorT.J. Tuthill, T.J. Tuthill Biochemistry and Molecular Biology, University of Leeds, UKSearch for more papers by this authorP. Kite, P. Kite Aseptica Ltd, Seattle, WA, USASearch for more papers by this authorR.A. Killington, R.A. Killington Biochemistry and Molecular Biology, University of Leeds, UKSearch for more papers by this authorP.D. Marsh, P.D. Marsh Department of Oral Biology, Leeds Dental Institute, University of Leeds, UKSearch for more papers by this author First published: 17 July 2007 https://doi.org/10.1111/j.1365-2672.2007.03491.xCitations: 20 D. Devine, Department of Oral Biology, Leeds Dental Institute, Clarendon Way, Leeds LS2 9LU, UK. E-mail: d.a.devine@leeds.ac.uk AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: We examined the efficacy of tetrasodium EDTA in eradicating biofilms derived from salivary inocula or pure cultures of Candida albicans on discs of polymethyl methacrylate (PMMA) denture base or on toothbrushes that had been used normally for 4–8 weeks. Its efficiency in virus neutralization was also determined. Methods and Results: Overnight (16 h) treatment with 4% (w/v) tetrasodium EDTA solution reduced salivary and C. albicans biofilm viable counts by ≥99%. Biofilm removal was confirmed using confocal laser scanning microscopy. Presence/absence of sucrose during biofilm formation had no effect on killing efficacy. Prolonged treatment of PMMA with tetrasodium EDTA did not influence subsequent formation of C. albicans biofilms or affect surface roughness of the PMMA, but it reduced subsequent biofilm formation from a salivary inoculum. Infectivities of herpes simplex virus and polio virus suspensions were reduced by >99·99% by treatment for 1 and 2 h, respectively. Conclusions: Tetrasodium EDTA solution efficiently disinfected toothbrushes and PMMA discs, with the detachment of biofilms, and rapidly neutralized both nonenveloped and enveloped viruses. Significance and Impact of the Study: Dentures and toothbrushes become contaminated by bacterial biofilms and by viruses. There is a need for disinfection methods that are rapidly effective, cost-effective, nontoxic and easily implemented. These studies indicate that tetrasodium EDTA solution has disinfection applications in the oral care field. Introduction Disinfection of commonly used dental products that harbour large numbers of viable micro-organisms may also protect from infection and help increase oral and general health. For example, biofilms form on dentures and can lead to painful infections with the oral yeast Candida albicans (Radford et al. 1999). Also, toothbrushes become heavily colonized with oral and environmental microorganisms over time (Verran and Leahy-Gilmartin 1996; Quirynen et al. 2003; Sammons et al. 2004), and these can include important opportunistic pathogens not normally found in the mouth, as well as viruses such as hepatitis C (Lock et al. 2006). Those most vulnerable to such biofilms are the elderly, who frequently have poor standards of oral and denture hygiene (Kuc et al. 1999; Kulak-Ozkan et al. 2002), and the immunocompromised. There is a need for easily implemented disinfection methods that are efficient, cost-effective, rapid, nontoxic and suitable for a wide range of materials. The effectiveness of tetrasodium EDTA has been demonstrated in the treatment of biofilms containing staphylococci (including methicillin-resistant Staphylococcus aureus, MRSA), other Gram-positives, Pseudomonas aeruginosa and enterobacteria (Kite et al. 2004; Percival et al. 2005). Tetrasodium EDTA also has proven activity against biofilms in catheters, with significant reductions in biofilm viable counts after treatment of haemodialysis catheters for 3 h and central venous catheters for 21 h (Kite et al. 2004; Percival et al. 2005). This agent has many advantages in terms of its