International Dental Journal (2003) 53.

249-254

The effect of Sterilex UltraRfor

disinfectionof dental unit waterlines
T. Larsen and N.-E. Fiehn
Copenhagen, Denmark

Aim: To evaluate the effect of a disinfectant agent based on hydrogen For several decades dental unit
peroxide (Sterilex UltraR, Sterilex Corporation, Maryland, USA) on the waterlines (DUWL) have been
microbiologicalwater quality in dental unit waterlines (DUWL). Setting: Six shown to contain bacteria in
older dental units were disinfected with Sterilex UltraRand another six numbers far beyond the recom-
units with sodium hypochlorite. The results were compared to six non- mended levels for human
disinfected units examined during a period without patient treatment. consumption, i.e. up to > l o "
Outcome measure: The number of colony forming units of bacteria (cfu)/ culturable bacteria/ml'-3. Also
ml of water was determined according to European Standards on water other microorganisms such as fungi
quality. Design: The water quality was initially determined during a six and amoebae have been isolated4s5.
week period, and subsequently in more detail for two weeks. Finally, the Among the many bacterial species
effect of prolongedadministrationof Sterilex UltraRwas investigatedfor six isolated, the high recovery (up to
weeks. Results: Instillation of Sterilex UltraRaccording to the recommen-
lo5 bacteria/ml) of respiratory
dations of the manufacturer initially reduced the number of bacteria in
DUWL to <lo2 cfu/ml. However, following daily, as well as prolonged
pathogens including Legionella sp.
administration of Sterilex UltraRa gradual recolonisation was observed and non-tuberculous Mycobacfetiam
resulting in bacterial numbers >lo4 cfu/ml in a number of units. Major sp. and opportunistic gram nega-
differencesbetween the number of cfu/ml in individualunits were observed. tive pathogens like Pseadomonas sp.
Conclusion: Neither daily nor extended administration of Sterilex UltraR have caused most concern6-8.
was capable of maintaining an acceptable water quality in these older Among these, Pseadomonas aemginosa
dental units. has been shown to be transferred
to patients during dental treatment
Key words: Bacteria, dental unit waterlines, disinfection, hypochlorite and to have caused infection in
patients'.'". Further, dentists have
been shown to have higher rates of
antibodies to Legionella compared
with the general public, and the
death of a dentist due to a Legionella
pneumonia was ascribed to expo-
sure to contaminated water at his
own ~linic".'~. So, though there is
no evidence of a major public
health problem, the risk of acquir-
ing an infection from contaminated
DUWL water does exist, especially
for the increasing number o f
immunocompromised individu-
a l ~ ~ Thus,
, ' ~ . procedures to control
the microbial colonisation in
DUWL should be employed.
Generally, bacterial numbers are
much higher in DUWL than in the
Correspondence to: Dr Tove Larsen, Associate Professor, Department of Oral Microbi- ordinary water supply in dental
ology, School of Dentisty,University of Copenhagen,Nsrre Alle 20, DK-2200 Copenhagen clinics8J4.The water quality dete-
N, Denmark. E-mail: tl@odont.ku.dk riorates in dental units where
Q 2003 FDI/World Dental Press
0020-6539/W04249-06
250
microbial biofilms build up in the
narrow water tubes',". The devel-
opment of biofilms is facilitated
by the ready adhesion of bacteria
to the hydrophobic polymeric plas-
tic tubing material used in dental
equipment and by the low flow
rate and the intermittent patterns
of use of water with over-night
and weekend stagnation4.l5.Thus, a
procedure for improving the
water quality in DUWL implying
prevention of biofilm formation
or elimination of biofilms seems
to be an appropriate method for
this purpose. Sterilex UltraR is a
disinfection agent based on hydro-
gen peroxide claimed to break up
biofilms in DUWL'. According to
preliminary results Sterilex UltraR
has shown a beneficial effect on
DUWL water qualitylb, and it has
received FDA clearance for market-
ing as a waterline cleaner'.
The purpose of the present study
was to evaluate the effect of Sterilex
UltraR o n the microbiological
water quality in dental units, which
have previously been found to build
up biofilms in their DUWL when
not disinfected.
Materials and methods
Disinfection of dental units
For the investigation of the water-
lines, twelve 15-year-old dental units
(FLEXR,Denmark) at the School
of Dentistry, University of Copen-
hagen were disinfected. The units
were used for routine patient treat-
ment during the study period.
Equipment to dispense Sterilex
UltraR (test agent) was mounted
on six units. The other six units
were disinfected with sodium
hypochlorite according to a routine
procedure previously d e ~ c r i b e d ' ~
(positive control). The results
obtained were compared to bacte-
rial numbers previously found in
six non-disinfected units (negative
control)". These were collected
during a period without patient
treatment (but with the equivalent
average daily flow of water), as
these units have been shown to
International Dental Journal (2003)Vol. 5WNo.4
build up biofilrns releasing bacteria
in numbers up to >105/ml when
not di~infected'~.
Sterilex UltraR(Sterilex Corpo-
ration, Maryland, USA) was used
according to the recommendations
of the manufacturer. One dose
(12.5g) of Sterilex UltraR was
dissolved in 250ml of water at
6OOC. This solution was applied to
the DUWL in the afternoon and
left overnight. In the morning
Sterilex UltraRwas let out and the
DUWL were rinsed with 11 of
water for 3 minutes.
Microbiologicalprocedures
All procedures were carried out
according to the European Standard
on 'Water quality - enumeration of
culturable micro-organisms' @ S /
E N I S 0 6222)'*.
Water samples (1Oml) were
taken in the morning from the
ultrasonic scaler, which is the last
instrument on the water lines of
the dental units. Sampleswere taken
in sterile tubes containing sodium
thiosulphate for neutralisation of
any remaining chlorine @S 2250)19
and immediately brought to the
laboratory. The samples were
10-fold serially diluted in peptone
diluent (IS0 8199)" to and
plated on yeast extract agar for
aerobic incubation. Two sets of
plates were left for incubation at
37°C for 44h and another two sets
at 21OC for 68h. The number of
colonies was counted at 2-3 times
magnification on an appropriate
dilution (containing 50-200 colo-
nies), and the number of colony
forming units cfu/ml was calcu-
lated (IS0 6222)'*.
Design
Part 1: Sterilex UltraRwas applied
on three consecutive days and
subsequently once a week for six
weeks. Sodium hypochlorite,
0.5ppm for 20 minutes followed
by rinsing with water, was applied
every night. Water samples were
taken before the first instillation of
Sterilex UltraR(baseline) and subse-
quently once a week immediately
before the next administration.
Part 2: Sterilex UltraR was
applied on three consecutive days
and subsequently once a week for
two weeks. Water samples were
taken every day during the second
week of administration.
Part 3: Sterilex UltraR was
applied for five weeks. The first
two weeks Sterilex UltraRwas left
in the DUWL for 4% days and the
following three weeks for 21/2 days.
Water samples were taken every
second morning three times a week
for six weeks.
At the end of the study photo-
graphs were taken of parts of the
water units.
Results
Part 1
Figure 1 shows the median number
of cfu/ml in the Sterilex UltraR
units and the hypochlorite units at
21OC and 37OC, respectively. After
the initial three administrations of
Sterilex UltraRthe number of cfu/
ml was considerably reduced.
After three weeks, however, the
numbers increased to baseline
levels (median >104/ml). This was
followed by another, though
minor, decrease, and at the final
examination after six weeks the
median was >lo4 cfu/ml. In the
units disinfected with sodium
hypochlorite there was less varia-
tion in the number of cfu/ml which
ranged from 3x10' to 3x103at 21OC
and from 7x10' to 2x104at 37°C.
In the Sterilex UltraRunits major
differences between the number of
cfu/ml in individual units were
observed during the recolonisation
period. This is illustrated in Figure
2a and 26.
Part 2
Figtre 3, and 3b show the gradual
recolonisation of the DUWL
during the second week after the
initial three days of Sterilex UltraR
administration ending at a median
number of cfu/ml of 4x104 and
 251

