- Email: [email protected]
A between-patient disinfection method to control water line contamination and biofilm inside dental units
Journal of Hospital Infection (2004) 56, 297–304
www.elsevierhealth.com/journals/jhin
A between-patient disinfection method to control water line contamination and biofilm inside dental units L. Montebugnolia,*, S. Chersonia, C. Pratia, G. Dolcib a
Department of Oral Science, University of Bologna, Via S. Vitale 59, 40125 Bologna, Italy University of Rome “La Sapienza”, Rome, Italy
b
Received 28 July 2003; accepted 16 December 2003
KEYWORDS Biofilm(s); Dental unit; Cross-contamination; Disinfection; Heterotrophs; Infection control
Summary The aim of the present study was to evaluate the efficacy of a between-patient disinfection procedures to maintain low bacterial counts in dental unit water line (DUWL) effluents, and control dental water line biofilms. Six dental units already in use, that had never been cleaned, were monitored for three weeks. During the first week only baseline contamination levels were assessed with no treatment of the system. In the second week lines were flushed with water for 30 s before treating each patient. During the third week, a disinfection procedure with 0.26% peracetic acid, followed by a water flush, was implemented before treating each patient. DUWL samples were collected both at the beginning and at the end of 216 dental procedures (72 during each period), plated on R2A agar and incubated at room temperature for seven days to obtain total bacterial counts in colony forming units per millilitre. To assess biofilm control, nine dental units (five never used and four old dental units with established biofilm) were used for 30 days in routine dental practice undergoing five between-patient DUWL disinfecting cycles every day. Water line samples were removed at baseline and at the end of the study and examined by scanning electron microscopy to determine the presence or absence of biofilms. A significant difference ðP , 0:01Þ in mean DUWL bacterial counts was found between the three sets of observations. Biofilms were not present in any of the new dental units and a demonstrable reduction in the biofilms from the four dental units with previous presence of established biofilms was observed at the end of the study. In this study, a between-patient disinfection procedure consisting of flushing DUWL with peracetic acid with use of water was efficacious in the control of both microbial contamination of dental treatment water and dental water line biofilms. Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Tel.: þ 39-51-278006; fax: þ39-51225208. E-mail address: [email protected] 0195-6701/$ - see front matter Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2004.01.015
298
Introduction It has been known for more than 30 years that treatment water, delivered by dental units during routine practice of dentistry, is highly contaminated by numerous species of pathogenic and nonpathogenic micro-organisms. The organisms enter dental units from the mains water source, or could be retracted into the dental unit water lines (DUWLs) from the oral cavity of patients during routine dental care,1 – 3 These heterotrophic bacteria persist in DUWLs as a multispecies biofilm on the lumenal surface, contaminating the dental unit water system. In 1996 the American Dental Association established a goal for treatment water to contain no more than 200 cfu/mL of heterotrophic unfiltered output.4 Several methods have been suggested through which the DUWL contamination by heterotrophic mesophilic organisms may be kept under this limit including flushing protocols and chemical treatment.5,6 Of the chemicals now available, peracetic acid has a rapid and broad-spectrum biocidal activity that could be useful in controlling DUWL contamination between patient treatments,7,8 but, as delivered, it has a series of side effects limiting its use in dentistry.9 – 11 However, a new chemical formulation (tetraacetylethylenediamine (TAED) in association with persalt) has been recently proposed as a non-hazardous means of generating peracetic acid in situ. This form of peracetic acid does not have the same side effects as pre-formed peracetic. acid.12,13 The aim of this study was to evaluate the efficacy of peracetic acid in controlling heterotrophic bacterial contamination when used between patient treatments and compare the results with those achieved through pure mechanical flushing with mains water, or in the absence of any treatment. An additional aim was to evaluate its long-term efficacy in controlling DUWL biofilms.
Method and materials To study the control of dental treatment water contamination, six dental units (Castellini Logos, Castellini S.p.A., Bologna, Italy) that had been in use for over one year were selected. These units were directly connected to mains water as a source for irrigation. None of these units had been treated to remove biofilm or reduce planktonic bacterial contamination. During the first week all units received no treatment (control), during the second week mechanical flushing with mains water for 30 s
L. Montebugnoli et al.
before each dental procedure (flushing) was implemented. During the third week a disinfecting cycle (disinfection) was initiated with each dental unit before dental treatment session. The disinfection cycle consisted of an automated procedure by a device integral to each dental unit (Autosteril, designed by Castellini S.p.A., Bologna, Italy) programmed to flush the disinfectant solution (contained in a built-in talk) through the water system and purge with water. The Autosteril procedure consisted of purging the residual dental treatment water loading the system with Ster4spray (manufactured by Farmed S.p.A and distributed by Castellini S.p.A., Bologna, Italy) for 5 min of contact, purging the chemical and finally flushing with water. Ster4spray is a fine powder containing a binary active system (TAED and sodium perborate), which is activated by dissolving in water at a temperature above 35 8C to from peracetyl ions at pH 8, equivalent to 0.26% in peracetic acid, ensuring a stable concentration for up to 24 h. The active ingredient is completely biodegradable and degrades to acetic acid, oxygen and water.13 Each dental unit was used for 12 dental procedures (requiring the use of the high-speed handpiece) every week for a total of 36 procedures. The number of dental procedures per day and per week was standardized between and within dental units. The total number of dental procedures during the three periods was 216 (72 each for control, flush and disinfection periods). An external evaluator observed and audited the time for each dental procedure and that of the handpiece use. Water samples were collected from the high-speed handpiece lines of each dental unit at the beginning and at the end of each dental procedure for a total of 432 samples. Each sample was processed to evaluate the total number of heterotrophic micro-organisms. Two millilitres of water from each unit were collected in a sterile tube with filter-sterilized sodium thiosulphate at a final concentration of 18 mg/L to oppose the growth-inhibiting effects of residual chlorine. Samples were plated on R2A Agar (Difco, Becton Dickinson, Le Pont de Claix, France) within 3 h of collection and incubated at room temperature (22 –24 8C) for seven days and the total number of heterotrophic micro-organisms counted. All absolute counts were converted to log10 values to normalize data. A one-way analysis of variance (ANOVA) was used to study differences between heterotrophic plate counts values, length of the dental session and duration of handpiece usage for the three periods. After fitting a general linear model, multiple regression ANOVA for repeated measures with split-plot design was used to
Water line contamination
evaluate differences between dental units, procedures (control, flush and disinfection periods) time (colony forming units per millilitre at the beginning and at the end of dental treatments regardless of the decontaminating procedure adopted) and the interaction of decontaminating procedure multiplied by the time; the post hoc test applied was Bonferroni t-test for multiple-comparisons at a ¼ 0:05: To evaluate biofilm removal, nine Castellini Logos dental units (five new dental units and four in use for 2, 3, 24 and 48 months) were used. All dental units were supplied by mains water and were used in routine dental practice for 30 days during which they were subjected to repeated betweenpatient DUWL disinfection cycles with Ster4spray (approximately 5 cycles/day). The disinfection cycle was identical to the protocol stated earlier. A 10 nm section of the water line tubing connected to the high-speed handpiece was removed from each dental unit at baseline and at the end of the study period, fixed in 3% glutaraldehyde in a 0.2 M sodium cacodylate buffer, treated with increasing concentration of ethyl alcohol (05 –95 and finally 100%), desiccated overnight using hexamethyldisilazane following the method of Perdigao et al.,14 mounted on a stud, coated with 10 nm gold and viewed using scanning electron microscopy (SEM; Jeol 5400, Jeol Japan, operating at 5 and 10 kV at magnification ranging £ 500 – 5000. Biofilm was quantified using the following criteria: (1) extension (% surface covered by biofilm at £ 500), (2) matrix thickness (microns), and (3) number of visible bacteria per 500 mm2 at £ 5000).
