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FEMS Microbiology Letters 235 (2004) 363–367
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In vitro modeling of dental water line contamination
and decontamination
D.A. Spratt
a
a,*
, J. Latif a, L.L. Montebugnoli b, M. Wilson
a
Division of Microbial Diseases, Eastman Dental Institute for Oral Health Care Sciences, UCL, London, UK
b
Department of Oral Science, University of Bologna, Italy
Received 18 March 2004; received in revised form 27 April 2004; accepted 10 May 2004
First published online 18 May 2004
Abstract
The contamination of dental unit water lines (DUWL) is an emerging concern in dentistry. The aim of this study was to use an in
vitro DUWL to model microbial contamination and evaluate the decontamination efficacy of tetraacetylethylenediamine (TAED)
solutions. A DUWL biofilm model used to simulate clinical conditions was used to generate a range of biofilms in DUWL. Three
distinct biofilms were generated: (1) biofilm from water, (2) biofilm from a mix of water + contaminating human commensal bacteria, (3) biofilm from water with contaminating oral bacteria added after biofilm formed. The contaminating oral species used were
Streptococcus oralis, Enterococcus faecalis and Staphylococcus aureus. Decontamination by simple water flushing or flushing with
TAED was evaluated (2, 5 and 10 min intervals). The DUWL tubes were split and samples were plated onto a range of media,
incubated and bacteria enumerated. Water flushing did not reduce the number of microorganisms detected. Bacteria were not
detected from any of the TAED sampling points for any of the biofilm types tested. Interestingly, if contamination was introduced
to new DUWL along with the waterborne species a biofilm was formed containing only the waterborne species. If however, an
existing biofilm was present before the introduction of ‘‘contaminating’’ bacteria then these could be detected in the biofilm. This
implies that if the DUWL are new or satisfactorily cleaned on a regular basis then the associated cross-contamination aspects are
reduced. In conclusion, TAED provides effective control for DUWL biofilms.
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Biofilm; Dental water line; Decontamination; TAED
1. Introduction
Bacteria exist in nature almost exclusively as biofilms
and are associated with up to 65% of human bacterial
infections [1]. Dental examples of biofilms include: on
teeth as subgingival and supragingival plaque and on the
lumen wall of dental unit water lines.
A biofilm can be defined as a community of microorganisms irreversibly attached to a surface, containing
exopolymeric matrix and exhibiting distinctive phenotypic properties [2]. When bacteria come in contact with
a surface they sense it and attempt to adhere to it.
*
Corresponding author. Tel.: +44-20-7915-1107; fax: +44-20-79151127.
E-mail address: [email protected] (D.A. Spratt).
Biofilms are characteristically up to 1000 times more
resistant to killing than their broth grown counterparts
[3].
The contamination of dental unit water lines
(DUWL) is an emerging concern in dentistry since the
proportion of elderly and immunocompromised patients
seeking dental care is increasing. DUWL contamination
is caused by the accumulation of microorganisms and
their products on the walls of the lumen of the tubing
used to dispense water.
Since biofilms on the lumen walls of DUWL act as a
permanent reservoir of microorganisms they have recently been identified as a source of potential bacterial
infection for the dental patient [4]. A further concern
regarding this is the cross-infection issue. It is possible
that bacteria derived from saliva and plaque from the
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.05.006
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D.A. Spratt et al. / FEMS Microbiology Letters 235 (2004) 363–367
mouth of one patient has the potential for re-inoculation
into another patients mouth at a later date. The contamination can be brought about by retraction of liquids
and aerosols due to the negative pressure generated in
the vicinity of the bur when a turbine stops rotating [5].
The volume of this can be as high as 0.9 ml [6] but is
obviously influenced by among other things the shape of
the turbine and the length of the bur used [6]. Anti-retraction devices are now fitted which greatly reduce the
volume of liquid retracted. However, given that biofilms
form on all the surfaces within the DUWL system and
the mechanical nature of these retraction devices, it may
be postulated that after continuous use over a long time
period that these devices will work sub-optimally.
The ‘‘contaminating’’ microorganisms from the oral
cavity whether they are fungal, bacterial or viral in nature may become established in the existing biofilm,
multiply and detach to potentially infect a future patient. Indeed, oral streptococci and Fusobacterium species have been detected in DUWL from a number of
dental surgeries [7]. The American Dental Association
has now set a guideline of 200 colony forming units per
millilitre (cfu/ml) of DUWL water [8] and in Europe the
European Union’s guideline for potable water is 100 cfu/
ml. A recent survey showed that of 55 dental surgeries
surveyed in the UK 83% and 95% of the DUWL tested
failed to meet these guidelines respectively [7].
