Reduction of bacterial ieveis in dentai unit wateriines

Nuala 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 with
independent water reservoir systems. Method and materials: Baseline water samples were tirst taken
from SIX units. Two ounces of the undiluted test product was run through lines, left overnight, and flushed
outthenext 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) in
treatment units declined from 23,389 (± 20,085) at baseline, t o 6 ( ± 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 proprietary
formulation, resulted in significantly reduced bacterial counts to levels that were consistently below the
American 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 unit
waterline, planktonic bacteria

year 2000,'' At the center of the controversy surround-

CLiNiCAL RELEVANCE: The findings of this study sug-
gest that intermittent overnight treatment of dental unit
wateriines 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.
he problem of dental unit waterline (DUWL) con-
T tamination has been recognized for many years,'"^
The American Dental Association (ADA) set a goal to
reduce the number ot" noncohform, mesophillc, het-
erotrophic bacteria in patient treatment water to 200
colony-forming units per milliliter (CFU/mL) by the
'Assistant Professor, Department of Community Dentistry, The University ot
Texas Healtfi Science Center at San Antonio, San Antonio, Texas,
'^President, OSHA Solutions, San Antonio, Texas; formerly. Associate
Protessor, Department of Generat Dentistry, and Director, Johnson 8
Johnson Fellowship in Infectious Disease Ccntrol, The University ot Texas
Health Science Center at San Antonio, San Antonio, Texas.
Reprint requests: Dr Nuala B, Porteous, The University of Texas Health
Science Center at San Antonio, 7703 Floyd Curl Dnve, San Antonio TX
78229, E-mail: porteous ©uthscsa,edu
ing tbe issue is biofilm formation,' A bioñlm may be
defined as "a structured community of bacterial cells
enclosed 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 hard
surface, from biomédical implants to rocks in streams.
Nutrients in aqueous environments are more abun-
dant near a solid surface, so bacteria tend to become
surface-bound as a strategy for survival, Tbese organ-
isms are believed to have an advantage over their
free-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 the
production of exopolysaccharides and/or specialized
structures that anchor the bacteria to each other or the
solid 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 a
protective slime layer, also known as the glycocalyx or
matrix polymer,'

630 Volume 35, Number B, 2004


The growth potential of any biofilm is dependent
on immediately available nutrients. In most species,
the glycocalyx is anionie, and it is an efficient means
of trapping essential minerals and nutrients from its
environment," The complex structure of biofilms pro-
duces an inherent resistance to antimicrobials because
the agents must penetrate the glycocalyx to deactivate
the sessile (those protected in the biofilm matrix) or-
ganisms. It has been shown that cells encased in the
biofilm grow more slowly and. thus, adsorb antimicro-
bials at a slow rate.'* At the same time, bacteria from
the stirface of the biofilm are continuously sbed into
tbe flowing water, contributing to the numbers of
planktonic bacteria,'"
Many approaches are employed to control biofilm
growtb, from tbe industrial arena to medical devices.
According to Donlan and Costerton," intervention
strategies 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 water
tbrough the lines and prolonged periods of stagnant
water, 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 commonly
used to treat tbe familiar biofilm-dental plaque, Tbese
include 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 of
antibacterial activity,'^ CHX has several uses in the
health care industry, from antimicrobial soaps to
scrubless presurgical wipes. It is an ingredient of many
formulations used in dental practice,^i-'" Because of
the demonstrated viddespread efficacy of CHX in the
health care industry, it has recently become available
on the market in proprietary formulations as a DUWL
cleaner.
The purpose of this study was to test the efficacy of
an 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 dental
clinic in a teacbing institution were used to test 0.12%
CHX in a proprietary formulation as a DUWL
cleaner, and three similar functioning units were used
as controls. All units were equipped with self-con-
Quintessence International
• Porte o u s / C o o ley •
tained water systems. Each unit contained five individ-
ual waterlines; two handpieces; an assistant water sy-
ringe; a unit syringe; and an ultrasonic. Beginning 1
week prior to the study, routine chemical treatment of
six units was stopped. Lines were flushed daily accord-
ing to the ADA^' and Centers for Disease Control and
Prevention (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 handpieces
were run to discharge water and air for a minimum of
20 to 30 seconds.
Baseline water samples were first taken from six
units (three test and three control). For the duration of
the study, the three control units continued to be
flushed daily, as described above, according to tbe
ADA and CDC recommendations,^'" No chemicals
were added to these three control units. The three test
units 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 25
mL of 0,12% CHX; tbis was run separately tbrough tbe
five individual waterlines on tbe three test units for 30
seconds, 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 was
flushed out of the lines with tap water. The initial treat-
ment was repeated for a total of 6 nights during a
2-week holiday period when the units were not in use.
As soon as normal activity resumed, units were treated
with 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 on
all six units, just before overnight weekly treatment of
the treatment units. Before sample collection, the
reservoir bottle on each of the treatment and control
units 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 2
minutes il' the unit was not in use. The end of each wa-
terline was wiped with an alcohol-soaked pad before
taking 100 mL of water aseptically from eacb unit in a
sterile collection bottle containing sodium thiosulfate
(idexx) to neutralize residual chlorine present,^' Tbis
was 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 were
made and tben vigorously agitated by vortex for 15
seconds. One tentb of a milliliter of each dilution was
plated on R2A agar plates in triplicate using the
spread plate method, and left to incubate at room tem-
perature (22 to 28''C) for 7 days," Bacterial colonies
were then counted. An average of the three plates for
each unit was first calculated, converted to CFU/mL,
631
• Porteous/Cooley