low cost, ease of use and efficacy against biofilms. As tetrasodium EDTA has proven to be effective in controlling catheter biofilms, we hypothesize that it will be effective in controlling biofilms on dental materials. We investigated the utility of tetrasodium EDTA to disinfect commonly used and usually heavily contaminated dental products, such as denture base material and toothbrushes. Effects on Candida species were assessed because of the role of these organisms in denture-associated infections, and on mutans streptococci and lactobacilli, because of their cariogenic potential. Materials and methods Reagents Tetrasodium EDTA (supplied by Aseptica, Inc., Seattle, WA, USA) was prepared at a concentration of 4% (w/v) in sterile distilled water. Its activity was halted in all experiments using aqueous 1 M CaCl2. Artificial saliva (±0·2%, w/v sucrose as required) was prepared as described by Sissons et al. (1991) and contained, per litre of distilled water, 10 g proteose peptone, 5 g tryptone, 5 g yeast extract, 2·5 g hog gastric mucin, 2·5 g KCl, 2 g sucrose, 0·21 g arginine, 60 mg urea, 50 mg haemin and 10 mg menadione. Proteose peptone, tryptone, yeast extract, mucin, KCl and arginine were dissolved in distilled deionized water and sterilized by autoclaving. Stock solutions of haemin and menadione (in 95% ethanol) and of urea and sucrose were filter-sterilized through 0·2 μm filters (Nunc International, USA) prior to addition. Reduced transport fluid (RTF) was prepared as described by Hoover and Newbrun (1977) and contained, per litre distilled water: 0·45 g K2HPO4, 0·45 g KH2PO4, 0·90 g NaCl, 0·1875 g (NH4)2SO4, 0·40 g Na2CO3, 0·20 g dithiothreitol, 10 ml of 0·1 M EDTA. The pH was adjusted to 8·0 and RTF was filter-sterilized and stored at 4°C. Preparation of polymethyl methacrylate (PMMA) denture base discs Wax blanks were made by pouring molten dental wax, approximately 3-mm deep, into a six-well tissue culture plate. Following storage in a freezer, they were invested in plaster, and the wax was boiled out to leave a mould, which was filled with the setting PMMA dough and cured. The PMMA discs were removed from the plaster and polished using a denture-polishing wheel. As PMMA absorbs water, discs were left to equilibrate in water for 5–6 days at 37°C and were then pressed into the wells of a six-well tissue culture plate. Treatment of biofilms on PMMA Immediately before each experiment, unstimulated whole saliva was collected from the same three individuals and pooled. Candida albicans ACTCC 90028 was stored in 30% (v/v) glycerol at −80°C and was cultivated from frozen stocks on Sabouraud agar (Oxoid, UK) incubated aerobically for 48 h at 37°C. For preparation of inocula, C. albicans ACTCC 90028 was transferred from Sabouraud agar into 10 ml of Sabouraud broth (Oxoid) and incubated aerobically at 37°C for 16 h, reaching a density of approx 3·0 × 107 colony-forming units per ml (CFU per ml). For biofilm formation, pairs of equilibrated PMMA discs were each inoculated with 250 μL of pooled salivary inoculum or with a pure culture of C. albicans ACTCC 90028; one disc of the pair was for future treatment with tetrasodium EDTA and one to function as a control. All experiments were carried out on at least three occasions. The discs were incubated statically in air plus 5% CO2 at 37°C for 2 h and unattached organisms removed by rinsing and gentle agitation with artificial saliva using a Pasteur pipette. The surfaces of the discs were covered with 250 μl of artificial saliva and they were incubated for a further 18 h in 5% CO2 at 37°C; unattached organisms and artificial saliva were again removed by rinsing and agitating gently with artificial saliva using a Pasteur pipette. Discs were finally covered with 250 μl of fresh artificial saliva ± 0·2% (w/v) sucrose and incubated for a