loo000 3x10' at 21OC and 37OC, respec-

tively. Again,variation between the
10000 units was observed.
z
2u
e
1000 -
-+
Hypochlorite, 21OC
Sterilex Ultra, 37OC
Part 3
Figure 4 shows the median number
E of cfu/ml during the six weeks of
10 prolonged administration of
Sterilex UltraR.In two of five weeks
1 the median cfu was >104/ml, and
0 1 2 3 4 5 6 this number was found in individual
week units during all weeks of the inves-
tigation. During the final part of
Fiaue 7. The median number of colony forming units (du)/ml at 21"Cand 37°Cin Sterilex
UltraRunits and hypochlorite units, respectively,during the first part of the study
the study leakage of water from
the water tubes and O-rings at the
water supply to the units was
noticed. Concurrently, white and
I

3
1000000 pink crystalline deposits occurred
at the check-valves and connections
of the units (Figt/re5).

1000 Discussion
The recognition of the potential
100
risk of infection when treating
-6
10 patients with water from DUWL
has led to the use of different
I 1 1 - 1 I I I I
approaches to control colonisation
0 1 2 3 4 5 6 and improve the water quality.
week These have included mechanical
methods like filtration and flushing
Figue2a. The number of colony forming units (cfu)/rnl in individual units at 21"C in Sterilex
UltraRunits during the first part of the study.
of the DUWL and different kinds
of chemical di~infection'.~. Seen
from an occupational and patient
safety perspective the mechanical
1000000 methods would be most advanta-
geous as contact with chemical
loo000 agents is potentially hazardous.
Unfortunately, neither flushing nor
10000
filtration have so far proved effec-
1000 tive in maintaining bacterial
numbers at a low level4.". The best
100 +5 results have been obtained when
10 applying chemical disinfectants,
especially chlorine compounds,
1 which have been studied most
0 1 2 3 4 5 6 extensively'. However, chlorine can
week
cause corrosion of metal parts of
the unit and thus damage the equip-
Rgum2b. The number of colony forming units (du)/ml in individual units at 37°Cin Sterilex ment4. So, new and better
UltraRunits during the first part of the study. disinfective agents are still sought.
Among the potential agents is
Sterilex UltraR,which has received
FDA clearance for marketing as a
waterline cleaner. This clearance,
though, does not imply a guarantee
Larsen and Fiehn: Sterilex UltraRanddental unit waterlines
252