299
Table I The results of fitting a general linear statistical model relating log10 colony forming units per millilitre to three predictive factors and their interaction using analysis of variance Source
F-ratio
P-value
Dental units Procedures Time Proceduresa £ time
9.63 799.53 0.06 11.59
0.0001 0.0001 0.801 0.0001
a
Significant differences in log10 cfu/mL were found between dental units, procedures and the interaction of procedures multiplied by the time.
cedure before dental treatment (control), or flush with tap water (flush) or DUWL disinfection cycles (disinfection). Among the water samples tested 143 out of 144 samples showed bacterial counts higher than 200 cfu/mL (ADA’s goal for the year 2000) in the control period, 142 out of 144 in the flush period and only 12 out of 144 in the disinfection period. Figure 2 shows the mean values of samples collected in the three different study periods. Means (^ SD) log10 colony-forming units per millilitre from effluent water samples at the beginning of dental procedures were 4.39 ^ .66 (range 2.0 to 5.79) for the control period, 3.73 ^ .65 (range 2.3 to 4.81) for the flush period and 1.02 ^ .94 (range not-detectable to 2.86) during the disinfection period. The mean log10 colony-forming units per millilitre at the end of dental procedures were 3.94 ^ .50 (range 2.6 to 5.53) in the control period, 4.03 ^ .64 (range 2.0 to 5.14) for the flush period and 1.22 ^ .98 (range not detectable to 3.20) for the disinfection period. The time-related decrease
Results No significant differences between the three groups were detected with reference to duration (in minutes) of dental treatment sessions (control 33.7 ^ 12.4, flush 30.9 ^ 11.4, and disinfection 32.0 ^ 13.8, F ¼ 0:84; P . 0:05). The duration of use of handpiece in minutes also showed no significant difference within each dental session (control 5.9 ^ 1.9, flush 6.2 ^ 2.0, and disinfection 5.5 ^ 2.4, F ¼ 1:12; P . 0:05). Multiple regression ANOVA showed significant differences in the log10 reduction of colony forming units between dental units, treatment procedures and the interaction between procedures and time (P , 0.05; Table I). Figure 1 shows log10 colony-forming units per millilitre means and 95% confidence intervals in samples collected during dental sessions from dental units undergoing no decontaminating pro-
Figure 1 Means of colony forming units per millilitre (in Log10) and 95% confidence intervals in samples collected during all dental sessions from dental units undergoing no decontaminating procedure before dental treatments (control), DUWL flushing (flushing), or DUWL disinfecting cycles (disinfection) before dental treatments.
300
L. Montebugnoli et al.
Figure 2 Means of colony forming units per millilitre values (in Log10) of samples collected at the beginning and at the end of dental sessions during the three different periods (control, flushing, and disinfection).
in mean bacterial counts during the control period ðF ¼ 22:12Þ and the time-related increase in mean bacterial counts during the flushing period ðF ¼ 20:01Þ were statistically significant, while no significant variation with time was found during the disinfection period ðF ¼ 1:72Þ; no significant difference was detected between control and flushing periods in the mean contamination levels at the end of the respective dental treatment sessions. Table II shows the results of SEM using the three parameters for quantification of biofilm. No biofilm was observed in all new dental units after 30 days of use in routine dental practice (Figure 3). The 24month-old units showed complete removal of biofilm (Figure 4). A partial reduction in the biofilm matrix was observed in one of the remaining three dental units, and with a small percentage decrease in one out of three. A greater decrease in the number of bacteria in biofilms was seen in all tube specimens from old dental units after 30 days of
Figure 3 (A) Scanning electron microscopic images of dental unit water line lumens taken from one of five new dental units before the installation and (B) after one month of use undergoing repeated between-patient disinfecting cycles with peracetic acid. The network of fine lines is probably related to the wall texture of the tubing; no sign of biofilm can be noticed in any specimen.
Table II SEM observations of tube specimens Dental unit
1 2 3 4 5 6 7 8 9
Age
New New New New New Two months Three months 24 months 48 months
Internal surface covered (%)
Biofilm matrix thickness
Number of bacteria inside biofilm
Baseline
End
Baseline
End
Baseline
End
0 0 0 0 0 10 90 60 100
0 0 0 0 0 10 90 0 60
0 0 0 0 0 0.3 –0.5 0.3 –0.5 0.8 –1 2.8 –3.2
0 0 0 0 0 0.1– 0.3 0.3– 0.5 0 2.8– 3.2
0 0 0 0 0 500–550 400–450 400–450 600–650
0 0 0 0 0 35 15 0 45
Water line contamination
Figure 4 (A) Scanning electron microscopic images of dental unit water line lumens taken from the two-monthold dental unit: high numbers of bacteria are present inside a sparse amount of biofilm before the beginning of the study. (B) Only a few bacteria are visible after one month of undergoing repeated between-patient disinfecting cycles with peracetic acid.
repeated between-patient DUWL, disinfection cycles with Ster4spray (Figures 5 –7).