further 4 h in 5% CO2 at 37°C, providing a 24 h biofilm. PMMA discs bearing 24 h biofilms were covered with 500 μl of a sterile solution of 4% (w/v) tetrasodium EDTA or sterile-distilled water (control). After 16 h (overnight) at room temperature (20–22°C), 500 μl of 1 M CaCl2 was added for at least 30 s to neutralize the tetrasodium EDTA. Total biofilms were recovered from PMMA discs into 1 ml of RTF by scraping the surface with a sterile plastic loop. Any remaining attached organisms were harvested by swabbing the disc with a sterile cotton wool swab, the top of which was cut off using sterile scissors and added to the 1 ml suspension. Microorganisms were then dispersed by vortex mixing and viable counts determined after serial dilution in RTF. To examine the effects of repeated exposure to tetrasodium EDTA on subsequent biofilm formation, uninoculated PMMA discs were immersed in 4% (w/v) tetrasodium EDTA or sterile, distilled water for 6 days (with daily changing of tetrasodium EDTA) prior to inactivation of the agent with 1 M CaCl2. The disc surfaces were rinsed and inoculated with saliva or C. albicans and biofilms allowed to grow as described. Treatment of biofilms on toothbrushes Commercial toothbrushes (Wisdom Ultraflex®) were used normally by adult volunteers for 4 weeks and were transported to the laboratory in clean, sealed nylon bags. The toothbrush heads were halved using a heated sterile scalpel. One half was used for determination of total bacterial viable counts, whereas the remaining half was treated by immersion for 16 h in either 8 ml of 4% (w/v) tetrasodium EDTA or 8 ml of sterile distilled water in a Universal bottle. Tetrasodium EDTA was inactivated by replacing the solution with 1 M CaCl2. Bacteria from the tufts (removed using sterile scissors) and half-toothbrush head were harvested into 1 ml RTF by vortex mixing with 3 mm glass beads and viable counts determined. Microbiological analyses Saliva, pure cultures or harvested biofilms were serially diluted in RTF and 100 μl spread on a variety of media (each in duplicate) for viable count determinations. Bacteriological media were prepared according to the manufacturers’ instructions. Columbia agar (Oxoid) containing 5% (v/v) horse blood (CBA) was employed for the determination of total bacterial counts, Sabouraud broth and agar media (Oxoid) for the cultivation of C. albicans, Trypicase yeast cystine agar (LabM) containing 20% (w/v) sucrose and 0·1 U ml−1 bacitracin (TYCSB) for mutans streptococci, and Rogosa agar for lactobacilli. Total anaerobes on CBA were enumerated after incubation for 4–7 days in a Don Whitley Mark III Anaerobic Work Station (Don Whitley Scientific, UK) under an atmosphere of 80% N2, 10% CO2 and 10% H2. TYCSB plates were also incubated anaerobically for 3–4 days. Sabouraud medium was incubated aerobically at 37°C for 24–48 h, and Rogosa agar plates were incubated for 3–4 days at 37°C in an aerobic incubator under 5% CO2. Virus neutralization Herpes simplex virus (HSV) and poliovirus (PV) were included as models for killing of pathogenic enveloped and nonenveloped viruses, respectively, as described by Devine et al. (2001). Baby hamster kidney (BHK) cells were used for growth of herpes simplex virus and HeLa cells for PV. BHK cells were grown in adherent culture in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% (v/v) newborn calf serum, at 37°C and 5% CO2. Cells were infected with HSV at low multiplicity of infection, incubated for 3 days and progeny virus clarified from cell debris by low-speed centrifugation. HeLa cells were grown in suspension culture to a density of 5 × 105 cells per ml at 37°C in DMEM containing 5% (v/v) foetal calf serum, 25 mM HEPES and lacking CaCl2. Cells were harvested by low-speed centrifugation, washed in PBS and re-suspended in serum-free DMEM