Biofouling

The Journal of Bioadhesion and Biofilm Research

ISSN: 0892-7014 (Print) 1029-2454 (Online) Journal homepage: http://www.tandfonline.com/loi/gbif20

Biofilm formation in an experimental water

distribution system: the contamination of
non-touch sensor taps and the implication for
healthcare

Ginny Moore, David Stevenson, Katy-Anne Thompson, Simon Parks, Didier

Ngabo, Allan M. Bennett & Jimmy T. Walker

To cite this article: Ginny Moore, David Stevenson, Katy-Anne Thompson, Simon Parks, Didier
Ngabo, Allan M. Bennett & Jimmy T. Walker (2015) Biofilm formation in an experimental water
distribution system: the contamination of non-touch sensor taps and the implication for
healthcare, Biofouling, 31:9-10, 677-687, DOI: 10.1080/08927014.2015.1089986

To link to this article: http://dx.doi.org/10.1080/08927014.2015.1089986

Published online: 07 Oct 2015.

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 Biofouling, 2015
Vol. 31, Nos. 9–10, 677–687, http://dx.doi.org/10.1080/08927014.2015.1089986

Biofilm formation in an experimental water distribution system: the contamination of

non-touch sensor taps and the implication for healthcare
Ginny Moore*, David Stevenson, Katy-Anne Thompson, Simon Parks, Didier Ngabo, Allan M. Bennett and
Jimmy T. Walker
Biosafety Investigation Unit, Public Health England, Salisbury, UK
(Received 3 July 2015; accepted 28 August 2015)

Hospital tap water is a recognised source of Pseudomonas aeruginosa. UK guidance documents recommend measures to
control/minimise the risk of P. aeruginosa in augmented care units but these are based on limited scientific evidence. An
experimental water distribution system was designed to investigate colonisation of hospital tap components. P. aeruginosa
Downloaded by [Universidad de Buenos Aires] at 11:40 07 June 2016

was injected into 27 individual tap ‘assemblies’. Taps were subsequently flushed twice daily and contamination levels
monitored over two years. Tap assemblies were systematically dismantled and assessed microbiologically and the effect of
removing potentially contaminated components was determined. P. aeruginosa was repeatedly recovered from the tap
water at levels above the augmented care alert level. The organism was recovered from all dismantled solenoid valves
with colonisation of the ethylene propylene diene monomer (EPDM) diaphragm confirmed by microscopy. Removing the
solenoid valves reduced P. aeruginosa counts in the water to below detectable levels. This effect was immediate and
sustained, implicating the solenoid diaphragm as the primary contamination source.
Keywords: Pseudomonas aeruginosa; tap water contamination; non-touch sensor taps; biofilm; model water distribution
system

Introduction Several outbreaks of P. aeruginosa have been attribu-

Water and water systems harbour a diversity of microor-
ganisms and whilst the majority are considered harmless
to healthy individuals, some, such as Pseudomonas
aeruginosa, can cause serious opportunistic infections,
particularly in individuals with a weakened immune sys-
tem (Lyczak et al. 2000). There are reports to suggest
that waterborne P. aeruginosa may account for 3% of all
healthcare-associated infections (Anaissie et al. 2002;
Hidron et al. 2008). Consequently, hospitals and other
healthcare facilities need to control the risk posed by
water systems to at-risk patients.
Neonates in whom the immune system is not yet
developed are particularly vulnerable (Cipolla et al.
2011; Jefferies et al. 2012). Between November 2011
and January 2012, 25 babies admitted to four different
hospitals in Northern Ireland acquired P. aeruginosa
whilst being treated in the neonatal intensive care unit
(NICU); nine babies developed a symptomatic infec-
tion and four died (Troop 2012). Each of the four
incidents was associated with a different strain (or
strains) of P. aeruginosa, indicating that transmission
between units had not occurred. However, 18 (72%)
of the 25 babies acquired the same strain of
P. aeruginosa as that recovered from water samples
and/or swabs taken from handwash basins located
within their NICU.
ted to contaminated tap water (Ferroni et al. 1998;
Vianelli et al. 2006; Aumeran et al. 2007; Durojaiye
et al. 2011). Once established within a faucet or else-
where within the water system, P. aeruginosa is difficult
to eradicate and this is partially attributable to the bio-
film forming properties of the organism (Høiby et al.
2001). Biofilms are complex, structured, specialised
communities of adherent microorganisms surrounded by
extracellular polymeric substances. They are fundamen-
tally different from populations in suspension in terms of
cell surface characteristics, metabolism and chemical
structure (Donlan 2002). These physiological changes
together with delayed penetration of chemical agents
through the biofilm matrix result in an increased toler-
ance to antimicrobial agents, including disinfectants and
other chemical biocides (Bridier et al. 2011).
Following the incidents in Northern Ireland, 30 repre-
sentative tap assemblies were removed from the neonatal
units and sent to Public Health England (PHE) Porton,
where they were dismantled and analysed. Scanning
electron microscopy revealed extensive biofilm formation
on sections of the plastic flow straighteners (Walker
et al. 2014). These flow straighteners, in comparison to
other tap components, were the most likely to be con-
taminated with P. aeruginosa and were associated with
the highest P. aeruginosa counts, implicating them as a