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 and
control units for the 12-week study period was subse-
quently calculated for statistical analysis.
RESULTS
The mean number of CFU/mL and standard deviation
in treatment and control units each week during the
study period are presented in Table 1. Mean CFU/mL
in treatment units declined after the initial intensive
treatment. After the first 7 days of weekly overnight
treatment, there was a dramatic decline in counts to
14 CFU/mL. There was then an increase again for 2
weeks that reached levéis higher than the ADA-rec-
ommcnded level of 200 CFU/mL, but still within tbe
Environmental Protection Agency (EPA) Drinking
Water Standard of 500 CFU/mL." After tbis tempo-
rary increase, counts dropped again to very low levels
in week 4 and stayed consistently low for the remain-
ing 8 weeks of the study period.
The overall mean CFU/mL and standard deviation
over 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 less
than 1 log in week 4; and remaining at that level, or
lower, for the remainder of the study period.

TABLE 1 Mean CFU/mL and standard deviation in

treatment and control units over 12 weeks DISCUSSION

Treatment units Contro units This study demonstrated that 0.12% CHX, in a propri-
Time Mean SD Mean SD etary formulation, used as an intermittent, overnight
Baseline 23388,89 20084.00 4122.22 6393.80 DUWL cleaner was eftective in reducing bacterial lev-
Atter IT- 5791.11 2364.16 13594.44 9395.38 els in treated units, in comparison to untreated con-
Weeki 14,44 13,88 3244.44 3950.29 trols, and in maintaining low counts for a prolonged
Week 2 330.00 401.34 2383.33 2155.68 period of time. The reason for the increase in counts
Weeks 258.89 255.09 855.56 435.04 in weeks 3 and 4. after a decline in week 2, is un-
Week 4 5.56 9.62 387.78 379.10 known, but it can be speculated, based on tbe avail-
Week 5 11.11 19.25 482.22 573.27 able evidence to support the acfion of 0.12% CHX as
Week 6 11.11 11.71 444.44 339.48
an oral rinse. In the oral environment, it is believed
Week 7 11.11 8.39 284 44 164.80
Weeks 17.78 25.02 447,78 251.76
tbat 0.12% CHX bas both bacteriostafic and bacterici-
Week 9 58.89 99.13 417,78 261.82
dal properties, and that the mechanisms of action are
Week 10 15.56 13.47 1743.33 1792.13 multifactorial." Electrostatic interactions occur be-
Week 11 13.33 15.28 603.33 372.87 tween CHX and the oral tissues, which are botb re-
Week 12 4.44 1.92 918.89 1225.27 versible and pH-dependent, thus allowing CHX to be
'IT = initiai treatment. slowly released over a period of time, preventing mul-
SD = standard deuation. tiplication and adherence of organisms.'^'**