At present, the guidelines for ‘‘clearing’’ this biofilm
contamination are a 30 s flush with water prior to patient
treatment followed by a 30 s rinse between patients [9].
Over the years a number of methods have been used
for the decontamination of DUWLs. In general the
bactericidal agents of choice e.g. NaOCl are not compatible with the materials used in the manufacture of the
DUWL systems and adverse reactions tend to occur
with respect to the steel fittings and various washers
within the system. Recently the antimicrobial activity of
a peracetic acid has been revisited since a new chemical
formulation has been proposed; tetraacetylethylenediamine (TAED) in association with sodium perborate.
TAED is an excellent low temperature bleach activator.
It is used as part of bleach system in a detergent formulation with a peroxygen source e.g. sodium percarbonate and sodium perborate to provide bleach
activation at low temperature. TAED is biodegradable
and non-sensitising. It is typically applied in domestic
laundry detergents, automatic dish washing, bleach
boosters and laundry soak treatments. Indeed due to
superior acidification performance, TAED has been
successfully used in textile industry, pulp and paper
processing etc. It has also has been suggested as a useful
DUWL decontaminant and cross-infection control
agent [10].
In this study we use in vitro biofilm model to assess
TAED with respect to DUWL decontamination/crossinfection (Fig. 1).
Fig. 1. In vitro model for DUWL biofilm formation and decontamination testing.
The aims of this study were to compare tap water
flushing with TAED flushing, model addition of human
commensal bacteria to the system and determine the
subsequent decontamination.
2. Materials and methods
An in vitro DUWL model simulating clinical conditions (Fig. 1) was used to compare simple water flushing
with TAED treatment. A 3% solution of TAED and
sodium perborate (Castellini spa, Bologna, Italy) at pH
8 equivalent to 0.26% peracetic acid was freshly made
and used in all experiments [10].
Two centimetre lengths of unused DUWL tubes
(polyurethane, Midwest Quad straight, 8400 , A-dec, Oregon, USA) were connected in series with silicone tubing
to a water reservoir and a programmable peristaltic
pump. Tap water (chlorinated) containing approximately 3 103 cfu/ml at point of use was passed through
an ‘‘in use’’ DUWL and used to inoculate the system (3 l
reservoir). This water was then pumped (peristaltic
pump, Watson Marlow, Falmouth, UK) around the
system for 8 h of simulated chair side use (alternating 15
min flow at 90 ml min1 and 15 min stagnation), 16 h of
stagnation, 8 h simulated chair side use and a further 16
h stagnation period etc. The system was run for three or
six days. Reproducibility within and between runs was
assessed. The system, containing five removable DUWL
sections, was run on three separate occasions for six
days. DUWL sections containing the biofilms were removed split and a sterile swab used to sample the lumen
and immediately placed into neutralising broth (Difco
Labs Ltd, Surrey, UK). Samples were serially diluted
and plated onto R2A agar incubated at room temperature for seven days and cfu/biofilm calculated.
Prior to decontamination or water flushing (90 ml/
min, equivalent to normal dental unit conditions) one
section of the DUWL was removed as a baseline control. Following introduction of the test decontaminant
(or water flushing), sections of DUWL were removed at
D.A. Spratt et al. / FEMS Microbiology Letters 235 (2004) 363–367
2, 5 and 10 min intervals ðn ¼ 4Þ. The DUWL tubes
(control and test) were split using a sterile scalpel and
forceps and a sterile swab used to sample the lumen and
immediately placed into neutralising broth.
Further modifications to this protocol were performed to model contamination of the DUWL by oral
bacteria entering the system by retraction during patient
treatment; either as a function of simple retraction or as
a result of the failure of an anti-retraction device. The
‘‘contaminating’’ bacteria used were Streptococcus oralis, Enterococcus faecalis and Staphylococcus aureus.
These were resuspended in water and added to the 3 l
water reservoir (107 cfu of each) either (i) during initial
biofilm formation or (ii) three days after initial biofilm
formation. A range of media were used throughout the
study to detect both waterborne species and ‘‘contaminating’’ species. Samples were serially diluted and plated
onto R2A agar for waterborne and environmental isolates, Blood Agar (BA) for total count or aerobic nonwater isolates, Mitis Salivarius agar (MS) to detect any
streptococci in the samples especially the S. oralis used,
Mannitol Salts Agar (MSA) to detect the S. aureus and
Bile Aesculin Agar (BAE) to detect the enterococci.
These were incubated in the appropriate conditions and
bacteria were enumerated [11]. Additionally these were
used as controls when no contaminants were added to
the system.