containing virus at a multiplicity of infection of 10 in a volume of one-hundredth of the original culture volume. Virus was allowed to adsorb to the cells at room temperature for 1 h with gentle agitation before infection was initiated by diluting ten-fold with prewarmed complete medium at 37°C. Infected cells were pelleted 7 h postinfection, lysed by freeze/thaw and clarified virus supernatants purified by ultracentrifugation in CsCl gradients. For titration of HSV, samples were diluted in BHK Growth Medium and the appropriate concentration of BHK cells was added. Universal bottles were incubated at 37°C for 30 min and shaken to allow adsorption of the virus. CMC-DTC overlay medium (Sigma) was added, poured into Petri dishes and incubated in a CO2 incubator for 3 days. Following addition of formol saline for 1 h, overlay was removed and cells were stained with gentian violet for 1 h; plaques were examined and counted using a stereo light microscope. For titration of PV, medium was aspirated from six-well plates containing confluent monolayers of HeLa cells. Virus suspensions serially diluted in DMEM Growth Medium were added (0·5 ml) and allowed to adsorb to the cell monolayer for 1 h at 37°C with gentle rocking. The inoculum was aspirated and molten agarose solution (0·6% w/v in 0·9× DMEM; 3 ml) was added to each well followed by incubation at 37°C in 5% CO2 for 2–3 days. Virus plaques were counted after fixing and staining with formol saline solution containing 0·5% (w/v) gentian violet. Virus suspensions (50 μl) at concentrations between 1 × 109 and 1 × 1010 PFU ml−1 were incubated at room temperature in sealed sterile tubes with 1 ml of 4% (w/v) tetrasodium EDTA or PBS (control) for various times. Preliminary experiments had shown that neutralization of the agent with CaCl2 had an adverse effect upon virus infectivity, and thus this step was not included and tetrasodium EDTA was inactivated through dilution. Following incubation, the remaining viral infectivity was determined by plaque titration (below). Confocal laser scanning microscopy Images of treated and untreated PMMA discs and toothbrushes were recorded using a confocal laser scanning microscope (TCS SP2/AOBS, Leica, Germany). A reflection image of unstained sample was generated using an Ar/ArKr laser with an objective magnification of 10× and numerical aperture set at 0·3 (×10/0·3 NA), selecting a scan format 512 × 512 pixels and scan speed of 400 Hz. The stage was moved vertically (z-direction) between the first and last detectable light reflection and a z-series of optical sections were generated. For measurement of the average surface roughness (Ra) of PMMA discs, the z-series were converted to greyscale (topographical) images and five regions of interest (ROIs) per disc, each of 750 μm × 750 μm, were created and Ra quantified. Ra is defined (BS EN ISO 4287: 2000) as the arithmetic average of the profile ordinates within the measured section (average height). Results Effects of tetrasodium EDTA on biofilms on PMMA Both salivary and C. albicans inocula produced biofilms with high total viable counts after 24 h (Tables 1 and 2), with or without addition of sucrose after 20 h (Table 2). Confocal laser scanning microscopy (CLSM) confirmed that the harvesting procedure had removed most or all of the biofilms from the PMMA disc surfaces (Fig. 1). The proportions of mutans streptococci, lactobacilli and Candida spp. in salivary inocula and biofilms (sucrose added at 20 h) on six discs of PMMA were determined by culture on selective and nonselective media (Table 1). Biofilms contained lower proportions of mutans streptococci and higher proportions of Candida spp. than did the salivary inocula, but counts and proportions of lactobacilli were similar. A repeated-measures anova with three measures (mutans streptococci × Candida