Discussion DUWLs, are highly contaminated both by heterotrophic bacteria originating from mains water piped to the dental unit and by oral micro-organisms present within the lumens of water line tubing during dental procedures due to suck-back associated with failure of anti-retraction devices in dental units.3,15 – 17 Although heterotrophic bacteria from the mains water are the most representative micro-organisms recovered in dental treatment water, there have been a small number of reports
301
Figure 5 (A) Scanning electron microscopic images of dental unit water line lumens taken from the threemonth-old dental unit: high numbers of bacteria are seen inside a biofilm matrix before the beginning of the study. (B) A thick matrix is still visible after one month of use undergoing repeated between-patient disinfecting cycles with peracetic acid, but only a few bacteria inside it can be detected.
showing oral micro-organisms; therefore the risk of cross-infection between patients exists.18 Several methods have been proposed in order to prevent the colonization of DUWL by heterotrophic organisms, as well as to control cross-contamination, but the most effective procedure has not been determined. The lack of standardized protocols to process DUWLs and water samples creates confusion and alters perceptions of water quality.19 The type of instrument selected to obtain DUWL samples, the great time-variability between samples within each dental unit, as well as the type of culture media, incubation times and temperatures are all proven factors that greatly influence both the number and the type of
302
L. Montebugnoli et al.
Figure 6 (A) Scanning electron microscopic images of dental unit water line lumens taken from the 24-monthold dental unit: a well established biofilm with bacteria inside can be detected before the beginning of the study. (B) No sign of biofilm after one month of undergoing repeated between-patient disinfecting cycles with peracetic acid.
Figure 7 (A) Scanning electron microscopic images of dental unit water line lumens taken from the 48-monthold dental unit: a strongly established biofilm is detectable before the beginning of the study. (B) A thick matrix is still visible after one month of undergoing repeated between-patient disinfecting cycles with peracetic acid, but only a few bacteria can be detected.
organisms recovered from a DUWL sample. As such they affect the evaluation of the efficacy of a disinfecting procedure, and when comparing different decontaminating procedures.20,21 This study was designed using the ‘worst-case scenario’ approach to testing the efficacy of the decontamination protocols. High-speed handpiece tubing has the smallest bores, which have highest potential for contamination.19 We measured contamination in each dental unit multiple times over each period to establish its mean contamination value, and applied the three different decontaminating procedures on the same dental units. Finally an appropriate cultural medium/method was selected.22 – 24 This study has confirmed that DUWLs are highly contamined if they have been in use over a period of
time without being subjected to methods of biofilm and treatment water contamination control. There have been no studies in the past demonstrating between-patient methods of chemical treatment to control both heterotrophic mesophilic organisms in mains water, and also those organisms originating from patients. Between-patient treatment addresses contamination from the mains water source, as well as potential cross-contamination between patients by oral microbes. The amount of contamination varied significantly from one dental unit to another. Although the rate of contamination appeared to decrease with every procedure performed within a day as a consequence of flushing water, less than 1% of samples reached levels below 200 cfu/mL.4 Flushing of DUWLs for 30 s before each patient provided significantly lower mean
Water line contamination
bacterial counts during the early phase in spite of over 98% of samples reaching counts higher than 200 cfu/mL. However, DUWL contamination increased with time, possibly due to detachment of micro-organisms from the biofilm, reaching mean values equivalent to those obtained in absence of any decontaminating procedure. These results are similar to data from the literature demonstrating that mechanical flushing alone is not efficacious in controlling microbial contamination.25,26 Conversely interesting results have been recently obtained by disinfecting water lines with peracetic acid left standing in DUWLs for 5 min before each dental procedure.27 In the present study all dental units showed adequate control of treatment water contamination both at the beginning and at the end of tile procedures. The mean colony-forming units per millilitre values were well below the ADA standard, with only about 9% being greater than 200 cfu/mL. SEM analysis of specimens treated with Sterspray showed no formation of biofilm in the new units, and a decrease in the number of bacteria in the biofilm samples of the older units with previously established biofilms. These data are in agreement with other studies, which adopted different treatment procedures.28 – 30 The use of chemicals introduced into water systems either continuously or intermittently to control DUWL contamination and biofilm inside dental units has been proposed.31,32 Continuous treatment has involved several chemical agents including chlorine compounds,33 chlorhexidine gluconate, 34 hydrogen peroxide, 35 povidone – iodine36 and commercial mouth rinses.37,38 Although continuous treatment with low-grade disinfectants offers a lower potential for recolonization of water lines, it may damage equipment, affect the healthcare worker or interfere with any dental materials.39,40 Furthermore, all the above-mentioned agents possess low biocidal activity when used at lower concentrations and do not deal with micro-organisms sucked back during dental procedures.3 Although DUWL decontamination and inhibition of biofilm formation have been achieved by overnight or pre-working treatments with chemical solutions, these procedures cannot ensure the control of micro-organisms sucked back during dental treatment.41 – 43 TAED with a peroxygen source at near neutral pH is claimed to provide a non-hazardous means of generating peracetic acid in situ. It is quite simple to prepare, has low inventory costs and low cost of working solution (about $7/L; $1/cycle); further it is non-damaging to instruments and is completely washed out from DUWL after 5 min contact. Preliminary data from a previous study con-
303
firmed the relevant biocidal in vitro activity of the test formulation, and data from the present study underline its efficacy in controlling heterotrophic DUML contamination during dental practice and biofilm formation inside DUWLs.12,27 Unlike preformed peracetic acid, glutaraldehyde or chlorine, solutions of TAED activated peroxide release no irritating fumes or unpleasant odours. In addition, TAED is traditionally formulated with a persalt to form solid, concentrated products with a long shelf life. In an attempt to control cross-contamination between patients, the test formulation could be very useful in dental units that incorporate automated devices to disinfect DUWLs with minimal effort from the dental staff.
