Reduction of bacterial ieveis in dentai unit wateriinesNuala B, Porteous, BDS, MPHVRobert L. Cooley, DMD, MS=

Objectives; To test the etticacy ol an intermittent use, dental unit waterline cleaner, containing 0,12%chlorhexidine, in a proprietary formulation, to rsduco bacterial levels in three functioning dental units withindependent water reservoir systems. Method and materials: Baseline water samples were tirst takenfrom SIX units. Two ounces of the undiluted test product was run through lines, left overnight, and flushedoutthenext morning,This was repeated for 6 nights initially, and once a week thereafter for 12 weeks.Three control dental units did not have chemicals added. Weekly samples were collected in bottles con-taining sodium thjosulphate on the atternoon before overnight treatment, plated en R2A agar, and incu-bated at room temperature for 7 days. Results: Mean colony-forming units per millimeter (CFU/mL) intreatment units declined from 23,389 (± 20,085) at baseline, t o6 (± 10) in week 4, to 5 (±2) in week 12,Statistical analysis showed a significant difference between treatment and control units. Conclusion:Intermittent treatment of dental unit wateriines with 0,12% chlcrhexidine gluccnate (CHX), in a proprietaryformulation, resulted in significantly reduced bacterial counts to levels that were consistently below theAmerican Dental Association's goal of 200 CFU/mL tor 8 weeks, (Quintessence Int 2004;35:630-634)

Key words: antimicrobial intermittent-use cleaner, biofilm, chlorhexidine, decontamination, dental unitwaterline, planktonic bacteria

CLiNiCAL RELEVANCE: The findings of this study sug-gest that intermittent overnight treatment of dental unitwateriines with 0,12% chlorhexidine, in a proprietary fcr-muiation, is an effective method to reduce bacterial lev-els. Regular monitoring of chemically treated water qual-ity is advised due to unknown long-term adverse effects.

The problem of dental unit waterline (DUWL) con-tamination has been recognized for many years,'"^

The American Dental Association (ADA) set a goal toreduce the number ot" noncohform, mesophillc, het-erotrophic bacteria in patient treatment water to 200colony-forming units per milliliter (CFU/mL) by the

'Assistant Professor, Department of Community Dentistry, The University otTexas Healtfi Science Center at San Antonio, San Antonio, Texas,

'^President, OSHA Solutions, San Antonio, Texas; formerly. AssociateProtessor, Department of Generat Dentistry, and Director, Johnson 8Johnson Fellowship in Infectious Disease Ccntrol, The University ot TexasHealth Science Center at San Antonio, San Antonio, Texas.

Reprint requests: Dr Nuala B, Porteous, The University of Texas HealthScience Center at San Antonio, 7703 Floyd Curl Dnve, San Antonio TX78229, E-mail: porteous ©uthscsa,edu

year 2000,'' At the center of the controversy surround-ing tbe issue is biofilm formation,' A bioñlm may bedefined as "a structured community of bacterial cellsenclosed in a self-produced polymeric matrix and ad-herent to an inert or living surface."^ Biofllms are ubiq-uitous where any liquid comes in contact with a hardsurface, from biomédical implants to rocks in streams.Nutrients in aqueous environments are more abun-dant near a solid surface, so bacteria tend to becomesurface-bound as a strategy for survival, Tbese organ-isms are believed to have an advantage over theirfree-floating, or planktonic, counterparts.^

The initial, reversible stage of biofilm formation, bac-terial adhesion, depends on environmental conditions,such as electrostatic, hydrophobic interactions and hy-drodynamic forces. The second stage of adhesion is theproduction of exopolysaccharides and/or specializedstructures that anchor the bacteria to each other or thesolid surface. This stage is irreversible, and once it oc-curs, biofilm maturation begins, creating a complex sur-face as extracellular components oí the attached bacte-ria react with environmental substances to form aprotective slime layer, also known as the glycocalyx ormatrix polymer,'