*Corresponding author. Email: ginny.moore@phe.gov.uk

© Crown Copyright 2015. Reproduced with the permission of the Controller of Her Majesty’s Stationery Office and Public Health England
 678 G. Moore et al.
Figure 1. The experimental water distribution system
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(EWDS).
possible source of the neonatal infections (Walker et al.
2014). As a consequence, the UK Department of Health
commissioned PHE to investigate the formation of bio-
film on tap components similar in type to those removed
from the neonatal units in Northern Ireland. The aim of
this study was to design and build an experimental water
distribution system and, with particular reference to
P. aeruginosa, investigate the contamination of different
tap components, including different types of flow
straightener.
Materials and methods
Design of experimental water distribution system
(EWDS)
Borehole-supplied water (total hardness: 248 mg
CaCO3 l−1; total chlorine: 0.25–0.61 mg l−1) was stored
in two domestic-style copper indirect combination cylin-
ders (capacity: 115 l (bottom tank), 40 l (top tank); RM
Cylinders, Castleford, UK). A heating element in one of
the tanks constantly heated the water to 60°C; the other
held water at room temperature. The water storage tanks
were connected to a centrifugal, negative head, supply-
on-demand shower pump (Salamander Pumps, Sunder-
land, UK) which, operating at 3 bar, supplied the hot
and cold water to the EWDS via two separate copper
lines (diameter 22 mm). The water system incorporated
bypass valves to allow for the flushing of each line with-
out the use of the taps, both to drain and to allow recir-
culation of unmixed hot and cold water back to the
respective storage tanks. Recirculation allowed a constant
supply of water at a controlled temperature to be main-
tained throughout the system, preventing the constant on/
off operation of the water pump, which can lead to
inconsistent supply pressures and flow rates.
Twenty-seven tap ‘assemblies’ each comprising a
thermostatic mixer valve (Pegler Prestex P402 15 mm
TMV3; Pegler Yorkshire Group Ltd, Doncaster,
UK), solenoid valve (Phoenix Contact Ltd, Telford, UK),
15 mm gate valve (Screwfix Direct Ltd, Yeovil, UK),
binder-style test point (Macro Construction, Portsmouth,
UK) and stainless steel non-touch sensor tap (Aquarius
deck mounted short neck tap with copper tails; Dart
Valley Systems, Paignton, UK) were connected to the
circulating hot and cold water supply via 15 mm diame-
ter copper piping (Figure 1). To allow automation of the
EWDS, each solenoid valve (disconnected from its
associated infra-red sensor) was connected to a bespoke
30-channel Siemens electronic control panel (AJK Ser-
vices Ltd, Tidworth, UK) programmed to sequentially
open each solenoid valve for 30 s.
Operation of experimental water distribution system
Twice daily (Monday–Friday), the hot and cold bypass
valves were opened to allow water to circulate the
EWDS. When the temperature of the returning hot water
was ~ 60°C, the control panel was manually switched on
and each tap assembly automatically flushed for 30 s.
The TMV and gate valve (pre-set during installation)
ensured the temperature and flow rate of the water from
each outlet was ~ 43°C and ~ 4.5 l min−1 respectively.
Contamination of experimental water distribution
system
The EWDS was contaminated with Pseudomonas
aeruginosa PA14 (variable number tandem repeat
(VNTR) profile 12, 2, 1, 5, 5, 2, 4, 5, 12) (Martin et al.
2013). An overnight culture of P. aeruginosa was diluted
100-fold using sterile deionised water. Ten ml of the
resulting suspension (~107 colony forming units (CFU)
ml−1) were injected directly into each tap assembly via its
associated binder point. To encourage P. aeruginosa
colonisation of the distribution system, the water within
the EWDS was allowed to stagnate for five days, after
which time the twice daily flushing regimen was resumed.
Collection and microbiological analysis of water
samples
Water samples were taken to assess colonisation and to
monitor contamination levels over time. In the UK, aug-
mented care units are advised that if test results are satis-
factory (ie if P. aeruginosa is not detected) water sampling
can be undertaken on a six-monthly basis. For the pur-
poses of this study and to assess how contamination levels
may fluctuate between any two sampling occasions, water
samples were taken over a two year period.
 Biofouling 679