References 1. Molinari JA. Dental infection control at the year 2000. J Am Dent Assoc 1999;130:1291—1298. 2. Smith AJ, Hood J, Bagg J. Water, water everywhere but not a drop to drink? Br Dent J 1999;186:12—14. 3. Walker JT, Bradshaw DJ, Bennett AM. Microbial biofilm formation and contamination of denial unit water systems in general dental practice. Appl Environ Microbiol 2000;66: 3363—3367. 4. Shearer BG. Biofilm and the dental office. J Am Dent Assoc 1996;127:181—189. 5. US Department of Health and Human Services. Recommended infection control practices for dentistry. MMWR 1993;42:RR-8. 6. Meiller TF, Depaola LG, Kelley JI. Dental unit waterlines: biofilms, disinfection and recurrence. J Am Dent Assoc 1999; 130:65—72. 7. Rutala WA, Weber DJ. Disinfection of endoscopes: review of new chemical sterilants used for high-level disinfection. Infect Control Hosp Epidemiol 1999;20:69—76. 8. Morin P. Identification of the bacteriological contamination of a water treatment line used for haemodialysis and its disinfection. J Hosp Infect 2000;45:218—224. 9. Griffiths PA, Babb JR, Fraise AP. Mycobactericidal activity of selected disinfectants using quantitative suspension test. J Hosp Infect 1999;41:111—121. 10. Middleton AM, Chadwick MV, Gaya H. Disinfection of bronchoscopes, contaminated in vitro with Mycobacterium tuberculosis, Mycobacterium avium-intracellulare and Mycobacterium chelonae in sputum, using stabilized, buffered peracetic acid solution (Nu-Cidex). J Hosp Infect 1997; 37:137—143. 11. Stanley P. Destruction of a glutaraldehyde-resistant mycobacterium by a peroxygen disinfectant. Am J Infect Control 1998;26:185. 12. Montebugnoli L, Vasconi L, Dolei G. Evaluation of a new chemical formulation for a rapid cold sterilization of dental instrument. J Dent Res 2000;79:3399. 13. Davis DM, Deary ME. Kinetics of the hydrolysis and perhydrolysis of tetraacetylethylenediamine, a peroxide bleach activator. J Chem Soc Perkin Trans 1991;2: 15491—15552. 14. Perdigao J, Lambrechts P, Van Meerbeek B. Morphological field emission—SEM study of the effect of six phosphoric acid
304
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
etching agents on human dentin. Dent Mater 1996;12: 262—271. Montebugnoli L, Dolci G. Effectiveness of two devices designed to prevent fluid retraction in a high-speed handpiece. J Prosthet Dent 2000;84:225—228. Putnins EE, Di Giovanni D, Bhullar AS. Dental unit water line contamination and its possible implications during periodontal surgery. J Periodontol 2001;372:393—400. Berlutti F, Testarelli L, Vaia F, De Luca MD, Dolei G. Efficacy of anti-retraction devices in preventing bacterial contamination of dental unit water lines. J Dent 2003;31:105—110. Mills S. The dental unit water controversy: defusing the myths, defining the solutions. J Am Dent Assoc 2000;131: 1427—1450. Noce L, DiGiovanni D, Putnins EF. An evaluation of sampling and laboratory procedures for determination of heterotrophic plate counts in dental unit waterlines. J Can Dent Assoc 2000;66:262—272. Barbeau J, Tanguay R, Faucher E, Avezard C, Trudel L, Cote L. Multiparametric analysis of water line contamination of dental units. Appl Environ Microbiol 1996;62:3954—3959. Zanetti F, Stampi S, DeLuea G, et al. Water characteristics associated with the occurrence of Legionella pneumophila in dental units. Eur J Oral Sci 2000;108:22—28. Williams HN, Kelley J, Folineo D. Assessing microbial contamination in clean water dental units and compliance with disinfection protocol. J Am Dent Assoc 1994;125: 1205—1211. Karpay RI, Plamondon TJ, Mills SE. Validation of an in-office dental unit monitoring technique. J Am Dent Assoc 1998; 129:207—211. Williams HN, Quinby H, Romberg E. Evaluation and use of a low nutrient medium and reduced incubation temperature to study bacterial contamination in the water supply of dental units. Can J Microbiol 1994;40:127—131. Williams HN, Baer ML, Kelley JL. Contribution of biofilm bacteria to the contamination of the dental unit water supply. J Am Dent Assoc 1995;126:1255—1260. Santiago JI, Huntington MK, Johnston MA. Microbial contamination of dental unit water lines: short and long-term effects of flushing. Gen Dent 1994;45:528—535. Montebugnoli L, Dolci G. A new chemical formulation for control of dental unit water line contamination: an ‘in vitro’ and clinical study. BMC Oral Health 2002;2:1—4. Kim PJ, Cederberg RA, Puttaiah R. A pilot study of 2 methods for control of dental unit biofilms. Quintessence Int 2000;31: 41—48.
L. Montebugnoli et al.
29. Wirthlin MR, Marshall GW. Evaluation of ultrasonic scaling unit water line contamination after use of chlorine dioxide mouthrinse lavage. J Periodontol 2001;72:401—410. 30. Meiller TF, Kelley JI, Baqui AA, et al. Laboratory evaluation of anti-biofilm agents for use in dental unit water lines. J Clin Dent 2001;12:97—103. 31. Fayle SA, Pollard MA. Decontamination of dental unit water system: a review of current recommendations. Br Dent J 1996;181:369—372. 32. Marais JT, Brozel VS. Electro-chemically activated water in dental unit water lines. Br Dent J 1999;187:154—158. 33. Karpay RI, Plamondon TJ, Mills SE. Combining periodic and continuous sodium hypochlorite treatment to control biofilms in dental unit water systems. J Am Dent Assoc 1999; 130:957—965. 34. Blake GC. The incidence and control of infection in dental spray reservoirs. Br Dent J 1963;115:413—420. 35. Kellet M, Holbrook WP. Bacterial contamination of dental handpieces. J Dent 1980;8:249—253. 36. Mills SE, Lauderdale PW, Mayhew RB. Reduction of microbial contamination in dental units with povidone—iodine 10%. J Am Dent Assoc 1986;113:280—284. 37. Meiller T, Baqui A, DePaola I, Overholser CD. Disinfection of dental unit water lines using Listerine antiseptic. J Dent Res 1995;74:153. 38. Eleazer PD, Schuster GS, Weathers DR. A chemical treatment, regimen to reduce bacterial contamination in dental waterlines. J Am Dent Assoc 1997;128:617—623. 39. Bull RJ, Birnbaum LS, Cantor KP. Water chlorination: essential process of cancer hazard? Fundam Appl Toxicol 1995;28:155—166. 40. Roberts HW, Karpay RI, Mills SE. Dental unit water line antimicrobial agents’ effect on dentin bond strength. J Am Dent Assoc 2000;131:179—183. 41. Waslker JT, Bradshaw DJ, Fulford MR, Marsh PD. Microbiological evaluation of a range of disinfectant product to control mixed-species biofilm contamination in a laboratory model of a dental unit water system. Appl Environ Microbiol 2003;69:3327—3332. 42. Smith AJ, McHugh S, Aitken I, Hood J. Evaluation of the efficacy of Alpron disinfectant for dental unit water lines. Br Dent J 2003;194:64—65. 43. Tuttlebee CM, O’Donnelll MJ, Keane CT, et al. Effective control of dental chair unit waterline biofilm and marked reduction of bacterial contamination of output water using two peroxide-based disinfectants. J Hosp Infect 2002;52: 192—205.
