Waterlines
Reducing Bacterial Counts in Dental Unit Waterlines: Distilled Water vs. Antimicrobial Agents
James D. Kettering, PhD; Carlos A. Muñoz-Viveros, DDS, MSD;
Joni A. Stephens, RDH, EdS; W. Patrick Naylor, DDS, MPH, MS; Wu Zhang,
MD
Copyright 2002 Journal of the California Dental Association.
Authors
James D. Kettering, PhD, is professor of microbiology and molecular genetics at the Schools of Medicine and Dentistry at Loma Linda University.
Carlos A. Muñoz-Viveros, DDS, MSD, is professor and director of the Center for Dental Research at LLU School of Dentistry.
Joni A. Stephens, RDH, EdS, is a professor of dental hygiene at LLU School of Dentistry.
W. Patrick Naylor, DDS, MPH, MS, is an adjunct professor in the Department of Restorative Dentistry at LLU School of Dentistry.
Wu Zhang, MD, is an assistant professor in the Department of Dental Educational Services at LLU School of Dentistry
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Background. This study evaluated five chemical disinfectants to compare their abilities to improve dental unit waterline quality and assess their effects, if any, on the biofilm layer. Methods. Sixty new dental units, with a closed-circuit water system, were used to compare microbial levels in DUWLs treated with five antimicrobials: Listerine, Bio 2000, Rembrandt, Dentosept, and sodium fluoride to a control group of sterile distilled water alone over a six-week period. For all units, the waterlines were filled with solution, left overnight, and then flushed for 30 seconds with sterile distilled water the following morning prior to patient treatment. Waterlines were examined for biofilm buildup using scanning electron microscopy and colony-forming-unit counts. Results. The sodium fluoride and the four chemical antimicrobials reduced the microbial count to 200 cfu/ml or less. Only samples taken from dental units receiving the control treatment (distilled water with no added antimicrobial) failed to meet ADA’s stated goal. Examination of the SEMs revealed an apparent decrease in the biofilm mass but not elimination, despite repeated treatment with the four antimicrobial materials. Conclusions. Even in a closed-circuit water system, distilled water alone cannot reduce microbial contamination of dental treatment water from dental unit waterlines to the 200 cfu/ml ADA stated goal. However, water treated with Listerine mouthrinse, Rembrandt mouthrinse, Bio 2000, 0.5 percent sodium fluoride, and Dentosept, it did meet microbial reduction goal. The biofilm apparently was reduced in volume, but not entirely eliminated. Clinical Significance. The ADA goal of a maximum of 200 cfu/ml was achieved using any of five chemical antimicrobials and distilled water in a closed-water system. Despite the successful reduction in microbial contamination of the dental treatment water, the biofilm was not completely eliminated. Biofilm elimination and prevention would be needed through some other means. |
A number of reports have been published on the issues surrounding microbial contamination of dental unit waterlines and techniques to achieve the American Dental Association stated goal of 200 colony forming units for dental treatment water.1-16 Kettering and colleagues15 demonstrated the critical role water source selection plays in achieving this 200 cfu/ml goal. In this study, bacterial contamination of dental unit waterlines was affected by water source selection (tap water vs. sterile distilled water), the system design (open system drawing tap water vs. a closed system using bottled, sterile distilled water), and whether the waterlines were treated with chemical disinfectants.16
Unfortunately, the actual mechanics for consistently obtaining and maintaining dental unit waterline quality have been left to the research community to determine. It has even been argued that dental unit manufacturers should provide the profession with remedies to meet the waterline quality standards rather than dental clinicians doing so.18 Regardless of where the answers to this problem might come from, the costs and responsibilities for addressing this multifaceted subject remain with the end user -- the clinician.
Even if dental units were redesigned to prevent microbial contamination of dental treatment water, conversion to these new systems would be both slow and costly. So research must continue to evaluate techniques and products that can provide water quality with existing dental equipment that meets or exceeds the ADA goal. Achieving this goal reduction will not only benefit the dental profession but also reassure patients as to the quality and safety of dental treatment water.
In Kettering and colleagues, 16 0.12 percent chlorhexidine gluconate (Bio2000, now marketed under the name of BioBLUE, manufactured by Micrylium Laboratories, Inc, Phoenix, Ariz.) was found to be effective in achieving the recommended reduction of microbial contaminants to 200 cfu/ml in dental unit waterlines while a 5.25 percent sodium hypochlorite (0.31 percent dilution of household bleach) was not. But several questions remained. Do differences exist between antimicrobial products? If so, which products can be used for DUWL treatment? What effects, if any, do these chemical products have on the biofilm that forms over time in dental unit waterlines? This study was undertaken to assess the effectiveness of sodium fluoride and four commercially available materials marketed as antimicrobial agents.