Microbiological analysis of tap components

Flow straighteners/faucet aerators
Each of the 27 taps was fitted with a flow straightener or
one of two faucet aerators (Figure 2; Neoperl Ltd,
Malvern, UK). After remaining in situ for two years, the
external surface (outlet face) of each flow straightener/
aerator was sampled (whilst still in situ) using a sterile
cotton-tipped swab (Sterilin Ltd, Newport, UK) moist-
ened with sterile thiosulphate Ringers (Oxoid). The swab
was snapped into 1 ml sterile thiosulphate Ringers and
vortexed for 1 min to release any bacteria from the swab
bud. The resulting suspension was diluted 10-fold and
100 μl aliquots of each dilution were plated on to non-
selective agar (R2A) and agar selective for P. aeruginosa
(C-N agar) and Pseudomonas spp. (C-F-C agar; Oxoid).
Each flow straightener/aerator (and associated rubber
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seal) was removed from the tap outlet and rinsed with
50 ml of sterile irrigation water to remove any loosely
attached bacteria. The flow straightener/aerator was then
placed in a plastic container containing 5 ml of sterile
thiosulphate Ringers and 10 sterile glass beads and vor-
texed for 2 min. The resulting suspension was diluted
100-fold and 100 μl aliquots of each dilution were plated
on R2A, C-N and C-F-C agar plates. The remaining
(undiluted) suspension was passed through a membrane
filter (0.2 μm) which was then placed on the surface of a
C-N agar plate. Finally, each flow straightener/aerator
was transferred to 10 ml of nutrient broth (Oxoid) and
incubated at 37°C for 48 h. An aliquot of broth was then
spread over the surface of a C-N agar plate.

Figure 2. Tap outlets were fitted with a flow straightener (A)

or one of two faucet aerators (B and C).

The first sample of water (500 ml) delivered from each

tap was collected in sterile, disposable polyethylene bottles
containing sodium thiosulphate (20 mg l−1) to neutralise
chlorine residues. The presence of P. aeruginosa was
determined by passing 100 ml of each water sample
through a polyethersulfone membrane disc filter (47 mm,
0.2 μm; Pall Life Sciences, Portsmouth, UK) and transfer-
ring the filter to Pseudomonas agar supplemented with
cetrimide and sodium nalidixate (C-N agar, Oxoid Ltd,
Basingstoke, UK). To determine the heterotrophic plate
count, the water samples were diluted 100-fold and ali-
quots (100 μl) of each dilution plated onto non-selective
Figure 3. Break down of the solenoid valve into its component
agar (R2A; Oxoid). Water taken directly from the hot and parts: (1) inlet port; (2) outlet port; (3) EPDM diaphragm;
cold water tanks was sampled in the same manner. (4) armature, push rod and seal; (5) outer casing for magnetic coil.
 680 G. Moore et al.
Binder points and solenoid valves
The binder points and solenoid valves associated with 10
tap assemblies (randomly selected using Microsoft Excel
(2012) (RANDBETWEEN function)) were removed and
dismantled using manual hand tools (Figure 3). Each
component part was rinsed with sterile irrigation water
(to remove loosely attached bacteria) before being sam-
pled using two sterile, pre-moistened cotton-tipped
swabs. Each pair of swabs was transferred to a plastic
container containing 10 ml of sterile water and 10 sterile
glass beads and vortexed for 2 min to release any bacte-
ria from the swab bud. The resulting suspension was
diluted 100-fold and 100 μl aliquots of each dilution
were plated on to R2A and C-N agar plates. The remain-
ing (undiluted) suspension was passed through a mem-
brane filter (0.2 μm) which was then placed on the
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surface of a C-N agar plate.
Segments of the different surface materials associated
with an unused flow straightener, aerator and solenoid
valve were analysed using a Phillips XL30 FEG
scanning electron microscope (SEM) (FEI UK Ltd,
Cambridge). Corresponding segments taken from compo-
nents removed from the EWDS were also analysed.
Removal and replacement of tap components
The 10 binder points and five of the 10 solenoid valves
that had been removed for sampling were replaced with
new components. The remaining five solenoid valves
and all 27 flow straighteners/aerators were permanently
removed from the EWDS; the former replaced with
copper piping (15 mm). The twice daily flushing regimen
was resumed and weekly water samples were taken to
assess any immediate and continual effect of removing
or replacing potentially contaminated tap components.
Analysis of results
All C-N plates were incubated at 37°C for 48 h.
Presumptive P. aeruginosa colonies were subcultured
and confirmed using the API 20NE biochemical test kit
(bioMérieux, Basingstoke, UK). Representative isolates
were sent to the Antimicrobial Resistance and Healthcare
Associated Infections Reference Unit (London, UK) for
typing. VNTR analysis at nine loci was carried out as
previously described (Turton et al. 2010).
All C-F-C and R2A plates were incubated at 30°C
for three to five days, after which time the number of
colony forming units were enumerated. Predominant
colonies were subcultured and identified using the API
20NE test kit.
Data analysis was performed using SigmaPlot for
Windows (version 12.0). Intergroup comparisons were
made using the Mann–Whitney rank sum test or the
Kruskal–Wallis one way analysis of variance in
combination with Dunn’s multiple comparison test.
Sample data were considered statistically significant
when p < 0.05.
Results
Colonisation of the experimental water distribution
system
Following contamination of the EWDS (and after the
water had been allowed to stagnate for five days), the
median number of P. aeruginosa colonies recovered from
the first sample of water delivered from each tap was
1.1 × 103 CFU 100 ml−1. All taps tested positive for
P. aeruginosa with counts ranging from 1 CFU 100 ml−1
to 4.5 × 105 CFU 100 ml−1. Sampling was next carried
out two days after the twice-daily flushing regimen had
been resumed. On this occasion, there was no detectable
P. aeruginosa in water taken from seven of the 27 taps
(ie P. aeruginosa counts were <1 CFU 100 ml−1). These
seven tap outlets tested negative for P. aeruginosa for the
duration of the study period. The remaining 20 tap
assemblies had become colonised. The median hetero-
trophic plate count was 3.5 × 105 CFU 100 ml−1 whilst
median P. aeruginosa counts one week and six months
after contamination of the EWDS were 690 CFU
100 ml−1 and 304 CFU 100 ml−1 respectively (Figure 4).
The type of flow straightener/aerator fitted within the out-
let had a small but significant effect upon contamination
levels (p = 0.006). The median number of P. aeruginosa
colonies recovered from water delivered via the flow
straightener was 197 CFU 100 ml−1 (n = 210) whilst the
more complex aerator (outlet fitting B; Figure 2) was
associated with the lowest P. aeruginosa counts
(145 CFU 100 ml−1; n = 208; p < 0.05). P. aeruginosa
persisted within the EWDS and continued to be recovered
from the 20 tap outlets at levels above the alert value for
augmented care (ie > 10 CFU 100 ml−1). One year after
contamination of the EWDS, median P. aeruginosa
counts were 267 CFU 100 ml−1. At no point during the
investigation was P. aeruginosa detected in the source
water.
Microbiological analysis of tap components
Flow straighteners/faucet aerators
The median number of bacteria recovered from the swab
samples (ie the external surface (outlet face) of the flow
straighteners/faucet aerators) was 10 CFU (n = 27). Het-
erotrophic plate counts ranged from <10 CFU to 4.72 ×
104 CFU. P. aeruginosa (10 CFU) was recovered from a
single surface swab. The type of flow straightener/aerator
had no significant effect upon heterotrophic plate count
(p > 0.05) or the prevalence of P. aeruginosa.
 Biofouling 681
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Figure 4. Median (IQR) recovery of P. aeruginosa from water delivered from the EWDS. The alert value for augmented care is also
illustrated (- - - -).