www.elsevierhealth.com/journals/jhin
A between-patient disinfection method to control water line contamination and biofilm inside dental units L. Montebugnolia,*, S. Chersonia, C. Pratia, G. Dolcib a
Department of Oral Science, University of Bologna, Via S. Vitale 59, 40125 Bologna, Italy University of Rome “La Sapienza”, Rome, Italy
b
Received 28 July 2003; accepted 16 December 2003
KEYWORDS Biofilm(s); Dental unit; Cross-contamination; Disinfection; Heterotrophs; Infection control
Summary The aim of the present study was to evaluate the efficacy of a between-patient disinfection procedures to maintain low bacterial counts in dental unit water line (DUWL) effluents, and control dental water line biofilms. Six dental units already in use, that had never been cleaned, were monitored for three weeks. During the first week only baseline contamination levels were assessed with no treatment of the system. In the second week lines were flushed with water for 30 s before treating each patient. During the third week, a disinfection procedure with 0.26% peracetic acid, followed by a water flush, was implemented before treating each patient. DUWL samples were collected both at the beginning and at the end of 216 dental procedures (72 during each period), plated on R2A agar and incubated at room temperature for seven days to obtain total bacterial counts in colony forming units per millilitre. To assess biofilm control, nine dental units (five never used and four old dental units with established biofilm) were used for 30 days in routine dental practice undergoing five between-patient DUWL disinfecting cycles every day. Water line samples were removed at baseline and at the end of the study and examined by scanning electron microscopy to determine the presence or absence of biofilms. A significant difference ðP , 0:01Þ in mean DUWL bacterial counts was found between the three sets of observations. Biofilms were not present in any of the new dental units and a demonstrable reduction in the biofilms from the four dental units with previous presence of established biofilms was observed at the end of the study. In this study, a between-patient disinfection procedure consisting of flushing DUWL with peracetic acid with use of water was efficacious in the control of both microbial contamination of dental treatment water and dental water line biofilms. Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Tel.: þ 39-51-278006; fax: þ39-51225208. E-mail address: [email protected] 0195-6701/$ - see front matter Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2004.01.015
298
Introduction It has been known for more than 30 years that treatment water, delivered by dental units during routine practice of dentistry, is highly contaminated by numerous species of pathogenic and nonpathogenic micro-organisms. The organisms enter dental units from the mains water source, or could be retracted into the dental unit water lines (DUWLs) from the oral cavity of patients during routine dental care,1 – 3 These heterotrophic bacteria persist in DUWLs as a multispecies biofilm on the lumenal surface, contaminating the dental unit water system. In 1996 the American Dental Association established a goal for treatment water to contain no more than 200 cfu/mL of heterotrophic unfiltered output.4 Several methods have been suggested through which the DUWL contamination by heterotrophic mesophilic organisms may be kept under this limit including flushing protocols and chemical treatment.5,6 Of the chemicals now available, peracetic acid has a rapid and broad-spectrum biocidal activity that could be useful in controlling DUWL contamination between patient treatments,7,8 but, as delivered, it has a series of side effects limiting its use in dentistry.9 – 11 However, a new chemical formulation (tetraacetylethylenediamine (TAED) in association with persalt) has been recently proposed as a non-hazardous means of generating peracetic acid in situ. This form of peracetic acid does not have the same side effects as pre-formed peracetic. acid.12,13 The aim of this study was to evaluate the efficacy of peracetic acid in controlling heterotrophic bacterial contamination when used between patient treatments and compare the results with those achieved through pure mechanical flushing with mains water, or in the absence of any treatment. An additional aim was to evaluate its long-term efficacy in controlling DUWL biofilms.
Method and materials To study the control of dental treatment water contamination, six dental units (Castellini Logos, Castellini S.p.A., Bologna, Italy) that had been in use for over one year were selected. These units were directly connected to mains water as a source for irrigation. None of these units had been treated to remove biofilm or reduce planktonic bacterial contamination. During the first week all units received no treatment (control), during the second week mechanical flushing with mains water for 30 s
L. Montebugnoli et al.
before each dental procedure (flushing) was implemented. During the third week a disinfecting cycle (disinfection) was initiated with each dental unit before dental treatment session. The disinfection cycle consisted of an automated procedure by a device integral to each dental unit (Autosteril, designed by Castellini S.p.A., Bologna, Italy) programmed to flush the disinfectant solution (contained in a built-in talk) through the water system and purge with water. The Autosteril procedure consisted of purging the residual dental treatment water loading the system with Ster4spray (manufactured by Farmed S.p.A and distributed by Castellini S.p.A., Bologna, Italy) for 5 min of contact, purging the chemical and finally flushing with water. Ster4spray is a fine powder containing a binary active system (TAED and sodium perborate), which is activated by dissolving in water at a temperature above 35 8C to from peracetyl ions at pH 8, equivalent to 0.26% in peracetic acid, ensuring a stable concentration for up to 24 h. The active ingredient is completely biodegradable and degrades to acetic acid, oxygen and water.13 Each dental unit was used for 12 dental procedures (requiring the use of the high-speed handpiece) every week for a total of 36 procedures. The number of dental procedures per day and per week was standardized between and within dental units. The total number of dental procedures during the three periods was 216 (72 each for control, flush and disinfection periods). An external evaluator observed and audited the time for each dental procedure and that of the handpiece use. Water samples were collected from the high-speed handpiece lines of each dental unit at the beginning and at the end of each dental procedure for a total of 432 samples. Each sample was processed to evaluate the total number of heterotrophic micro-organisms. Two millilitres of water from each unit were collected in a sterile tube with filter-sterilized sodium thiosulphate at a final concentration of 18 mg/L to oppose the growth-inhibiting effects of residual chlorine. Samples were plated on R2A Agar (Difco, Becton Dickinson, Le Pont de Claix, France) within 3 h of collection and incubated at room temperature (22 –24 8C) for seven days and the total number of heterotrophic micro-organisms counted. All absolute counts were converted to log10 values to normalize data. A one-way analysis of variance (ANOVA) was used to study differences between heterotrophic plate counts values, length of the dental session and duration of handpiece usage for the three periods. After fitting a general linear model, multiple regression ANOVA for repeated measures with split-plot design was used to
Water line contamination
evaluate differences between dental units, procedures (control, flush and disinfection periods) time (colony forming units per millilitre at the beginning and at the end of dental treatments regardless of the decontaminating procedure adopted) and the interaction of decontaminating procedure multiplied by the time; the post hoc test applied was Bonferroni t-test for multiple-comparisons at a ¼ 0:05: To evaluate biofilm removal, nine Castellini Logos dental units (five new dental units and four in use for 2, 3, 24 and 48 months) were used. All dental units were supplied by mains water and were used in routine dental practice for 30 days during which they were subjected to repeated betweenpatient DUWL disinfection cycles with Ster4spray (approximately 5 cycles/day). The disinfection cycle was identical to the protocol stated earlier. A 10 nm section of the water line tubing connected to the high-speed handpiece was removed from each dental unit at baseline and at the end of the study period, fixed in 3% glutaraldehyde in a 0.2 M sodium cacodylate buffer, treated with increasing concentration of ethyl alcohol (05 –95 and finally 100%), desiccated overnight using hexamethyldisilazane following the method of Perdigao et al.,14 mounted on a stud, coated with 10 nm gold and viewed using scanning electron microscopy (SEM; Jeol 5400, Jeol Japan, operating at 5 and 10 kV at magnification ranging £ 500 – 5000. Biofilm was quantified using the following criteria: (1) extension (% surface covered by biofilm at £ 500), (2) matrix thickness (microns), and (3) number of visible bacteria per 500 mm2 at £ 5000).