Materials and Methods
Sixty new dental units with a closed-circuit water system (Adec, model Decade 1021) were used in this study. Water samples were collected from the air/water syringe and the high-speed handpiece waterlines at baseline and two, four, and six weeks. Prior to the initiation of the study, the dental units were used with tap water for two weeks without any other treatment. At the start of the study, the water bottle containers of each dental unit were sterilized. The day the samples were collected from the waterlines, the lines were flushed for 30 seconds, and approximately 50 ml of water was collected in a sterile, plastic container. All the samples were collected at noon. When the collection was completed, no handpiece or syringe tip was used because the authors did not want to introduce another potential source of contamination and/or variable. For all units, the waterlines were filled with solution, left overnight, and then flushed for 30 seconds with sterile distilled water the following morning prior to patient treatment. During the day, all the dental units used sterile distilled water in the bottle containers. No municipal tap water was used at any time during this portion of the investigation. Careful attention was paid to avoid possible contamination of the water when the samples were collected. After collection, the water samples were immediately sent to the microbiology laboratory. The specimens were stored at 4 degrees Celsius and cultured within 24 hours.
The dental units were randomly assigned to one of the following six groups (Table 1) with 10 units in each group:
* Group 1 -- sterile distilled water with no antimicrobial (control group);
* Group 2 -- original Listerine mouthrinse;
* Group 3 -- 0.5 percent sodium fluoride;
* Group 4 -- Rembrandt mouthrinse;
* Group 5 -- Bio2000 (Now called BioBLUE) (0.12 percent chlorhexidine gluconate); and
* Group 6 -- Dentosept.
Microbial Culturing Protocol
Each water container was labeled but coded so the evaluators were blind to its contents. The labeled water samples were mixed by vortexing for 30 seconds. Fifty μl of liquid was removed and added to 100 ml of sterile, deionized water (Suspension 1). One hundred μl was removed from suspension 1 and added to a second 100 milliliters of sterile water (Suspension 2). Each addition was mixed thoroughly. The 100 ml suspensions were filtered through separate 47 mm membrane filters (MicroFunnel, Gelman Sciences, Ann Arbor, Mich.), and the filter was removed aseptically to a sterile R2A agar plate (R2A Agar, Difco, Becton Dickinson Microbiology Systems, Sparks, Md.). The plates were incubated at room temperature for five days. Bacterial colonies were counted by one investigator and dilution factors applied (20 for Suspension 1 and 2000 for Suspension 2) to obtain cfu/ml values.
In an effort to evaluate the presence or absence of a biofilm, 2 cm sections of waterline were removed from two samples from each group, air dried for 24 hours, and sputter-coated with gold for viewing in a scanning electron microscope. The samples were examined in a Phillips scanning electron microscope XL30, and photomicrographs were taken at 8000x magnification.
Statistical Methods
A one-way analysis of variance was performed to evaluate the change in cfu/ml over time for each of the five treatment groups (p<0.05). When differences were found, a Student-Newman-Kuels all pairwise multiple comparison was used to identify those differences.
Results
The study results for the comparison of outcomes for Groups 1 to 6 are presented in Table 2 and illustrated graphically in Figures 1 and 2 for the dental handpiece and the air/water syringe waterlines, respectively. All three mouthrinses (Listerine, sodium fluoride, and Rembrandt) and the two antimicrobials (Bio2000 and Dentosept) reduced the bacterial count to 200 cfu/ml or less. All values were well within the American Dental Association recommended level of 200 cfu/ml (Table 1). Only samples taken from dental units receiving the control treatment of distilled water with no added antimicrobial (Group 1) failed to meet the ADA goal. At the end of six weeks, the cfu count for Group 1 declined substantially, but the count was well more than the ADA standard.
Over the course of the entire six-week test period, all scores for the test groups were at or near zero cfu’s, except for Week 2 and Week 4 for the Bio2000. This anomaly during Week 2 for the Bio2000 resulted in a microbial count of 46,348 cfu/ml for the air/water syringe and 8,060 cfu/ml for the handpiece after two weeks. In contrast, all of the other samples for the other four test groups had essentially zero colony counts. This outcome was believed to be due to a lapse in sterile technique rather than a breakdown of the chemical product. All five treatment group samples essentially returned to zero bacteria counts from Week 3 to Week 6.
Examination of the SEMs revealed an apparent decrease in the biofilm mass, but a portion of the biofilm remained intact despite repeated treatment with the four chemical antimicrobials (Figures 3 through 9).