The median number of bacteria recovered from the
flow straighteners/aerators (together with the associated
rubber seal) was 2.55 × 105 CFU (n = 27). Heterotrophic
plate counts ranged from <50 CFU to 1.60 × 106 CFU
with significantly fewer bacteria being recovered from
the flow straighteners than from the aerators (p = 0.019).
In all cases, the most predominant colony was identified
as being Sphingomonas paucimobilis. Stenotrophomonas
maltophilia (50 CFU) was recovered from one of the
faucet aerators. However, no presumptive P. aeruginosa
colonies were recovered from any of the direct or filtered
samples, although P. aeruginosa was recovered from one

Figure 5. Median (IQR) number of bacteria recovered from the surface of different parts of the solenoid valve.
 682 G. Moore et al.
of the enriched flow straighteners. The type of flow
straightener/aerator had no significant effect upon
prevalence of P. aeruginosa (p > 0.05).
Binder points and solenoid valves
The heterotrophic and P. aeruginosa plate counts from
different component parts of the solenoid valve are pre-
sented in Figure 5. The results have been standardised by
dividing the number of colonies recovered by the surface
area sampled and are expressed as CFU cm−2. The med-
ian number of bacteria recovered from the solenoid valves
was 3.4 × 104 CFU cm−2. Heterotrophic plate counts ran-
ged from 2.9 × 103 CFU cm−2 to 5.1 × 105 CFU cm−2.
The diaphragm was significantly more contaminated
(1.7 × 105 CFU cm−2) than the outer casing for the mag-
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netic coil (5.75 × 103 CFU cm−2; p < 0.05). The median
number of P. aeruginosa colonies recovered from the
solenoid valves was 8.7 CFU cm−2. Counts ranged from
0.3 CFU cm−2 to 1.25 × 103 CFU cm−2 with significantly
more P. aeruginosa being recovered from the diaphragm
(79 CFU cm−2) than from the inlet and outlet ports
Figure 6. Recovery of P. aeruginosa from the water delivered from the EWDS: the immediate and continual effect of removing or
replacing the solenoid valve.
(p < 0.05; Figure 5). Isolates colonising the solenoid valve
had the same VNTR profile (ie were the same strain) as
that recovered from the water. P. aeruginosa was not
recovered from any of the binder points.
Removal and replacement of tap components
Removing the flow straightener/aerator from the tap
outlet had no significant effect upon the median number
of P. aeruginosa colonies recovered from the tap water
(p > 0.05). P. aeruginosa continued to be recovered from
tap assemblies in which the original solenoid valve had
remained in situ and, two years after contamination of the
EWDS, P. aeruginosa counts associated with eight of 10
such taps were still above the alert level for augmented
care (Figure 6). In contrast, removing the solenoid valve
and replacing it with either copper piping or a new
component immediately reduced the P. aeruginosa count
in water delivered from the tap outlet to below detectable
levels (ie < 1 CFU 100 ml−1; Figure 6). Removing the
solenoid valve from a tap assembly already negative for
P. aeruginosa (tap 16) had no immediate effect on the