spp. × lactobacilli) and two conditions (saliva × biofilm) were used to investigate these relationships. There were significant main effects of measure and condition (F1,5 = 7·56, P < 0·05, F1,5 = 7·20, P < 0·05, respectively). Further analysis of the simple effects indicated that there were significant differences between the proportions of mutans streptococci in saliva compared to biofilm populations (mean = 14·21, F2,4 = 6·55, P < 0·05), and proportions of Candida spp. in saliva compared to biofilms (mean = −0·11, F2,4 = 6·55, P < 0·05). Table 1. Bacterial composition of pooled salivary inocula and 24 h biofilms formed on PMMA denture base material discs Count Salivary inoculum (n = 6) 24 h biofilm* (n = 6) Range log10 CFU per ml Mean CFU per ml (log10 ± SD) Mean % of total count† Range log10 CFU per ml Mean CFU per ml (log10 ± SD) Mean % of total count Total viable count 6·2–7·1 6·6 ± 0·4 100 6·2–7·9 6·8 ± 0·6 100 Mutans streptococci 5·2–5·9 5·6 ± 0·2 17·4 ± 12·6 4·2–5·8 5·1 ± 0·6 3·2 ± 3·1‡ Candida spp. 0–3·5 0·6 ± 1·3 0·01 ± 0·01 0–3·6 2·5 ± 1·8 0·11 ± 0·09‡ Lactobacilli 0–3·8 1·8 ± 1·8 0·06 ± 0·08 0–3·8 1·6 ± 1·6 0·06 ± 0·14 *Total biofilm was harvested into 1 ml of RTF. †The percentage of the total count was calculated for each disc and the means of these values calculated. ‡Significantly different comparing saliva and biofilm values (P < 0·05). Table 2. Effects of 16 h treatment with tetrasodium EDTA on viable counts of 24 h biofilms formed on PMMA denture base material discs Inoculum Sucrose added* Mean log10 biofilm viable count† (CFU per ml) ± SD after 16 h treatment with Mean % reduction by tetrasodium EDTA‡ distilled water (n = 3) 4% (w/v) tetrasodium EDTA (n = 3) Pooled saliva − 6·5 ± 0·3 3·5 ± 0·5 99·8 ± 0·12 + 6·4 ± 0·6 3·2 ± 0·2 99·9 ± 0·09 C. albicans − 6·3 ± 0·2 4·4 ± 0·4 98·8 ± 0·50 + 6·3 ± 0·0 4·3 ± 0·0 98·8 ± 0·16 *0·2% (w/v) sucrose in artificial saliva added to biofilms after 20 h incubation. †Total biofilm was harvested into 1 ml RTF. ‡The percentage was calculated for each pair of discs and the means of these values calculated. Figure 1Open in figure viewerPowerPoint CLSM images of PMMA discs: (a) with 24 h biofilm of Candida albicans; bar represents 30 μm; (b) following harvesting of 24 h biofilm of C. albicans; bar represents 150 μm; and (c) following treatment of 24 h biofilm of C. albicans with 0·4% (v/v) tetrasodium EDTA for 16 h; bar represents 150 μm. Treatment of salivary or C. albicans biofilms with tetrasodium EDTA for 16 h resulted in greater than 99% reductions in biofilm viable counts compared with control counts obtained after incubation in distilled water (Table 2). CLSM confirmed that little biofilm remained on the PMMA disc surfaces following 16 h tetrasodium EDTA treatment (Fig. 1). The effect of prolonged exposure to tetrasodium EDTA on the subsequent formation of biofilms was assessed. Discs that had been treated with tetrasodium EDTA or sterile water for 6 days were treated with 1 mM CaCl2, inoculated with pooled saliva or C. albicans and incubated for 24 h for biofilm formation. The viable count on each test disc was compared with that on a control disc set up at the same time with the same inoculum and expressed as a percentage of the control viable count (Table 3). Viable counts of salivary biofilms on tetrasodium EDTA-treated discs reached 58–69% (mean 62·8 ± 4·8%) of the counts achieved on water-treated discs inoculated with the same pooled saliva. The tendency for C. albicans to form biofilms on denture base material discs was unaffected by prolonged exposure of discs to tetrasodium EDTA prior to inoculation. A paired t-test indicated that biofilm formation from salivary inocula was significantly lower than for C. albicans (t = −17·013, P = <0·005) on discs pretreated with tetrasodium EDTA for 6 days. Table 3. Biofilm