299
Table I The results of fitting a general linear statistical model relating log10 colony forming units per millilitre to three predictive factors and their interaction using analysis of variance Source
F-ratio
P-value
Dental units Procedures Time Proceduresa £ time
9.63 799.53 0.06 11.59
0.0001 0.0001 0.801 0.0001
a
Significant differences in log10 cfu/mL were found between dental units, procedures and the interaction of procedures multiplied by the time.
cedure before dental treatment (control), or flush with tap water (flush) or DUWL disinfection cycles (disinfection). Among the water samples tested 143 out of 144 samples showed bacterial counts higher than 200 cfu/mL (ADA’s goal for the year 2000) in the control period, 142 out of 144 in the flush period and only 12 out of 144 in the disinfection period. Figure 2 shows the mean values of samples collected in the three different study periods. Means (^ SD) log10 colony-forming units per millilitre from effluent water samples at the beginning of dental procedures were 4.39 ^ .66 (range 2.0 to 5.79) for the control period, 3.73 ^ .65 (range 2.3 to 4.81) for the flush period and 1.02 ^ .94 (range not-detectable to 2.86) during the disinfection period. The mean log10 colony-forming units per millilitre at the end of dental procedures were 3.94 ^ .50 (range 2.6 to 5.53) in the control period, 4.03 ^ .64 (range 2.0 to 5.14) for the flush period and 1.22 ^ .98 (range not detectable to 3.20) for the disinfection period. The time-related decrease
Results No significant differences between the three groups were detected with reference to duration (in minutes) of dental treatment sessions (control 33.7 ^ 12.4, flush 30.9 ^ 11.4, and disinfection 32.0 ^ 13.8, F ¼ 0:84; P . 0:05). The duration of use of handpiece in minutes also showed no significant difference within each dental session (control 5.9 ^ 1.9, flush 6.2 ^ 2.0, and disinfection 5.5 ^ 2.4, F ¼ 1:12; P . 0:05). Multiple regression ANOVA showed significant differences in the log10 reduction of colony forming units between dental units, treatment procedures and the interaction between procedures and time (P , 0.05; Table I). Figure 1 shows log10 colony-forming units per millilitre means and 95% confidence intervals in samples collected during dental sessions from dental units undergoing no decontaminating pro-
Figure 1 Means of colony forming units per millilitre (in Log10) and 95% confidence intervals in samples collected during all dental sessions from dental units undergoing no decontaminating procedure before dental treatments (control), DUWL flushing (flushing), or DUWL disinfecting cycles (disinfection) before dental treatments.
300
L. Montebugnoli et al.
Figure 2 Means of colony forming units per millilitre values (in Log10) of samples collected at the beginning and at the end of dental sessions during the three different periods (control, flushing, and disinfection).
in mean bacterial counts during the control period ðF ¼ 22:12Þ and the time-related increase in mean bacterial counts during the flushing period ðF ¼ 20:01Þ were statistically significant, while no significant variation with time was found during the disinfection period ðF ¼ 1:72Þ; no significant difference was detected between control and flushing periods in the mean contamination levels at the end of the respective dental treatment sessions. Table II shows the results of SEM using the three parameters for quantification of biofilm. No biofilm was observed in all new dental units after 30 days of use in routine dental practice (Figure 3). The 24month-old units showed complete removal of biofilm (Figure 4). A partial reduction in the biofilm matrix was observed in one of the remaining three dental units, and with a small percentage decrease in one out of three. A greater decrease in the number of bacteria in biofilms was seen in all tube specimens from old dental units after 30 days of
Figure 3 (A) Scanning electron microscopic images of dental unit water line lumens taken from one of five new dental units before the installation and (B) after one month of use undergoing repeated between-patient disinfecting cycles with peracetic acid. The network of fine lines is probably related to the wall texture of the tubing; no sign of biofilm can be noticed in any specimen.
Table II SEM observations of tube specimens Dental unit
1 2 3 4 5 6 7 8 9
Age
New New New New New Two months Three months 24 months 48 months
Internal surface covered (%)
Biofilm matrix thickness
Number of bacteria inside biofilm
Baseline
End
Baseline
End
Baseline
End
0 0 0 0 0 10 90 60 100
0 0 0 0 0 10 90 0 60
0 0 0 0 0 0.3 –0.5 0.3 –0.5 0.8 –1 2.8 –3.2
0 0 0 0 0 0.1– 0.3 0.3– 0.5 0 2.8– 3.2
0 0 0 0 0 500–550 400–450 400–450 600–650
0 0 0 0 0 35 15 0 45
Water line contamination
Figure 4 (A) Scanning electron microscopic images of dental unit water line lumens taken from the two-monthold dental unit: high numbers of bacteria are present inside a sparse amount of biofilm before the beginning of the study. (B) Only a few bacteria are visible after one month of undergoing repeated between-patient disinfecting cycles with peracetic acid.
repeated between-patient DUWL, disinfection cycles with Ster4spray (Figures 5 –7).