presence/absence of P. aeruginosa. However, removing
or replacing the solenoid valve ensured that there was no
detectable P. aeruginosa in water taken from any such
tap for the remainder of the study.
Discussion
A range of model systems, including chemostats (Rogers
et al. 1994), Propella reactors (Manuel et al. 2007) and
rotating disc reactors (Murga et al. 2001) have been used
to investigate the formation of biofilm on different
plumbing materials. Such laboratory studies are control-
lable and reproducible but do not necessarily replicate
‘in-use’ conditions. Recreating a water distribution sys-
tem under laboratory conditions can be challenging but
can provide a realistic model in which to investigate
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microbial contamination and control (Muraca et al. 1987;
Waines et al. 2011).
The experimental water distribution system (EWDS)
used in the current study was designed to simulate that of
a hospital ward and was based upon tap components
similar in type to those removed from the neonatal units
in Northern Ireland (Troop 2012; Walker et al. 2014).
Following contamination of the EWDS, there was no
detectable P. aeruginosa in water taken from seven of the
27 taps, reflecting perhaps the natural variability observed
in hospital water systems. However, 20 tap assemblies
Figure 7. SEM images (magnification 4,000×) of biofilm on a section of the plastic flow straightener (A) and aerator (B) after two -
years in situ. Higher numbers of bacteria were recovered from the aerator (B). Images of an unused flow straightener (C) and aerator
(D) are included for comparison.
Biofouling 683
became colonised and the water delivered from the tap
outlets was contaminated with P. aeruginosa at a level
that, if detected in an augmented care unit, would result
in the isolation of the outlet and a requirement for
remediation work. The absence of P. aeruginosa in both
the source and recirculated water suggested that the
source of the contamination, rather than being systemic,
was at or close to the outlet. This was similar to that
observed in the neonatal units where despite high counts
in pre-flush samples, P. aeruginosa was not detected in
samples collected post-flush, indicative of a problem
related to the local water outlet(s).
Flow straighteners taken from the neonatal units were
shown to be heavily colonised with P. aeruginosa (median
CFU: 5.2 × 104; n = 41) and those described as being
‘complex’ were associated with significantly higher
P. aeruginosa counts than those more simplistic in design
(Walker et al. 2014). In the current study, the type of outlet
fitting had a small but significant effect upon the number
of P. aeruginosa colonies recovered from the water. The
aerator considered the most complex (outlet fitting B;
Figure 2) was associated with the lowest P. aeruginosa
counts (p < 0.05) suggesting that bacteria may have
become trapped and retained within the complex internal
configuration of the fitting, potentially leading to the
formation of biofilm on and/or within the aerator.
Certainly, SEM analysis of the two types of fitting showed
 684 G. Moore et al.
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Figure 8. Visual appearance of outlet fittings taken from a
neonatal unit (A) and the EWDS (B) after remaining in situ for
four months and two years, respectively. Images demonstrate a
significant difference in biofilm formation.
Figure 9. Macroscopic and microscopic (magnification 4,000×) images of biofilm on an EPDM diaphragm removed from the
EWDS. Arrows indicate individual bacterial cells within the biofilm.
marked differences in terms of biofilm structure (Figure 7)
and when removed from the outlet, significantly more
bacteria were recovered from aerators than from the less
complex flow straighteners (p = 0.019). However, despite
the presence of high numbers of P. aeruginosa in the water
delivered from the outlets and despite remaining in situ for
two years, only two of the 27 outlet fittings were contami-
nated with P. aeruginosa (≤ 10 CFU) and both isolates
had a VNTR profile (8, 2, 5, 3, 4, 2, 7, 2, 10) that differed
from those recovered from the water.
It is known that P. aeruginosa can enter a viable but
non-culturable state (Bédard et al. 2014), thus, the num-
ber of colonies recovered from the outlet fittings may
not represent the actual level of colonisation. However,
the weakness of any laboratory model is the inherent
difficulty in extrapolating results to the ‘real-world’ envi-
ronment (Muraca et al. 1987). The visual appearance of
the flow straighteners removed from the EWDS differed
considerably from those taken from the neonatal units
after four months in situ (Mayes et al. 2014) (Figure 8).
Local environmental factors including the concentration
of carbon, nitrogen and phosphorus in the water (Frias
et al. 2001; Chu et al. 2005), the disinfection process
(Lund and Ormerod 1995) and the condition of the pipe
material (Pederson 1990) can impact bacterial growth
and biofilm formation. Fundamentally however, whilst a
laboratory model can simulate a hospital water distribu-
tion system, it is not possible to recreate the clinical
environment.
In the current study, colonisation of the EWDS was
achieved by injecting P. aeruginosa directly into each tap
assembly. In the ‘real-world’ environment, retrograde
contamination of faucets by patients may be a more likely