formation following 6-day exposure of PMMA denture base material to tetrasodium EDTA Inoculum Mean log10 biofilm viable count* (CFU per ml) ± SD on discs treated for 6 days with Mean % on test compared with control discs† Distilled water (n = 6) 4% (w/v) tetrasodium EDTA (n = 6) Saliva 7·28 ± 0·42 7·21 ± 0·03 62·8 ± 4·8‡ C. albicans 7·10 ± 0·28 6·97 ± 0·03 93·3 ± 6·2‡ *Total biofilm was harvested into 1 ml of RTF. †Mean percentage reduction in counts on test compared with control disc. ‡Significantly different (P ≤ 0·005) The difference in salivary biofilm formation following tetrasodium EDTA treatment was not due to changes in the surface of the PMMA discs, as assessed by surface roughness. The mean value (n = 10) of Ra prior to tetrasodium EDTA application was 3·32 μm (±1·18 μm) and after application for 6 days, it was 3·55 μm (±0·92 μm). A paired t-test showed that there was no statistically significant difference between these values of Ra. Effects of tetrasodium EDTA on biofilms on toothbrushes Toothbrushes had substantial bacterial accumulations with total counts ranging from 9·6 × 102 to 1·7 × 106 CFU per ml per half-toothbrush. Three half-toothbrushes were treated with distilled water and four with tetrasodium EDTA (Table 4). Overnight (16 h) treatment with tetrasodium EDTA reduced total viable counts by more than 99% (Table 4) and CLSM confirmed removal of biofilms (Fig. 2). The control procedure of leaving brushes in sterile distilled water for the same time resulted in smaller reductions in viable count (mean 42%). One-way anova indicated that the mean percentage reduction of viable counts by immersion of control toothbrushes in water for 16 h was significantly less than the mean percentage reduction of viable counts on brushes that had been treated for 16 h with tetrasodium EDTA (F1,5 = 22·4, P < 0·01). Table 4. Reduction by 16 h treatment with 4% (w/v) tetrasodium EDTA of total bacterial viable counts recovered from toothbrushes Treatment (16 h) Mean log10 biofilm viable count* (CFU per ml) ± SD on toothbrushes Mean % reduction (posttreated compared with pretreated) Pretreatment Posttreatment Sterile distilled water† 4·52 ± 1·33 4·25 ± 1·17 42·12 ± 20·41§ 4% (w/v) tetrasodium EDTA‡ 3·99 ± 0·80 1·38 ± 1·39 99·35 ± 0·83§ *Total biofilm was harvested into 1 ml of RTF. †n = 4. ‡n = 3. §Significantly different (P ≤ 0·01). Figure 2Open in figure viewerPowerPoint CLSM images of toothbrushes used for 4 weeks: (a) multiple tufts (toothbrush 1) prior to treatment; bar represents 300 μm; (b) a single tuft (toothbrush 2) prior to treatment; bar represents 58 μm; (c) multiple tufts (toothbrush 2) following 16 h treatment with 0·4% (v/v) tetrasodium EDTA; bar represents 150 μm; and (d) a higher power image of one of the tufts in (c); bar represents 52 μm. Neutralization of viruses by tetrasodium EDTA Tetrasodium EDTA significantly reduced the infectivity of PV, eliciting a >5 log reduction after 2 h incubation when compared to the control (Table 5). After 20 h, viral infectivity was reduced by >6 log, to levels below the limit of sensitivity for the assay. Tetrasodium EDTA reduced the infectivity of HSV to below the threshold of detection in the assay, corresponding to a minimum 6 log reduction, after 1 h (Table 5). Table 5. Effects of tetrasodium EDTA on infectivity of suspensions of herpes simplex virus and poliovirus Virus Exposure time (h) Mean virus titre* (PFU per ml†) ± SD after treatment with Mean % reduction by tetrasodium EDTA‡ Phosphate-buffered saline 4% (w/v) tetrasodium EDTA (n = 3) Poliovirus 2 5·0 × 109±1·53 × 108 1·4 × 104±3·06 × 103 99·9997 20 3·1 × 109±1·47 × 108 99·9999 Herpes simplex virus 1 4·0 × 109±1·20 × 108 99·9999 *Virus suspensions (1 × 109–1 × 1010 PFU per ml) were incubated at room temperature with 4% (w/v) tetrasodium EDTA or phosphate-buffered