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• Porte o us/Coo ley •

The growth potential of any biofilm is dependenton immediately available nutrients. In most species,the glycocalyx is anionie, and it is an efficient meansof trapping essential minerals and nutrients from itsenvironment," The complex structure of biofilms pro-duces an inherent resistance to antimicrobials becausethe agents must penetrate the glycocalyx to deactivatethe sessile (those protected in the biofilm matrix) or-ganisms. It has been shown that cells encased in thebiofilm grow more slowly and. thus, adsorb antimicro-bials at a slow rate.'* At the same time, bacteria fromthe stirface of the biofilm are continuously sbed intotbe flowing water, contributing to the numbers ofplanktonic bacteria,'"

Many approaches are employed to control biofilmgrowtb, from tbe industrial arena to medical devices.According to Donlan and Costerton," interventionstrategies act in one or more of the following ways:prevent biofilm attachment; minimize cell attachment;kill cells; or remove the device." Removal of the cont-aminated devices may be an option for medical de-vices, but not so for DUWLs, Witb such a large sur-face-to-volume ratio, meaning laminar flow of watertbrough the lines and prolonged periods of stagnantwater, prevention of biofilm formation is a challenge,'Strategies to control biofilms in DUWLs must tbere-fore focus on minimizing cellular adhesion and attach-ment and killing cells.

Antimicrobials used in oral rinses are commonlyused to treat tbe familiar biofilm-dental plaque, Tbeseinclude essential oils, quaternary ammonium com-pounds, triclosan, and bisbiguanides.'^ Tbe best-known bisbiguanide, chlorhexidene gluconate (CHX),with a strong cationic charge, has a broad spectrum ofantibacterial activity,'^ CHX has several uses in thehealth care industry, from antimicrobial soaps toscrubless presurgical wipes. It is an ingredient of manyformulations used in dental practice,^i-'" Because ofthe demonstrated viddespread efficacy of CHX in thehealth care industry, it has recently become availableon the market in proprietary formulations as a DUWLcleaner.

The purpose of this study was to test the efficacy ofan intermittent use, commercially available product,containing 0,12% CHX (Bioblue, Micrylium Labora-tories) to reduce bacterial levels in DUWLs.

METHOD AND MATERIALS

Three functioning dental units at an outpafient dentalclinic in a teacbing institution were used to test 0.12%CHX in a proprietary formulation as a DUWLcleaner, and three similar functioning units were usedas controls. All units were equipped with self-con-

tained water systems. Each unit contained five individ-ual waterlines; two handpieces; an assistant water sy-ringe; a unit syringe; and an ultrasonic. Beginning 1week prior to the study, routine chemical treatment ofsix units was stopped. Lines were flushed daily accord-ing to the ADA '̂ and Centers for Disease Control andPrevention (CDC)̂ ^ recommendations: At the begin-ning of each day, all lines were flushed for 2 to 3 min-utes. After use on each patient, high-speed handpieceswere run to discharge water and air for a minimum of20 to 30 seconds.

Baseline water samples were first taken from sixunits (three test and three control). For the duration ofthe study, the three control units continued to beflushed daily, as described above, according to tbeADA and CDC recommendations,^'" No chemicalswere added to these three control units. The three testunits were treated according to the manufacturer's rec-ommendations. This required an inifial, intensive treat-ment intended to remove or disrupt existing biofilm:Tbe self-contained reservoir bottle was filled with 25mL of 0,12% CHX; tbis was run separately tbrough tbefive individual waterlines on tbe three test units for 30seconds, and then left in the lines overnight. The fol-lowing morning, the self-contained bottle was re-moved, filled witb tap water, and the product wasflushed out of the lines with tap water. The initial treat-ment was repeated for a total of 6 nights during a2-week holiday period when the units were not in use.As soon as normal activity resumed, units were treatedwith a single overnight treatment once a week.