scenario (Reuter et al. 2002; Rogues et al. 2007).
Colonisation of taps via splash contamination from the
hand basin (Pitten et al. 2001) and/or during the washing
of soiled hands (Grundmann et al. 1993) has also been
reported. A tap outlet associated with a hand basin fre-
quently used for rinsing patient care items or for disposing
patient secretions is also more likely to be contaminated
than an outlet associated with a basin that is not misused
(Balm et al. 2013). Such observations have resulted in a
recommendation that aerators/flow straighteners be
removed, replaced and/or regularly disinfected (Weber
et al. 1999; Kappstein et al. 2000; Balm et al. 2013).
However, if, as in the current study, the outlet fitting is not
the primary or only source of P. aeruginosa (or other
potential pathogen), such measures may have little effect
in terms of microbial or infection control.
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Tap handles can become heavily contaminated and
non-touch sensor taps have been introduced in many
hospitals both for water economy and in the belief that
avoidance of hand contact will reduce cross infection
(Zingg and Pittet 2012). However, it has been reported
that in comparison to manually operated taps, the water
delivered from sensor taps is more likely to be contami-
nated with P. aeruginosa and/or other waterborne patho-
gens (Halabi et al. 2001; Merrer et al. 2005; Sydnor et al.
2012) and be associated with higher bacterial counts
(Hargreaves et al. 2001). It has been suggested that the
comparatively low flow rate coupled with a lack of ther-
mal control and the presence of synthetic rubber materials
may facilitate the survival of bacteria and encourage bio-
film growth (Halabi et al. 2001; Walker et al. 2014).
In a previously reported study, water samples taken
from different sections of non-touch sensor taps implied
the outlet and the solenoid valve were highly contaminated
with P. aeruginosa (Halabi et al. 2001). In the current
study, individual tap components were systematically
removed from the EWDS, dismantled and sampled
directly. High numbers of bacteria were recovered from
the solenoid valve, particularly from the diaphragm.
Macroscopic, microscopic and microbiological analysis of
the ethylene propylene diene monomer (EPDM) surface
confirmed the presence of biofilm (Figure 9) and the
colonisation by P. aeruginosa (Figure 5).
Replacing sensor taps with conventional manual taps
has eliminated P. aeruginosa from hospital tap outlets
(Merrer et al. 2005; Berthelot et al. 2006) and resulted in
the termination of outbreaks (Durojaiye et al. 2011). The
solenoid valve is integral to a non-touch tap system and,
in the current study, its removal from a tap assembly
reduced the P. aeruginosa count in water delivered from
the tap outlet to below detectable levels. This effect was
immediate and sustained and implicated the solenoid dia-
phragm as the source of the contamination, confirming
EPDM as an unsuitable material for use in water distribu-
tion systems (Moritz et al. 2010; Waines et al. 2011).
Biofouling 685
Conclusions
The aim of this investigation was to build an experimen-
tal water distribution system to investigate the formation
of biofilm on different tap components. Following
artificial contamination of the EWDS, the solenoid valve
became colonised with P. aeruginosa and the EPDM
diaphragm was identified as a major weakness within a
non-touch tap system. These findings support recom-
mendations that sensor taps be removed from augmented
care units. However, there are benefits to installing auto-
matic taps and manufacturers should be made aware of
the potential risks associated with EPDM and challenged
to propose new design solutions incorporating alternative
materials. The EWDS was operated over a two year per-
iod and during this time high numbers of P. aeruginosa
were repeatedly recovered from the water delivered from
the outlets. Despite this, under laboratory conditions,
colonisation of the flow straighteners and aerators was
minimal, implying clinical practice and/or behaviour may
play a major role in the contamination of outlet fittings.
Thus, in addition to engineering interventions, strategies
should be developed to reduce the potential for retrograde
contamination and these should be effectively communi-
cated to clinical and non-clinical staff. P. aeruginosa is a
ubiquitous environmental microorganism and its control
within the healthcare environment should be the responsi-
bility of all.
Acknowledgements
The authors would like to thank Paul Day, Trevor Williams
and Liam Oliver for their contribution to the design and build
configuration of the EWDS; Dart Valley Systems for supplying
the non-touch sensor taps and solenoids; Neoperl Ltd for sup-
plying the outlet fittings; Howard Tolley for carrying out the
scanning electron microscopy; Jane Turton for carrying out the
VNTR analysis and Caroline Lymbery for additional technical
assistance.
Conflict of interest disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was funded by the Department of Health. The views
expressed in this publication are those of the authors and not
necessarily those of PHE or any other Government Agency.
References
Anaissie EJ, Penzak SR, Dignani MC. 2002. The hospital water
supply as a source of nosocomial infections: a plea for
action. Arch Intern Med. 162:1483–1492.
Aumeran C, Paillard C, Robin F, Kanold J, Baud O, Bonnet R,
Souweine B, Traore O. 2007. Pseudomonas aeruginosa
and Pseudomonas putida outbreak associated with contami-
nated water outlets in an oncohaematology paediatric unit.
J Hosp Infect. 65:47–53.
 686 G. Moore et al.
Balm MN, Salmon S, Jureen R, Teo C, Mahdi R, Seetoh T,
Teo JT, Lin RT, Fisher DA. 2013. Bad design, bad prac-
tices, bad bugs: frustrations in controlling an outbreak of
Elizabethkingia meningoseptica in intensive care units. J
Hosp Infect. 85:134–140.
Bédard E, Charron D, Lalancette C, Déziel E, Prévost M.
2014. Recovery of Pseudomonas aeruginosa culturability