saline, pH 7·4 (control), for times specified. Tetrasodium EDTA was inactivated by dilution in phosphate-buffered saline and remaining viral infectivity was determined by plaque titration. †PFU: plaque-forming units. ‡The percentage of the virus titre in the control phosphate-buffered saline suspension represented by the titre in the tetrasodium EDTA-treated suspension. §Sensitivity limit of the assay. Discussion There is a need for simple but effective methods to disinfect commonly used oral care items, and the current studies were undertaken to assess the possible applications of tetrasodium EDTA solution, which has previously been shown to be capable of eradicating preformed biofilms on catheters (Kite et al. 2004; Percival et al. 2005). We showed that tetrasodium EDTA solution was also effective in eradicating bacterial and yeast biofilms formed on PMMA denture base and on toothbrushes, and it efficiently neutralized enveloped and nonenveloped viruses. Dentures are commonly colonized by organisms derived from saliva, oral mucosa and skin (Theilade and Budtz-Jørgensen 1988), and denture plaque accumulations are associated with inflammation and infections. The importance of controlling biofilms on dentures is particularly important in relation to the oral and general health of the elderly, the group that most frequently wears dentures. Denture wearing can result in increased carriage of cariogenic bacteria (Marsh et al. 1992), opportunistic pathogens such as C. albicans (Bergendal et al. 1979; Bergendal 1982) and in increased frequency of isolation of pathogens that are not normal inhabitants of the mouth, for example, staphylococci (including MRSA), enterobacteria and Mycobacterium tuberculosis (Theilade and Budtz-Jørgensen 1988; Marsh et al. 1992; Kuc et al. 1999; Eguchi et al. 2003; Smith et al. 2003). Denture-associated stomatitis is common (Theilade and Budtz-Jørgensen 1988) and is associated with poor denture hygiene (Arendorf and Walker 1979). Candida albicans has long been implicated as a causative agent (Theilade and Budtz-Jørgensen 1988; Barbeau et al. 2003), but the condition can occur in its absence when Gram-positive bacteria (streptococci, staphylococci, actinomyces and lactobacilli) may form a pathogenic community that contributes to the aetiology of the condition (Radford et al. 1999). The most effective and rational prevention of denture-associated stomatitis is through meticulous hygiene and removal of denture plaque. In 16 h, tetrasodium EDTA reduced salivary bacterial and C. albicans biofilm viable counts by more than 99% and removed most or all of the biofilms from the material surface. Sucrose, an important environmental parameter within the mouth, did not affect the efficacy of this killing. Prolonged (6 days) exposure of denture material base to tetrasodium EDTA had the added benefit of reducing subsequent biofilm formation by salivary microorganisms, although biofilm formation by C. albicans was unaffected. In contrast, some agents may increase the tendency for organisms to adhere to the material surface (Nikawa et al. 2003). In particular, increased surface roughness is important in determining the propensity of oral microorganisms to adhere to materials and form biofilms (Quirynen et al. 1990; Verran and Maryan 1997; Taylor et al. 1998; Radford et al. 1999; Morgan and Wilson 2001). Tetrasodium EDTA did not affect PMMA surface roughness. We detected microbial loads on toothbrushes similar to those described in previous studies (Caudry et al. 1995; Taji and Rogers 1998; Sammons et al. 2004). The transmission of organisms from toothbrushes may pose some risk to the most vulnerable individuals, such as the elderly and immuno- or medically-compromised. Toothbrushing can induce a transient bacteraemia (Schl
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