Water samples were taken from treatment and con-trol units after initial treatment and sampling was re-peated on the same day of the week for 12 weeks onall six units, just before overnight weekly treatment ofthe treatment units. Before sample collection, thereservoir bottle on each of the treatment and controlunits was filled with fresh tap water and then reat-tached to the unit. Lines were then fiushed for 20 sec-onds if the dental unit was in use that day, or for 2minutes il' the unit was not in use. The end of each wa-terline was wiped with an alcohol-soaked pad beforetaking 100 mL of water aseptically from eacb unit in asterile collection bottle containing sodium thiosulfate(idexx) to neutralize residual chlorine present,^' Tbiswas approximately 20 mL from eacb of the five lines.Water samples were immediately taken to the labora-tory. Ten-fold serial dilutions in phosphate buffer weremade and tben vigorously agitated by vortex for 15seconds. One tentb of a milliliter of each dilution wasplated on R2A agar plates in triplicate using thespread plate method, and left to incubate at room tem-perature (22 to 28''C) for 7 days," Bacterial colonieswere then counted. An average of the three plates foreach unit was first calculated, converted to CFU/mL,

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and recorded. The mean number of CFU/mL in treat-ment and control units for cacb week was then calcu-lated. The overall mean CFU/mL in treatment andcontrol units for the 12-week study period was subse-quently calculated for statistical analysis.

RESULTS

The mean number of CFU/mL and standard deviationin treatment and control units each week during thestudy period are presented in Table 1. Mean CFU/mLin treatment units declined after the initial intensivetreatment. After the first 7 days of weekly overnighttreatment, there was a dramatic decline in counts to14 CFU/mL. There was then an increase again for 2weeks that reached levéis higher than the ADA-rec-

ommcnded level of 200 CFU/mL, but still within tbeEnvironmental Protection Agency (EPA) DrinkingWater Standard of 500 CFU/mL." After tbis tempo-rary increase, counts dropped again to very low levelsin week 4 and stayed consistently low for the remain-ing 8 weeks of the study period.

The overall mean CFU/mL and standard deviationover the 12-week period, in treatment units was 63 (±158) and 1,018 (± 1,526) in the control units.Statistical analysis, using the one-tailed t test to com-pare the means of the two groups, showed a signifi-cant difference (P < .0005, df = 52) between treat-ment and control units.

Figure 1 illustrates the trend over time, from a base-line reading of 4 log CFU/mL to 3 logs after inifia!treatment; to 2 logs in weeks 2 and 3; declining to lessthan 1 log in week 4; and remaining at that level, orlower, for the remainder of the study period.

TABLE 1 Mean CFU/mL and standard deviation intreatment and control units over 12 weeks

Time

BaselineAtter IT-WeekiWeek 2WeeksWeek 4Week 5Week 6Week 7WeeksWeek 9Week 10Week 11Week 12

'IT = initiai treatmeSD = standard deu

Treatment units

Mean

23388,895791.11

14,44330.00258.89

5.5611.1111.1111.1117.7858.8915.5613.334.44

nt.ation.

SD

20084.002364.16

13,88401.34255.09

9.6219.2511.718.39

25.0299.1313.4715.281.92

Contro

Mean

4122.2213594.443244.442383.33855.56387.78482.22444.44284 44447,78417,78

1743.33603.33918.89

units

SD

6393.809395.383950.292155.68435.04379.10573.27339.48164.80251.76261.82

1792.13372.87

1225.27

DISCUSSION

This study demonstrated that 0.12% CHX, in a propri-etary formulation, used as an intermittent, overnightDUWL cleaner was eftective in reducing bacterial lev-els in treated units, in comparison to untreated con-trols, and in maintaining low counts for a prolongedperiod of time. The reason for the increase in countsin weeks 3 and 4. after a decline in week 2, is un-known, but it can be speculated, based on tbe avail-able evidence to support the acfion of 0.12% CHX asan oral rinse. In the oral environment, it is believedtbat 0.12% CHX bas both bacteriostafic and bacterici-dal properties, and that the mechanisms of action aremultifactorial." Electrostatic interactions occur be-tween CHX and the oral tissues, which are botb re-versible and pH-dependent, thus allowing CHX to beslowly released over a period of time, preventing mul-tiplication and adherence of organisms.'̂ '**

- Control- Treatment- ADA goal (200)

4 5 6 7Time (wk)

Fig 1 Mean CFU/mL (iog 10) ot heterotrophicmesophilio bacteria found in treatmeni and controiunits.