following copper- and chlorine-induced stress. FEMS
Microbiol Lett. 356:226–234.
Berthelot P, Chord F, Mallaval F, Grattard F, Brajon D,
Pozzetto B. 2006. Magnetic valves as a source of faucet
contamination with Pseudomonas aeruginosa? Intens Care
Med. 32:1271–1271.
Bridier A, Briandet R, Thomas V, Dubois-Brissonnet F. 2011.
Resistance of bacterial biofilms to disinfectants: a review.
Biofouling. 27:1017–1032.
Chu C, Lu C, Lee C. 2005. Effects of inorganic nutrients on
the regrowth of heterotrophic bacteria in drinking water
distribution systems. J Environ Manage. 74:255–263.
Downloaded by [Universidad de Buenos Aires] at 11:40 07 June 2016
Cipolla D, Giuffrè M, Mammina C, Corsello G. 2011. Prevention
of nosocomial infections and surveillance of emerging resis-
tances in NICU. J Matern-Fetal Neonatal Med. 24:23–26.
Donlan R. 2002. Biofilms: microbial life on surfaces. Emerg
Infect Dis. 8:881–890.
Durojaiye OC, Carbarns N, Murray S, Majumdar S. 2011. Out-
break of multidrug-resistant Pseudomonas aeruginosa in an
intensive care unit. J Hosp Infect. 78:154–155.
Ferroni A, Nguyen L, Pron B, Quesne G, Brusset M, Berche P.
1998. Outbreak of nosocomial urinary tract infections due
to Pseudomonas aeruginosa in a paediatric surgical unit
associated with tap-water contamination. J Hosp Infect.
39:301–307.
Frias J, Ribas F, Lucena F. 2001. Effects of different nutrients
on bacterial growth in a pilot distribution system. Antonie
van Leeuwenhoek. 80:129–138.
Grundmann H, Kropec A, Hartung D, Berner R, Daschner F.
1993. Pseudomonas aeruginosa in a neonatal intensive care
unit: reservoirs and ecology of the nosocomial pathogen. J
Infect Dis. 168:943–947.
Halabi M, Wiesholzer-Pitt M, Schober J, Mittermayer H. 2001.
Non-touch fittings in hospitals: a possible source of Pseu-
domonas aeruginosa and Legionella spp. J Hosp Infect.
49:117–121.
Hargreaves J, Shireley L, Shannon H, Bren V, Gordon F,
Lacher C, Esslinger V, Watne T. 2001. Bacterial contamina-
tion associated with electronic faucets: a new risk for
healthcare facilities. Infect Control Hosp Epidemiol.
22:202–205.
Hidron A, Edwards J, Patel J, Horan T, Sievert D, Pollock D,
Fridkin S. 2008. Antimicrobial-resistant pathogens associ-
ated with healthcare-associated infections: annual summary
of data reported to the national healthcare safety network at
the centers for disease control and prevention, 2006–2007.
Infect Control Hosp Epidemiol. 29:996–1011.
Høiby N, Krogh Johansen H, Moser C, Song Z, Ciofu O,
Kharazmi A. 2001. Pseudomonas aeruginosa and the
in vitro and in vivo biofilm mode of growth. Microbes
Infect. 3:23–35.
Jefferies JM, Cooper T, Yam T, Clarke SC. 2012. Pseudomonas
aeruginosa outbreaks in the neonatal intensive care unit - a
systematic review of risk factors and environmental
sources. J Med Microbiol. 61:1052–1061.
Kappstein I, Grundmann H, Hauer T, Niemeyer C. 2000. Aera-
tors as a reservoir of Acinetobacter junii: an outbreak of
bacteraemia in paediatric oncology patients. J Hosp Infect.
44:27–30.
Lund V, Ormerod K. 1995. The influence of disinfection
processes on biofilm formation in water distribution sys-
tems. Water Res. 29:1013–1021.
Lyczak JB, Cannon CL, Pier GB. 2000. Establishment of Pseu-
domonas aeruginosa infection: lessons from a versatile
opportunist. Microbes Infect. 2:1051–1060.
Manuel CM, Nunes OC, Melo LF. 2007. Dynamics of drinking
water biofilm in flow/non-flow conditions. Water Res.
41:551–562.
Martin K, Baddal B, Mustafa N, Perry C, Underwood A, Con-
stantidou C, Loman N, Kenna DT, Turton JF. 2013. Clus-
ters of genetically similar isolates of Pseudomonas
aeruginosa from multiple hospitals in the UK. J Med
Microbiol. 62:988–1000.
Mayes C, McCracken G, Rooney P. 2014. Minimising risk
from Pseudomonas aeruginosa using tap design. Arch Dis
Child Fetal Neonatal Ed. 99:A40–A40.
Merrer J, Girou E, Ducellier D, Clavreul N, Cizeau F, Legrand
P, Leneveu M. 2005. Should electronic faucets be used in
intensive care and hematology units? Intens Care Med.
31:1715–1718.
Moritz MM, Flemming H-C, Wingender J. 2010. Integration of
Pseudomonas aeruginosa and Legionella pneumophila in
drinking water biofilms grown on domestic plumbing mate-
rials. Int J Hyg Environ Health. 213:190–197.
Muraca P, Stout JE, Yu VL. 1987. Comparative assessment of
chlorine, heat, ozone, and UV light for killing Legionella
pneumophila within a model plumbing system. Appl Envi-
ron Microbiol. 53:447–453.
Murga R, Forster TS, Brown E, Pruckler JM, Fields BS,
Donlan RM. 2001. Role of biofilms in the survival of
Legionella pneumophila in a model potable-water system.
Microbiology. 147:3121–3126.
Pederson K. 1990. Biofilm development on stainless steel and
PVC surfaces in drinking water. Water Res. 24:239–243.
Pitten F-A, Panzig B, Schröder G, Tietze K, Kramer A. 2001.
Transmission of a multiresistant Pseudomonas aeruginosa
strain at a German university hospital. J Hosp Infect.
47:125–130.
Reuter S, Sigge A, Wiedeck H, Trautmann M. 2002. Analysis
of transmission pathways of Pseudomonas aeruginosa
between patients and tap water outlets. Crit Care Med.
30:2222–2228.
Rogers J, Dowsett A, Dennis P, Lee J, Keevil C. 1994. Influ-
ence of plumbing materials on biofilm formation and
growth of Legionella pneumophila in potable water sys-
tems. Appl Environ Microbiol. 60:1842–1851.
Rogues AM, Boulestreau H, Lasheras A, Boyer A, Gruson D,
Merle C, Castaing Y, Bebear CM, Gachie JP. 2007. Con-
tribution of tap water to patient colonisation with Pseu-
domonas aeruginosa in a medical intensive care unit. J
Hosp Infect. 67:72–78.
Sydnor ER, Bova G, Gimburg A, Cosgrove SE, Perl TM,
Maragakis LL. 2012. Electronic-eye faucets: legionella spe-
cies contamination in healthcare settings. Infect Control
Hosp Epidemiol. 33:235–240.
Troop P. 2012. Independent review of incidents of Pseu-
domonas aeruginosa infection in neonatal units in Northern
Ireland. Belfast (Northern Ireland): The Regulation and
Quality Improvement Authority.
Turton JF, Turton SE, Yearwood L, Yarde S, Kaufmann ME,
Pitt TL. 2010. Evaluation of a nine-locus variable-number