Discussion DUWLs, are highly contaminated both by heterotrophic bacteria originating from mains water piped to the dental unit and by oral micro-organisms present within the lumens of water line tubing during dental procedures due to suck-back associated with failure of anti-retraction devices in dental units.3,15 – 17 Although heterotrophic bacteria from the mains water are the most representative micro-organisms recovered in dental treatment water, there have been a small number of reports
301
Figure 5 (A) Scanning electron microscopic images of dental unit water line lumens taken from the threemonth-old dental unit: high numbers of bacteria are seen inside a biofilm matrix before the beginning of the study. (B) A thick matrix is still visible after one month of use undergoing repeated between-patient disinfecting cycles with peracetic acid, but only a few bacteria inside it can be detected.
showing oral micro-organisms; therefore the risk of cross-infection between patients exists.18 Several methods have been proposed in order to prevent the colonization of DUWL by heterotrophic organisms, as well as to control cross-contamination, but the most effective procedure has not been determined. The lack of standardized protocols to process DUWLs and water samples creates confusion and alters perceptions of water quality.19 The type of instrument selected to obtain DUWL samples, the great time-variability between samples within each dental unit, as well as the type of culture media, incubation times and temperatures are all proven factors that greatly influence both the number and the type of
302
L. Montebugnoli et al.
Figure 6 (A) Scanning electron microscopic images of dental unit water line lumens taken from the 24-monthold dental unit: a well established biofilm with bacteria inside can be detected before the beginning of the study. (B) No sign of biofilm after one month of undergoing repeated between-patient disinfecting cycles with peracetic acid.
Figure 7 (A) Scanning electron microscopic images of dental unit water line lumens taken from the 48-monthold dental unit: a strongly established biofilm is detectable before the beginning of the study. (B) A thick matrix is still visible after one month of undergoing repeated between-patient disinfecting cycles with peracetic acid, but only a few bacteria can be detected.
organisms recovered from a DUWL sample. As such they affect the evaluation of the efficacy of a disinfecting procedure, and when comparing different decontaminating procedures.20,21 This study was designed using the ‘worst-case scenario’ approach to testing the efficacy of the decontamination protocols. High-speed handpiece tubing has the smallest bores, which have highest potential for contamination.19 We measured contamination in each dental unit multiple times over each period to establish its mean contamination value, and applied the three different decontaminating procedures on the same dental units. Finally an appropriate cultural medium/method was selected.22 – 24 This study has confirmed that DUWLs are highly contamined if they have been in use over a period of
time without being subjected to methods of biofilm and treatment water contamination control. There have been no studies in the past demonstrating between-patient methods of chemical treatment to control both heterotrophic mesophilic organisms in mains water, and also those organisms originating from patients. Between-patient treatment addresses contamination from the mains water source, as well as potential cross-contamination between patients by oral microbes. The amount of contamination varied significantly from one dental unit to another. Although the rate of contamination appeared to decrease with every procedure performed within a day as a consequence of flushing water, less than 1% of samples reached levels below 200 cfu/mL.4 Flushing of DUWLs for 30 s before each patient provided significantly lower mean
Water line contamination
bacterial counts during the early phase in spite of over 98% of samples reaching counts higher than 200 cfu/mL. However, DUWL contamination increased with time, possibly due to detachment of micro-organisms from the biofilm, reaching mean values equivalent to those obtained in absence of any decontaminating procedure. These results are similar to data from the literature demonstrating that mechanical flushing alone is not efficacious in controlling microbial contamination.25,26 Conversely interesting results have been recently obtained by disinfecting water lines with peracetic acid left standing in DUWLs for 5 min before each dental procedure.27 In the present study all dental units showed adequate control of treatment water contamination both at the beginning and at the end of tile procedures. The mean colony-forming units per millilitre values were well below the ADA standard, with only about 9% being greater than 200 cfu/mL. SEM analysis of specimens treated with Sterspray showed no formation of biofilm in the new units, and a decrease in the number of bacteria in the biofilm samples of the older units with previously established biofilms. These data are in agreement with other studies, which adopted different treatment procedures.28 – 30 The use of chemicals introduced into water systems either continuously or intermittently to control DUWL contamination and biofilm inside dental units has been proposed.31,32 Continuous treatment has involved several chemical agents including chlorine compounds,33 chlorhexidine gluconate, 34 hydrogen peroxide, 35 povidone – iodine36 and commercial mouth rinses.37,38 Although continuous treatment with low-grade disinfectants offers a lower potential for recolonization of water lines, it may damage equipment, affect the healthcare worker or interfere with any dental materials.39,40 Furthermore, all the above-mentioned agents possess low biocidal activity when used at lower concentrations and do not deal with micro-organisms sucked back during dental procedures.3 Although DUWL decontamination and inhibition of biofilm formation have been achieved by overnight or pre-working treatments with chemical solutions, these procedures cannot ensure the control of micro-organisms sucked back during dental treatment.41 – 43 TAED with a peroxygen source at near neutral pH is claimed to provide a non-hazardous means of generating peracetic acid in situ. It is quite simple to prepare, has low inventory costs and low cost of working solution (about $7/L; $1/cycle); further it is non-damaging to instruments and is completely washed out from DUWL after 5 min contact. Preliminary data from a previous study con-
303
firmed the relevant biocidal in vitro activity of the test formulation, and data from the present study underline its efficacy in controlling heterotrophic DUML contamination during dental practice and biofilm formation inside DUWLs.12,27 Unlike preformed peracetic acid, glutaraldehyde or chlorine, solutions of TAED activated peroxide release no irritating fumes or unpleasant odours. In addition, TAED is traditionally formulated with a persalt to form solid, concentrated products with a long shelf life. In an attempt to control cross-contamination between patients, the test formulation could be very useful in dental units that incorporate automated devices to disinfect DUWLs with minimal effort from the dental staff.