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When used as an intermittent DUWL cleaner,0.12% CHX has been sbown to reduce the amount of,but not eliminate, the biofilm.'' These findings suggestthat the initial, intensive treatment caused a reductionin the planktonic form of bacteria. Continued treat-ment appears to bave disrupted the biofilm, causing atransient increase in planktonic bacteria during weeks2 and 3, after wbich counts remained steadily low.Confirmation of this observation requires furtberinvestigation.

Methods to test DUWL products have not beenstandardized, so vafid comparisons with each otherand with these current findings cannot be made.-^ Forexample, a study by Kettering et aP" tested the efficacyof 0.12% CHX in a clinical setting. Tbe investigatorsanalyzed samples from a handpiece and a water sy-ringe line only, and the study lasted for 6 weeks. Theauthors found that the ADA goal of 200 CFU/mL wasachieved only when lines were treated with disfilledwater and CHX. or CHX alone; units treated with tapwater and CHX showed no significant reduction inbacterial counts. Tbe same group of researcbers con-cluded in an earlier study that tap water should not beused as a water source for DUWLs.'" In institufionswith large numbers of functioning operatories, tbismay not be a very practical or cost-effective measure.Tbe findings of this study are inconsistent with theseUndings. This study was of longer duration (12 versus6 weeks) and used a "worst case scenario," wherebyequal volumes from every available waterline werepooled on the unit, even from the slow-speed hand-piece and ultrasonic lines, which were rarely used.

One of the reasons CHX, used in different concen-trations, has such widespread use in the health care in-dustry is that resistant organisms are uncommon, al-though there is some evidence that gram-negativebacteria with resistance to certain antibiotics also sbowincreased CHX resistance.^' It has also been demon-strated that subgingival bacteria treated with increasingconcentrafions of CHX adapt by undergoing structuraland/or biochemical changes.5° Long-term conse-quences of adding chemicals to DUWLs are stili inquestion, so continuous monitoring of treated lines isrecommended.^''^

Some In vitro studies have examined the effect ofCHX on dental materials. When compared with tapwater as a continuous-use DUWL product, it has beenshown to result in significantly lower enamel shearbond strength." A reduction in dentin bond strengthwas observed wben a mixture of distilled water and0,12% CHX was used as a rinsing agent after etching."Alternatively, a study that tested shear bond strengthof resin composite to dentin afier 2% CHX was usedas a cavity disinfectant, showed no significant effect.̂ ^Filler et aP^ found similar results in a study that tested

shear bond strength of resin composite to enamel thatwas treated with 0.12% CHX for 1 minute, four timesdaily for 7 days. Meiers and Shook" investigated theeffect of a 2% CHX cavity disinfectant on the shearbond strength of resin composite to dentin mediatedby two dentin-bonding agents. They concluded tbatthe results depended on specific characterisfics of thedenfin-bonding agents ratber tban CHX.

An unexpected observafion in this study was a bac-terial reduction in the control units during the studyperiod, noting tbat from weeks 4 through 9, counts re-mained within EPA Drinldng Water Standard of 500CFU/mL.''' This decline cannot be fully explained, butit may be attributable to a number of factors, such as:strict personnel compliance with flushing protocol;length of flow time of source tap water before reser-voir bottles were filled; and close monitoring of bacte-rial and chlorine levels of source tap water throughoutthe study period. Although results on the effect offiushing DUWLs with tap water vary, flushing hasbeen shown to be an effective means of reducing bothplanktonic bacterial load and biofilm formation,̂ ^ evenreducing counts of planktonic bacteria to zero after a5-minute flush period.'" It is generally agreed, how-ever, that it is not a pracfical means of DUWL decont-aminafion. Biofilm will continue to form during inac-tive periods, and bacterial levels will return to preflushlevels after 30 minutes of stasis.-"*

CONCLUSION

This study showed that intermittent treatment ofDUWLs with a commercially available 0.12% CHXproduct resulted in statistically significant reducedbacterial counts, in comparison to control units, tolevels that remained consistently below the ADA goalof 200 CFU/mL, for 8 consecutive weeks. However,due to tbe unknown long-term effects, it is advisableto continually monitor chemically treated DUWLquality. Further research on the efiect of 0.12% CHXon DUWL biofilm formation is currently under inves-tigation by tbe authors.

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