tandem-repeat scheme for typing of Pseudomonas aerugi-
nosa. Clin Microbiol Infect. 16:1111–1116.
Vianelli N, Giannini MB, Quarti C, Sabattini MB, Fiacchini M,
de Vivo A, Graldi P, Galli S, Nanetti A, Baccarani M.
2006. Resolution of a Pseudomonas aeruginosa outbreak
in a hematology unit with the use of disposable sterile
water filters. Haematologica. 91:983–985.
Waines PL, Moate R, Moody AJ, Allen M, Bradley G. 2011.
The effect of material choice on biofilm formation in a
model warm water distribution system. Biofouling.
27:1161–1174.
Downloaded by [Universidad de Buenos Aires] at 11:40 07 June 2016
Biofouling 687
Walker JT, Jhutty A, Parks S, Willis C, Copley V, Turton JF,
Hoffman PN, Bennett AM. 2014. Investigation of health-
care-acquired infections associated with Pseudomonas
aeruginosa biofilms in taps in neonatal units in Northern
Ireland. J Hosp Infect. 86:16–23.
Weber DJ, Rutala WA, Blanchet CN, Jordan M, Gergen MF.
1999. Faucet aerators: a source of patient colonization with
Stenotrophomonas maltophilia. Am J Infect Control.
27:59–63.
Zingg W, Pittet D. 2012. Electronic-eye faucets - curse or
blessing? Infect Control. 33:241–242.