Biofilm

Controlling Biofilm and Microbial Contamination in Dental Unit Waterlines

Thomas K. Lee, DDS; Emile J. Waked, DDS; Lawrence E. Wolinsky, PhD, DMD; Ronald S. Mito, DDS; and Richard E. Danielson, PhD

Copyright 2001 Journal of the California Dental Association.

Despite the fact that the ADA had set the goal of less than 200 colony-forming units per milliliter of unfiltered output water from dental unit waterlines to be achieved voluntarily by the year 2000, there is much confusion and resistance within the profession with regard to waterlines. Many in the profession are still wondering what the most effective means are to predictably achieve the goal. It is a well-established fact that bacterial biofilm can readily form within dental unit waterlines and degrade the microbial quality of the water in dental units regardless of the water source. These biofilms are primarily formed by various microcolonies of bacteria that attach to surfaces over time within the waterlines. An increasing number of medically compromised and immunocompromised patients being treated in dental offices and increased public awareness have brought about renewed interest in this issue. There are generally four categories of products that are available to address this issue: independent water systems, sterile water delivery systems, filtration, and chemical treatment protocols. A recent study at the University of California at Los Angeles demonstrates that the Ultra chemical treatment protocol can be an effective means of controlling biofilm in dental unit waterlines.

Since Blake first described the microbial contamination of dental unit waterlines in 1963,¹ there have been numerous studies that affirmed his findings and explored ways to control the contamination. It is now well-established that the microbial quality of the water in dental unit waterlines can easily be compromised regardless of the water source due to the presence of biofilm.^2-4 These biofilm are primarily formed by various microcolonies of bacteria and fungi that attach to the inner surfaces of dental unit waterlines over time, usually after no more than a few weeks at room temperature. Colonization of bacteria within the waterlines can occur through fluid retraction from the operating field through high-speed handpieces and air-water syringe lines when anti-retraction valves fail to work properly. However, even with properly working anti-retraction valves and sterile water as its source water, biofilm can eventually establish itself if no preventive and disinfection measures are taken. In fact, as long as there is water present for a few weeks, biofilm will form in all dental unit waterlines even if no patients have been treated with a particular dental unit (Figures 1 through 5).

A quick review of biofilm and resulting bacterial contamination in general would be beneficial for a better understanding of its significance in dentistry. Biofilm and its bacteria are everywhere in nature, as long as there is a stagnant body of water or moisture. The representation of biofilm dentists are most readily familiar with is the dental plaque present in everyone’s mouth. The slimy dark green stuff seen growing on any chronically wet surface is also biofilm. As such, biofilm formation in general is not unique to dentistry and is a major problem that has been dealt with in many other industries, such as the food and beverage industries. Another good example of biofilm hazard is public water fountains in parks and public buildings since water can also stagnate in them for long periods.

The topic of biofilm and resulting microbial contamination in dental unit waterlines did not gain widespread attention from the dental profession until recently. The increasing number of medically compromised and immunocompromised patients treated in dental offices and increased public awareness have created a renewed interest in this issue.⁵ The main concern in dentistry is that contaminated water from biofilm-laden dental unit waterlines may be ingested by patients, may contact open wounds, or may be aerosolized and inhaled by patients and care providers during routine dental visits.⁵ Although no scientific evidence to date has demonstrated any direct detrimental health effects from biofilm-contaminated-water exposure to patients or practitioners, there is indirect evidence that points to the need for improved water quality in dental offices.^5-7 Most health professionals would also agree that it is inconsistent with currently practiced infection control protocols to routinely introduce large amounts of microorganisms per milliliter into patients’ mouths when so much effort is made to disinfect and sterilize various dental instruments and equipment. There is a misconception among most dental professionals that the quality of the water going into the dental unit waterline is the same quality coming out the other end. This is simply not the case.