There did not appear to be any correlation between the amount of biofilm present and the number of viable microorganisms for any of the groups. All the SEM indicated a significant amount of biofilm in the water lines, despite the reduction in colony forming units (Figures 3 through 9). Figure 3 illustrates what the intaglio surface of new tubing received directly from the manufacturer should look like. At magnification of 8000x, this area of the tube is not smooth but has a rather an undulating surface, which might contribute to biofilm accumulation
Discussion
It is apparent that water source selection played a vital role in achieving consistent disinfection of water run through dental unit waterlines. Sterile distilled water in combination with a chemical antimicrobial was found to be an effective combination for achieving the ADA waterline quality goal of 200 cfu/ml or less. Results from this investigation indicated that all five products tested achieved the desired level of microbial decrease for both air/water syringe and dental handpiece waterlines. Moreover, the reductions were dramatic. Bacterial counts were maintained at the near zero level during this six-week period with the sole exception of Week 6. The high colony counts in Week 2 were believed to be due to a handling error that resulted in the contamination of the water sample. The source of the contamination was not known, but the authors presumed that it occurred during water collection or because someone may have touched the tube that goes in the bottle reservoir, accidentally contaminating the sample.
These findings provide additional support for the conclusion that water source selection may play a pivotal role in the disinfection of dental unit waterlines. It is believed that the American Dental Association recommended goal of 200 cfu/ml can, in fact, be achieved for dental treatment water with existing dental units. But to consistently achieve this reduction, three essential criteria must be met:
* The dental unit must have a closed water system,
* A chemical antimicrobial in conjunction with sterile distilled water must be used; and
* The closed-water system must be properly maintained to avoid contamination of the sterile distilled water and the dental unit water bottle.
There was no correlation between the amount of biofilm present and the reduction in colony forming units. Figure 3 illustrates the inside surface of new tubing at high magnification. The tube is not smooth but has a rather an undulating surface, which might contribute to biofilm accumulation.
Additional research is recommended to evaluate individual commercial products for patient acceptance of water taste, corrosion of dental equipment, prevention and/or elimination of the biofilm, long-term effectiveness of antimicrobials, effects of these products on restorative materials (the bond strength of composite resins, color changes of tooth-colored restorations, etc.), and the effects of routine use on the longevity of dental handpieces and ultrasonic scalers.
It should be noted that the products used in these study are marketed as antimicrobial agents and not as disinfectants and should be used according to the manufacturer’s intended use and directions
Conclusions
Based on the testing format and the materials used in this study, the following conclusions were drawn:
1. Sterile distilled water alone cannot reduce microbial contamination of dental treatment water from dental unit waterlines to the 200 cfu/ml ADA stated goal.
2. Dental units with a closed water system met or exceeded the American Dental Association goal of 200 cfu/ml when sterile distilled water was treated with any one of the following products: Listerine mouthrinse, 0.5% sodium fluoride, Rembrandt mouthrinse, Bio 2000 (Now BioBLUE), or Dentosept.
3. Microbial reduction of dental unit waterlines can be achieved using 100 percent of any of the five antimicrobial products tested and no distilled water, but such a protocol would be more costly than one involving a dilution with sterile distilled water.
4. Given the equivalent performance of the antimicrobial products tested in this study, strong consideration should be given to the cost of each product.
5. Additional research is recommended to assess patient acceptance of water taste, corrosion of dental equipment, prevention and/or elimination of the biofilm, long-term effectiveness of the antimicrobials, effects on restorative materials, and the impact of routine use on the longevity of dental handpieces and ultrasonic scalers.
6. A biofilm was detected in the waterline samples for all the dental units. While the biofilm may have been reduced in size (volume) as a result of treatment, it was not eliminated by any of the antimicrobial rinses used in this study.
References
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To request a printed copy of this article, please contact: James D. Kettering, PhD, Loma Linda University, Loma Linda, CA 92350, or jkettering@som.llu.edu.
Legends
Figure 1. Air/Water Syringe Waterline (Colony Forming Units)
Figure 2. High-Speed Handpiece Waterline (Colony Forming Units)
Figure 3. Scanning electron micrograph of new tubing prior to placement in the dental unit (8000x magnification).
Figure 4. SEM of biofilm buildup in the dental unit waterline after four weeks of use with tap water (50x magnification).
Figure 5. Biofilm buildup in the air/water syringe waterline tubing at baseline (8000x magnification).
Figure 6. Biofilm buildup in the handpiece waterline tubing after six weeks of treatment with Bio 2000 (8000x magnification).
Figure 7. Biofilm buildup in the handpiece waterline tubing after six weeks of treatment with distilled water (8000x magnification).
Figure 8. Biofilm buildup in the handpiece waterline tubing after six weeks of treatment with Listerine (8000x magnification).
Figure 9. Biofilm buildup in the handpiece waterline tubing after six weeks of treatment with Rembrandt (8000x magnification).