1OOOOOO of a germicidal effect or of biofilm

1OOOOO removal'. So the ability of this agent
still has to be investigated.
loo00 The present study evaluated the
effect of Sterilex UltraR on the
number of culturable bacteria in
100 the DUWL. The goal to be achieved
-6 was first brought forward by the
10 American Dental Association,
recommending that dental unit
1 water should contain no more than
0 1.1 1.2 1.3 1.4 1.5 2.1 200 cfu/ml". European standards
week are also due, e.g. a Danish Stand-
ngure38. The number of colony forming units (cfu)/ml in individualunits at 21"C in Sterilex ard has recently defined that the
UltraRunits during the second part of the study. number of cfu/ml in DUWL
should not be beyond the accepted
levels for drinking wateP. These
levels have been defined by the
1OOOOOO Danish Ministry for the Environ-
1OOOOO ment to be maximally 20cfu/ml at
37OC and 200cfu/ml at 21"C,
loo00 respectively, in the water supply22.
The DUWL of the dental units
lo00 under investigation in the present
100 *5 study have previously been shown
to build up biofilms releasing
10 bacteria in high numbers when not
disinfectedt4.In a preceding study
1
average bacterial numbers from
0 1.1 1.2 1.3 1.4 1.5 2.1 3x104-2x105cfu/ml at 21OC as well
week as at 37OC were found during a
Rgutw36.The number of colony forming units (cfu)/ml in individualunitsat 37% in Sterilex three week study period where the
UltraRunits during the second part of the study. units were left untreated". When
Sterilex UltraRwas applied to the
units in the present study the bacte-
rial numbers were immediately
1OOOOO I reduced to almost zero. This
reduction was accompanied by
clogging up of the outlets from the
DUWL indicating a beneficial
effect of the agent on material
deposited in the DUWL. However,
the agent did not cause total elimi-
nation of the deposits as material
continued to be released from the
DUWL during the study period,
though not quite to the same
1
1.1 1.3 1.S 1.1 1.3 3.1 3J 3.5 4.1 43 4.5 5.1 SJ W extent. Further, the number of
week bacteria found in the units increased
again, indicating that biofilms could
Hgum 4 The median number of colonyforming units (du)/ml in Sterilex UltraRunits at 21"C still form in the DUWL in spite of
and 37°C respectively, during the third part of the study
the weekly administration of Sterilex
UltraR.These results are in contrast
to a recent study by Linger et al.
who almost maintained the number
of cfu/ml in DUWL of 20 units at
zero for five weeks by weekly

International Dental Journal (2003)Vol. 53/No.4


Figurn 5. Depositsat the check-valvesand connections of the unit at the end of the study.
instillation of Sterilex UltraRz3.
Their
study was conducted on units that
were not used for patient treat-
ment thus omitting the possibility
for retraction of bacteria from the
oral cavity as a source of contami-
nation. Further, their units were of
a different manufacture with self-
contained water systems and
possibly using another material for
the piping that may influence the
recolonisation.
In the present study recolo-
nisation was also observed when
the administration of Sterilex UltraR
was extended as recommended by
the manufacturer in the third part
of the study, so this procedure was
no solution to the problem.
Furthermore, during this period
leakage of water accompanied by
deposition of crystalline material
was observed implying that this
procedure also caused technical
problems in the units. As stated
previously the units included in this
study were 15 years old and previ-
ously colonised by millions of
bacterial4.Whether the effect would
have been better if Sterilex UltraR
had been applied to new units
before biofilms were allowed to
build up is not known. This would
imply the instillation of Sterilex
UltraR right from installation as
newly installed units can release up
to > l o 5 cfu/ml within a few
weeks".
The number of cfu/ml found
in the units disinfected with sodium
hypochlorite (median up to 3x103
at 21°C and 2x104 at 37OC) was
higher than the average numbers
of 6x10' to 6x10' cfu/ml previ-
ously obtained using this method".
Probably the explanation to this is
that in the previous study the instilla-
tion of 0.5ppm sodium hypochlorite
every night was preceded by one
application of a very high concen-
tration (20ppm). Such a shock
treatment is a well described
method of reducing bacterial
numbers in water
has been shown to be effective in
dental unit^'^.
In conclusion, the administra-
tion of Sterilex UltraRaccording to
the recommendations of the manu-
facturer initially reduced the number
af bacteria in DUML to <lo2cfu/
ml. However, during the following
253
period a gradual recolonisation was
observed resulting in bacterial
numbers > lo4cfu/ml in a number
of units. Thus, Sterilex UltraRwas
not capable of maintaining an
acceptable water quality in these
older units.
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