In dentistry, the stagnant water problem is exponentially compounded by the nature of dental unit waterlines. The extensive use of long, thin plastic waterlines within a dental operatory results in an extremely high surface-to-volume ratio within the tubing. In other words, there is a very large inner surface area for the small volume of water that passes through dental unit waterlines, leading to extremely high surface area contact for the water in the system (Figure 6). Most dental procedures use relatively small amounts of water throughout the day, even during an active treatment day. Thus, only small amounts of water move through the tubing at any given time, contributing to easy formation of biofilm and ample time for the resulting microbial contamination of the water. Water sitting in such an environment for extended periods, e.g., overnight and weekends, at room temperature when the dental units are not in use would easily be a breeding ground for microbial proliferation and the resultant biofilm.⁵

In addition, friction near the tubing surface significantly slows down the movement of proximal water until the water flow at the surface is minimal. This hydrodynamic phenomenon, known as laminar flow, is another reason for the proliferation of the biofilm in dental unit waterlines since this phenomenon ensures prolonged contact time between the tubing inner surface and the proximal water.^5,8 Another contributing factor to consider is that the polyurethane and polyvinyl chloride materials primarily used for waterline tubing also happen to be good a substrates for biofilm formation because of their texture, as compared with the texture of metal piping, for example. The above-mentioned conditions add up to an ideal environment for biofilm formation within dental unit waterlines. Once a biofilm is established within a waterline, it quickly develops three-dimensional matrices that are quite resistant to most disinfecting efforts, and its inner layers are often able to survive germicidal assault. The biofilm is self-sustainable and does not require any further microbial contamination for future proliferation, and the water output from that unit becomes highly contaminated with bacteria that continuously proliferate and detach from the established biofilm.^3,4

The American Dental Association, through its Council on Scientific Affairs and Board of Trustees, in 1995 recommended that the research and dental manufacturing communities develop methods to control and eliminate biofilm in dental unit waterlines. The ADA set a goal of less than 200 cfu/ml in unfiltered output water from dental unit waterlines for all dental procedures by the year 2000.⁹ The ADA’s goal is more stringent than the drinking water standard, which is less than 500 cfu/ml; and the American Public Health Association along with the Organization for Safety and Asepsis Procedures have issued statements supporting the ADA goal.^10,11

The ADA and the Centers for Disease Control and Prevention guidelines recommend flushing waterlines for several minutes to remove suspended bacteria before the first patient of the day is treated, and for 20 to 30 seconds between patients to remove material that may have been retracted during treatments.^5,12,13 Such flushing may minimize the risk of colonization and cross infection; however, its effects are only temporary. It is now well-established that flushing alone fails to eliminate or prevent bacterial colonization.^3,14

In recent years, the Food and Drug Administration has approved various products intended to improve the microbial quality of dental unit water.5 These products generally fall into four categories: independent water systems, sterile water delivery systems, filtration, and chemical treatment protocols.

An independent water system is disconnected from the municipal (tap) water source and draws fluid from a reservoir bottle holding the practitioners’ choice of water or treatment solution. Most major dental unit suppliers now offer retrofitting for older dental units for this option, and it is not too costly. The major advantage of this design is that the practitioner’s independent water source of choice and various chemical treatments for the dental unit waterlines can be used. While regular flushing with a disinfectant solution is easy and practical with this system, the system can also be used to dry the internal surfaces of the waterlines by purging the system of the solution until the system is void of the water. Although waterlines are less susceptible to biofilm formation while when they are kept dry overnight, there is no literature to support that dental unit waterlines left dry overnight achieve biofilm control. It is also noteworthy that sterile water put into an ordinary independent water system would not maintain its sterility as it goes through the dental unit waterlines.

Sterile water delivery systems, required for surgical procedures, are expensive to purchase and operate, and are often less convenient to use than other available delivery systems in the general dentistry setting. For the sterile water delivery system to be effective, it must be kept independent of the dental unit waterlines and the waterlines must be either replaced or sterilized after each use. Although well-suited for many surgical procedures, sterile water delivery systems are neither practical nor necessary for most general dentistry procedures.

Filtration involves the use of 0.2 m m membrane filters. Although this method can be effective in filtering out most of the potentially harmful bacterial organisms, location of the filter within the waterlines and its maintenance are critical to its success since the filters have no effect on pre- or post-filter biofilm formation itself. Of particular concern is that the dental unit waterline segment beyond the filter remains vulnerable to colonization over time. Hence, the distance between the filter and the high-speed handpiece or the air-water syringe must be minimized or eliminated because the biofilm can easily establish itself in such a location and essentially render the filter ineffective. Filters can be expensive and often require shortened replacement intervals as the biofilm colonies potentially get worse at the pre-filter location and clog up the waterlines at the filter point. Also, high levels of bacterial endotoxins that cannot be filtered with the 0.2 m m membrane filters have been found in contaminated dental unit waterlines when filtration methods are exclusively used for a long time.