3. Results
The reproducibility both with a run and between runs
was assessed and showed minimal variation. The mean
cfu/biofilm for each run was 8 105 (SD ¼ 3 105 ) the
results are shown in Fig. 2. The effect of a 2 min water
flushing did not reduce the viable count of the biofilm.
Conversely, a 2 min flush of TAED reduced the viable
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Fig. 3. Comparison between tap water flushing and TAED flushing to
decontaminate a DUWL biofilm generated with ‘‘contaminating’’
bacteria added to system prior to biofilm formation showing viable
counts on a range of media (R2A agar, BA – Blood Agar, MS – Mitis
Salivarius agar, MSA – Mannitol Salts Agar and BAE – Bile Aesculin
Agar). Before flushing (j), after 2 min flushing with water ( ) and
after 2 min flushing with TEAD ( ). Columns represent mean values
ðn ¼ 5Þ and bars represent standard deviations.
count of the biofilm to below detectable limits (10 cfu/
ml). Longer flushing periods using TAED were initially
used (5 and 10 min) and again no viable bacteria were
detected.
Further studies were carried out with respect to crossinfection issues. The addition of ‘‘contaminating’’ bacteria to the system either BEFORE a biofilm was grown on
the lumen walls or AFTER was modelled. The decontamination protocols used above i.e. flushing with tap
water or TAED were carried out on both these biofilms
(Figs. 3 and 4). The results presented in Fig. 3 show that
when contaminating bacteria were added to the reservoir
before biofilm formation a ‘‘normal’’ water-derived
biofilm was grown and no ‘‘contaminating’’ bacteria
(S. oralis, E. faecalis or S. aureus) could be detected.
Conversely, the results presented in Fig. 4 show that if
contaminating bacteria were added to the system after
a biofilm was present then of all the species could be
detected albeit in low numbers (102 –103 per biofilm).
4. Discussion
Fig. 2. Comparison of reproducibility of viable counts per biofilm
within and between runs. Grey area represents boundaries between 105
and 106 cfu/ml.
The in vitro DUWL model was used to generate a
range of biofilms and to study a range of decontamination procedures. Using this system the concept of
flushing with tap water to eliminate biofilm was tested
using a 2 min flushing instead of the recommended
30 s flush, this fourfold increase in the flushing time
did not reduce the biofilm on the lumen walls of
DUWL compared to the no flushing control. These
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D.A. Spratt et al. / FEMS Microbiology Letters 235 (2004) 363–367
Fig. 4. Comparison between tap water flushing and TAED flushing to decontaminate a DUWL biofilm generated with ‘‘contaminating’’ bacteria
added to system after biofilm formation showing viable counts on a range of media (R2A agar, BA – Blood Agar, MS – Mitis Salivarius agar, MSA –
Mannitol Salts Agar and BAE – Bile Aesculin Agar). Before flushing (j), after 2 min flushing with water ( ) and after 2 min flushing with TEAD
( ). Columns represent mean values ðn ¼ 5Þ and bars represent standard deviations.
findings are in agreement with other studies carried out
in our laboratory on samples of water (pre- and postflushing) from ‘‘in use’’ DUWL (data not shown).
Conversely TAED was flushed through the DUWL for
2, 5 and 10 min and at all time points the biofilm was
reduced to below detectable limits. These data are in
agreement with Montebugnoli and Dolci [9] whose
preliminary study showed that TEAD achieved complete kill of heterotrophs in an in vitro model during
the 5 min contact time used. They also showed that in
clinical situation viable counts reduced from over 105
cfu/ml prior to TAED treatment to 102 cfu/ml after
treatment.
The cross-infection model was developed using three
human ‘‘normal flora’’ species namely S . oralis a human
oral commensal [12] and E. faecalis and S. aureus both
of which are often isolated from the oral cavity [13,14].
In addition, E. faecalis and S. aureus are opportunist
pathogens which are associated with cross-infection
issues.
Experiments modelling the establishment of these
‘‘contaminating’’ species in biofilms on the lumen of
DUWL were carried out. The most important finding
was that if contamination was introduced to new
DUWL along with the waterborne species a biofilm was
formed containing only the waterborne species, no
contaminating species were detected on any of the selective media used. If however an existing biofilm was
present before the introduction of ‘‘contaminating’’
bacteria then these could be detected in the biofilm at the
sampling times. This implies, perhaps simplistically, that
if the DUWL are new or satisfactorily cleaned on a
regular basis then the theoretical cross-contamination
aspects via detached DUWL biofilm are much reduced.
However, further testing in a clinical situation needs to
be carried out.
In conclusion, these studies show that in our model
TAED is more efficient than flushing alone when used to
control biofilm build up and may protect against possible cross-infection.
Acknowledgements
The authors would like to thank Castellini S.p.A.
who funded this work.
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