Fig 1 Mean CFU/mL (iog 10) ot heterotrophic

mesophilio bacteria found in treatmeni and controi
- Control units.
- Treatment
- ADA goal (200)

4 5 6 7
Time (wk)

632 Voiume 35, Number 8, 2004


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 suggest
that the initial, intensive treatment caused a reduction
in the planktonic form of bacteria. Continued treat-
ment appears to bave disrupted the biofilm, causing a
transient increase in planktonic bacteria during weeks
2 and 3, after wbich counts remained steadily low.
Confirmation of this observation requires furtber
investigation.
Methods to test DUWL products have not been
standardized, so vafid comparisons with each other
and with these current findings cannot be made.-^ For
example, a study by Kettering et aP" tested the efficacy
of 0.12% CHX in a clinical setting. Tbe investigators
analyzed samples from a handpiece and a water sy-
ringe line only, and the study lasted for 6 weeks. The
authors found that the ADA goal of 200 CFU/mL was
achieved only when lines were treated with disfilled
water and CHX. or CHX alone; units treated with tap
water and CHX showed no significant reduction in
bacterial counts. Tbe same group of researcbers con-
cluded in an earlier study that tap water should not be
used as a water source for DUWLs.'" In institufions
with large numbers of functioning operatories, tbis
may not be a very practical or cost-effective measure.
Tbe findings of this study are inconsistent with these
Undings. This study was of longer duration (12 versus
6 weeks) and used a "worst case scenario," whereby
equal volumes from every available waterline were
pooled 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-negative
bacteria with resistance to certain antibiotics also sbow
increased CHX resistance.^' It has also been demon-
strated that subgingival bacteria treated with increasing
concentrafions of CHX adapt by undergoing structural
and/or biochemical changes.5° Long-term conse-
quences of adding chemicals to DUWLs are stili in
question, so continuous monitoring of treated lines is
recommended.^''^
Some In vitro studies have examined the effect of
CHX on dental materials. When compared with tap
water as a continuous-use DUWL product, it has been
shown to result in significantly lower enamel shear
bond strength." A reduction in dentin bond strength
was observed wben a mixture of distilled water and
0,12% CHX was used as a rinsing agent after etching."
Alternatively, a study that tested shear bond strength
of resin composite to dentin afier 2% CHX was used
as a cavity disinfectant, showed no significant effect.^^
Filler et aP^ found similar results in a study that tested
Quintessence International
PorLeous/Cooley •
shear bond strength of resin composite to enamel that
was treated with 0.12% CHX for 1 minute, four times
daily for 7 days. Meiers and Shook" investigated the
effect of a 2% CHX cavity disinfectant on the shear
bond strength of resin composite to dentin mediated
by two dentin-bonding agents. They concluded tbat
the results depended on specific characterisfics of the
denfin-bonding agents ratber tban CHX.
An unexpected observafion in this study was a bac-
terial reduction in the control units during the study
period, noting tbat from weeks 4 through 9, counts re-
mained within EPA Drinldng Water Standard of 500
CFU/mL.''' This decline cannot be fully explained, but
it 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 throughout
the study period. Although results on the effect of
fiushing DUWLs with tap water vary, flushing has
been shown to be an effective means of reducing both
planktonic bacterial load and biofilm formation,^^ even
reducing counts of planktonic bacteria to zero after a
5-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 preflush
levels after 30 minutes of stasis.-"*
CONCLUSION
This study showed that intermittent treatment of
DUWLs with a commercially available 0.12% CHX
product resulted in statistically significant reduced
bacterial counts, in comparison to control units, to
levels that remained consistently below the ADA goal
of 200 CFU/mL, for 8 consecutive weeks. However,
due to tbe unknown long-term effects, it is advisable
to continually monitor chemically treated DUWL
quality. Further research on the efiect of 0.12% CHX
on DUWL biofilm formation is currently under inves-
tigation by tbe authors.
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Voiume 35. Number 8, 2004