Finally, chemical treatment protocols, depending on the nature of various germicidal agents, may be used intermittently as a "shock" treatment or continuously introduced into waterlines in small quantities.^15-17 This protocol requires having an independent reservoir system from which the solution of choice can be originated. Bleach (sodium hypochlorite) along with various proprietary chemicals, such as Bio-2000 (chlorohexidine, glycerin and alcohol-based), Dentacide (iodine-based), Bioclear (citric acid-based) and Ultra (alkaline peroxide-based) are some of the examples of chemical treatment protocols on the market for dental practitioners’ use.

Some dental equipment manufacturers recommend weekly cleaning by flushing the units with a 10 percent bleach solution for 10 minutes. This approach has been supported by various studies and the ADA, with the understanding that details of the protocol must be adhered to for its clinical efficacy.^15,16 However, bleach may have unintended corrosive effects on various parts of dental equipment and introduces the risk of patient exposure to harmful chemicals, such as trihalomethanes (suspected human carcinogen), in systems using continuous bacteriostatic chlorination.8

Another issue to keep in mind for various chemical treatments, especially the ones that are used continuously in small amounts, is the potential bacterial resistance in the long run and their interactions with other dental materials used in the clinical setting such as bonding agents. Attempts to simply kill the bacteria within biofilm is also ineffective because disinfectants or germicidal agents often fail to penetrate the complex matrix of the biofilm, resulting in viable bacteria within the matrix that can get dislodged and immediately re-contaminate the water or eventually contribute to bacterial resistance. Literature review indicates that biofilm and resulting bacterial contamination within dental unit waterlines are best managed when biofilm itself is somehow physically removed. In fact, that is how dentists best deal with the other form of biofilm well known to dentistry -- dental plaque. Dental plaque is mechanically removed by brushing, flossing, and scaling and root planning. Although it is impractical to brush the insides of the dental unit waterlines where the biofilm reside, a chemical treatment that will physically remove the biofilm would be ideal.

Only one product in the marketplace, Ultra (formerly marketed as Ultrakleen, Sterilex, Baltimore, MD), has received ADA’s Seal of Approval in controlling biofilm. Ultra achieves the ADA-set goal of less than 200 cfu/ml by physically removing the biofilm within the waterlines. This physical removal of the biofilm prevents or minimizes bacterial contamination of the dental unit waterlines. According to preliminary results of a recent study at UCLA (supported by a gift from A-dec, Inc.), the alkaline peroxide product Ultra was effective at physically removing the established biofilm (Figures 7 and 8). Microbial evaluations as in various heterotrophic plate counts indicated that this alkaline peroxide product is effective at maintaining the water supply under the ADA goal of 200 CFU/ml when used weekly, as directed by the manufacturer. Tables 1 and 2 represent preliminary data from the study.

The following are some of the results of the study:

* Flushing of the waterline before and after patient treatments failed to achieve the ADA goal.

* Bleach protocol achieved the ADA goal 54 percent of the time.

* Ultra achieved the ADA goal 100 percent of the time at one week after treatment.

When the recommended protocol is followed using Ultra, a water supply meeting the ADA set goal of less than 200 cfu/ml can be achieved, as long as the source water itself meets the ADA standard. Weekly treatments with this alkaline peroxide system (the manufacturer’s recommended protocol) will consistently achieve the water quality that is needed to satisfy the ADA goal of less than 200 cfu/ml. A-dec’s internal experiments have also indicated that Ultra is noncorrosive to various A-dec components within dental unit waterlines (personal communication with A-dec, Inc.).

Biofilm and the microbial contamination of dental unit waterlines are real issues that require real solutions in an everyday clinical setting. There are many ways to approach this problem as was reviewed in this article. It is recommended that each dental practitioner evaluate the clinical setting he or she is in and implement appropriate protocols to achieve the goal of delivering water that meets the ADA goal.

Authors

Thomas K. Lee, DDS, is an assistant clinical professor at the University of California at Los Angeles School of Dentistry. He serves as the director of the Advanced Education in General Dentistry postdoctoral residency program at UCLA.

Emile J. Waked, DDS, is an assistant adjunct professor in the Division of Public Health and Community Dentistry at the UCLA School of Dentistry. He also serves as the acting director of the UCLA-Venice Dental Center.