References 1. Molinari JA. Dental infection control at the year 2000. J Am Dent Assoc 1999;130:1291—1298. 2. Smith AJ, Hood J, Bagg J. Water, water everywhere but not a drop to drink? Br Dent J 1999;186:12—14. 3. Walker JT, Bradshaw DJ, Bennett AM. Microbial biofilm formation and contamination of denial unit water systems in general dental practice. Appl Environ Microbiol 2000;66: 3363—3367. 4. Shearer BG. Biofilm and the dental office. J Am Dent Assoc 1996;127:181—189. 5. US Department of Health and Human Services. Recommended infection control practices for dentistry. MMWR 1993;42:RR-8. 6. Meiller TF, Depaola LG, Kelley JI. Dental unit waterlines: biofilms, disinfection and recurrence. J Am Dent Assoc 1999; 130:65—72. 7. Rutala WA, Weber DJ. Disinfection of endoscopes: review of new chemical sterilants used for high-level disinfection. Infect Control Hosp Epidemiol 1999;20:69—76. 8. Morin P. Identification of the bacteriological contamination of a water treatment line used for haemodialysis and its disinfection. J Hosp Infect 2000;45:218—224. 9. Griffiths PA, Babb JR, Fraise AP. Mycobactericidal activity of selected disinfectants using quantitative suspension test. J Hosp Infect 1999;41:111—121. 10. Middleton AM, Chadwick MV, Gaya H. Disinfection of bronchoscopes, contaminated in vitro with Mycobacterium tuberculosis, Mycobacterium avium-intracellulare and Mycobacterium chelonae in sputum, using stabilized, buffered peracetic acid solution (Nu-Cidex). J Hosp Infect 1997; 37:137—143. 11. Stanley P. Destruction of a glutaraldehyde-resistant mycobacterium by a peroxygen disinfectant. Am J Infect Control 1998;26:185. 12. Montebugnoli L, Vasconi L, Dolei G. Evaluation of a new chemical formulation for a rapid cold sterilization of dental instrument. J Dent Res 2000;79:3399. 13. Davis DM, Deary ME. Kinetics of the hydrolysis and perhydrolysis of tetraacetylethylenediamine, a peroxide bleach activator. J Chem Soc Perkin Trans 1991;2: 15491—15552. 14. Perdigao J, Lambrechts P, Van Meerbeek B. Morphological field emission—SEM study of the effect of six phosphoric acid
304
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
etching agents on human dentin. Dent Mater 1996;12: 262—271. Montebugnoli L, Dolci G. Effectiveness of two devices designed to prevent fluid retraction in a high-speed handpiece. J Prosthet Dent 2000;84:225—228. Putnins EE, Di Giovanni D, Bhullar AS. Dental unit water line contamination and its possible implications during periodontal surgery. J Periodontol 2001;372:393—400. Berlutti F, Testarelli L, Vaia F, De Luca MD, Dolei G. Efficacy of anti-retraction devices in preventing bacterial contamination of dental unit water lines. J Dent 2003;31:105—110. Mills S. The dental unit water controversy: defusing the myths, defining the solutions. J Am Dent Assoc 2000;131: 1427—1450. Noce L, DiGiovanni D, Putnins EF. An evaluation of sampling and laboratory procedures for determination of heterotrophic plate counts in dental unit waterlines. J Can Dent Assoc 2000;66:262—272. Barbeau J, Tanguay R, Faucher E, Avezard C, Trudel L, Cote L. Multiparametric analysis of water line contamination of dental units. Appl Environ Microbiol 1996;62:3954—3959. Zanetti F, Stampi S, DeLuea G, et al. Water characteristics associated with the occurrence of Legionella pneumophila in dental units. Eur J Oral Sci 2000;108:22—28. Williams HN, Kelley J, Folineo D. Assessing microbial contamination in clean water dental units and compliance with disinfection protocol. J Am Dent Assoc 1994;125: 1205—1211. Karpay RI, Plamondon TJ, Mills SE. Validation of an in-office dental unit monitoring technique. J Am Dent Assoc 1998; 129:207—211. Williams HN, Quinby H, Romberg E. Evaluation and use of a low nutrient medium and reduced incubation temperature to study bacterial contamination in the water supply of dental units. Can J Microbiol 1994;40:127—131. Williams HN, Baer ML, Kelley JL. Contribution of biofilm bacteria to the contamination of the dental unit water supply. J Am Dent Assoc 1995;126:1255—1260. Santiago JI, Huntington MK, Johnston MA. Microbial contamination of dental unit water lines: short and long-term effects of flushing. Gen Dent 1994;45:528—535. Montebugnoli L, Dolci G. A new chemical formulation for control of dental unit water line contamination: an ‘in vitro’ and clinical study. BMC Oral Health 2002;2:1—4. Kim PJ, Cederberg RA, Puttaiah R. A pilot study of 2 methods for control of dental unit biofilms. Quintessence Int 2000;31: 41—48.
L. Montebugnoli et al.
29. Wirthlin MR, Marshall GW. Evaluation of ultrasonic scaling unit water line contamination after use of chlorine dioxide mouthrinse lavage. J Periodontol 2001;72:401—410. 30. Meiller TF, Kelley JI, Baqui AA, et al. Laboratory evaluation of anti-biofilm agents for use in dental unit water lines. J Clin Dent 2001;12:97—103. 31. Fayle SA, Pollard MA. Decontamination of dental unit water system: a review of current recommendations. Br Dent J 1996;181:369—372. 32. Marais JT, Brozel VS. Electro-chemically activated water in dental unit water lines. Br Dent J 1999;187:154—158. 33. Karpay RI, Plamondon TJ, Mills SE. Combining periodic and continuous sodium hypochlorite treatment to control biofilms in dental unit water systems. J Am Dent Assoc 1999; 130:957—965. 34. Blake GC. The incidence and control of infection in dental spray reservoirs. Br Dent J 1963;115:413—420. 35. Kellet M, Holbrook WP. Bacterial contamination of dental handpieces. J Dent 1980;8:249—253. 36. Mills SE, Lauderdale PW, Mayhew RB. Reduction of microbial contamination in dental units with povidone—iodine 10%. J Am Dent Assoc 1986;113:280—284. 37. Meiller T, Baqui A, DePaola I, Overholser CD. Disinfection of dental unit water lines using Listerine antiseptic. J Dent Res 1995;74:153. 38. Eleazer PD, Schuster GS, Weathers DR. A chemical treatment, regimen to reduce bacterial contamination in dental waterlines. J Am Dent Assoc 1997;128:617—623. 39. Bull RJ, Birnbaum LS, Cantor KP. Water chlorination: essential process of cancer hazard? Fundam Appl Toxicol 1995;28:155—166. 40. Roberts HW, Karpay RI, Mills SE. Dental unit water line antimicrobial agents’ effect on dentin bond strength. J Am Dent Assoc 2000;131:179—183. 41. Waslker JT, Bradshaw DJ, Fulford MR, Marsh PD. Microbiological evaluation of a range of disinfectant product to control mixed-species biofilm contamination in a laboratory model of a dental unit water system. Appl Environ Microbiol 2003;69:3327—3332. 42. Smith AJ, McHugh S, Aitken I, Hood J. Evaluation of the efficacy of Alpron disinfectant for dental unit water lines. Br Dent J 2003;194:64—65. 43. Tuttlebee CM, O’Donnelll MJ, Keane CT, et al. Effective control of dental chair unit waterline biofilm and marked reduction of bacterial contamination of output water using two peroxide-based disinfectants. J Hosp Infect 2002;52: 192—205.