Lawrence E. Wolinsky, PhD, DMD, is a full-time professor in the Division of Oral Biology and Oral Medicine at the UCLA School of Dentistry. He also serves as the director of UCLA’s Clinical Research Center and associate director of the Dental Research Institute.

Ronald S. Mito, DDS, is a professor and the associate dean for clinical sciences at the UCLA School of Dentistry. He also serves as a CDA trustee from the Western Los Angeles Dental Society.

Richard E. Danielson, PhD, received his doctorate from the University of California at Berkeley School of Public Health. He was section chief of the Environmental Microbial Diseases Laboratory for the California Department of Health Services and is now laboratory director of BioVir Laboratories, Inc., an environmental microbiology testing laboratory.

References

1. Blake GC, The incidence and control of bacterial infection in dental spray reservoirs. Br Dent J 115:413-6, 1963.

2. Williams HN, Baer ML, Kelley JI, Contribution of the dental unit water supply. J Am Dent Assoc 126(9):1255-60, 1995.

3. Mayo JA, Oertling KM, Andrieu SC, Bacterial biofilm: a source of contamination in dental air-water syringes. Clin Prev Dent 12:13-20, 1990.

4. Whitehouse RL, Peters E, et al, Influence of biofilm on microbial contamination in dental unit water. J Dent 19:290-5, 1991.

5. ADA Council on Scientific Affairs, Dental unit waterlines: Approaching the year 2000. J Am Dent Assoc 130:1653-64, 1999.

6. Mills SE, The dental unit waterline controversy: Defusing the myths, defining the solutions. J Am Dent Assoc 131:1427-41, 2000.

7. Pankhurst CL, Johnson NW, Woods RG, Microbial contamination of dental unit waterlines: the scientific argument. Int Dent J 48(4):359-66, 1998.

8. Costerton JW, Overview of microbial biofilms. J Ind Microbiolol 15:137-40, 1995.

9. ADA Council on Scientific Affairs and ADA Council on Dental Practice, Infection control for the dental office and dental laboratory. J Am Dent Assoc 127:672-80, 1996.

10. Bednarsh H, Bond W, presiders, Dental unit water: how good is good; how bad is bad? Symposium presented at: American Public Health Association Annual Session, Indianapolis, Nov 10, 1997.

11. Organization for Safety and Asepsis Procedures, Position paper on dental unit waterlines. OSAP, Annapolis, Md, 1997.

12. Centers for Disease Control and Prevention, Recommended infection-control practices for dentistry, 1993. Morb Mortal Wkly Rep 41(RR-8):1-12, 1993.

13. American Dental Association, Dentist’s desk reference: Materials, instruments and equipment, 2nd ed. ADA, Chicago, 1983, p 16.

14. Williams JF, Johnston AM, et al, Microbial contamination of dental unit waterlines: prevalence, intensity and microbiological characteristics. J Am Dent Assoc 124:59-65, 1993.

15. Fayle SA, Pollard MA, Decontamination of dental unit water systems: a review of current recommendations. Br Dent J 181:369-72, 1996.

16. Williams HN, Kelley J, et al, Assessing microbial contamination in clean water dental units and compliance with disinfection protocol. J Am Dent Assoc 125:1205-10, 1994.

17. Meiller TF, DePaola LG, et al, Dental unit waterlines: biofilms, disinfection and recurrence. J Am Dent Assoc 130:65-72, 1999.

To request a printed copy of this article, please contact/ Thomas K. Lee, DDS, Advanced Education in General Dentistry, 13-089 CHS, Box 951668, Los Angeles, CA 90095-1668 or at tlee@dent.ucla.edu

Figure 1. Scanning electron micrograph of new tubing in a dental unit waterline at 120x magnification.

Figure 2. SEM of new tubing in a waterline at 2,500x magnification.

Figure 3. SEM of biofilm buildup in a dental unit waterline at 120x magnification.

Figure 4. SEM of biofilm buildup in a waterline at 2,500x magnification.

Figure 5. SEM of biofilm buildup at 4,500x magnification.

Figure 6. An open dental unit box showing numerous plastic tubes.

Figure 7. SEM of dental unit waterline tubing after biofilm build-up and subsequent treatment by Ultra at 120x magnification.

Figure 8. SEM of dental unit waterline tubing after biofilm build-up and subsequent treatment by Ultra at 2,500x magnification.

Table 1. Treatment and Sampling Frequencies.

Table 2. Colony Counts for Control and Treatment Groups