Download A combination of cis-2-decenoic acid and antibiotics eradicates pre

Journal of Medical Microbiology (2014), 63, 1509–1516
DOI 10.1099/jmm.0.075374-0
A combination of cis-2-decenoic acid and
antibiotics eradicates pre-established
catheter-associated biofilms
Azadeh Rahmani-Badi,1 Shayesteh Sepehr,1 Parisa Mohammadi,1
Mohammad Reza Soudi,1 Hamta Babaie-Naiej1 and Hossein Fallahi2,3
Correspondence
1
Azadeh Rahmani-Badi
2
[email protected]
Department of Biology, Alzahra University, Tehran, Iran
Department of Biology, Razi University, Kermanshah, Iran
3
Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
Received 12 March 2014
Accepted 29 July 2014
The catheterized urinary tract provides ideal conditions for the development of biofilm populations.
Catheter-associated urinary tract infections (CAUTIs) are recalcitrant to existing antimicrobial
treatments; therefore, established biofilms are not eradicated completely after treatment and
surviving biofilm cells will carry on the infection. Cis-2-decenoic acid (CDA), an unsaturated fatty
acid, is capable of inhibiting biofilm formation by Pseudomonas aeruginosa and of inducing the
dispersion of established biofilms by multiple types of micro-organisms. Here, the ability of CDA
to induce dispersal in pre-established single- and dual-species biofilms formed by Escherichia
coli and Klebsiella pneumoniae was measured by using both semi-batch and continuous cultures
bioassays. Removal of the biofilms by combined CDA and antibiotics (ciprofloxacin or ampicillin)
was evaluated using microtitre plate assays (crystal violet staining). The c.f.u. counts were
determined to assess the potential of combined CDA treatments to kill and eradicate preestablished biofilms formed on catheters. The effects of combined CDA treatments on biofilm
surface area and bacteria viability were evaluated using fluorescence microscopy, digital image
analysis and live/dead staining. To investigate the ability of CDA to prevent biofilm formation,
single and mixed cultures were grown in the presence and absence of CDA. Treatment of preestablished biofilms with only 310 nM CDA resulted in at least threefold increase in the number of
planktonic cells in all cultures tested. Whilst none of the antibiotics alone exerted a significant
effect on c.f.u. counts and percentage of surface area covered by the biofilms, combined CDA
treatments led to at least a 78 % reduction in biofilm biomass in all cases. Moreover, most of the
biofilm cells remaining on the surface were killed by antibiotics. The addition of 310 nM CDA
significantly prevented biofilm formation by the tested micro-organisms, even within mixed
cultures, indicating the ability of CDA to inhibit biofilm formation by other types of bacteria in
addition to Pseudomonas aeruginosa. These findings suggested that the biofilm-preventive
characteristics of CDA make it a noble candidate for inhibition of biofilm-associated infections
such as CAUTIs, which paves the way toward developing new strategies to control biofilms in
clinical as well as industrial settings.
INTRODUCTION
The biofilm mode of growth is a basic survival strategy
deployed by bacteria in a wide range of environmental,
industrial and clinical settings (Stoodley et al., 2002). The
catheterized urinary tract provides ideal conditions for
the development of enormous biofilm populations.
Consequently, catheter-associated urinary tract infections
(CAUTIs) are amongst the most common infectious
Abbreviations: CAUTI, catheter-associated urinary tract infection; CDA,
cis-2-decenoic acid; EPS, extracellular polymeric substance.
075374 G 2014 The Authors
diseases of humans, and significantly burden the healthcare
system by increasing both morbidity and treatment costs
(Ulett et al., 2007). The initial infections are usually by single
bacterial species, such as Escherichia coli, Staphylococcus
epidermidis and Enterococcus faecalis (Stickler, 2008).
However, over time, a variety of organisms, including
Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus
mirabilis and Morganella morganii, colonize the bladder
urine and form poly-microbial communities that are
embedded in protective self-produced extracellular polymeric substances (EPSs) (Stickler, 2008). In order to
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1509
A. Rahmani-Badi and others
inactivate these encased cells, antimicrobial molecules must
diffuse through the biofilm EPSs and gain access to the
microbial cells in sufficient concentrations (Walters et al.,
2003). However, EPSs present a diffusional barrier for these
molecules either by influencing the rate of transport of the
molecule to the biofilm interior (e.g. ciprofloxacin and
ampicillin) (Anderl et al., 2000) or by reaction of the
antimicrobial material with the matrix material (Campanac
et al., 2002). Thus, micro-organisms that are sensitive to
antibiotics in their planktonic phenotype become resistant
in their biofilm mode of growth (Stickler, 2008). This
characteristic makes CAUTIs, like other biofilm-associated
infections, recalcitrant to treatment by using existing antimicrobial treatments, and it is a common and frustrating
experience that after treatment, established biofilms are not
completely eradicated and surviving biofilm cells carry on
the infection (Fux et al., 2005). To address the need for novel
and improved measures against biofilms, especially preestablished biofilms, a clear strategy is to study the biofilm
life cycle and identify key trigger points that regulate biofilm
development. To control biofilm, the last stage of biofilm
development presents several advantages, when a coordinated dispersal of biofilm cells is possible. Induction of
biofilm dispersal can potentially use the micro-organisms’
own energy to remove EPSs and disrupt pre-established
biofilms, which results in cells reverting to a planktonic
phenotype and restores their susceptibility to antibiotics. It
has been reported recently that Pseudomonas aeruginosa
produces a diffusible signal factor family signal, cis-2decenoic acid (CDA), which is capable of inhibiting biofilm
formation by Pseudomonas aeruginosa and of inducing the
dispersion of established biofilms by multiple types of
micro-organisms (bacteria as well as yeast) (Davies &
Marques, 2009). In fact, CDA is involved in inter-species
and inter-kingdom signalling where it can modulate the
behaviour of other micro-organisms that do not produce the
signal (Davies & Marques, 2009). Its broad spectrum of
activity in addition to the fact that it has no cytotoxic effects
on human cells at the nanomolar range (Jennings et al.,
2012) makes CDA a promising candidate for the control of
biofilms. However, there is limited information on the
potential of CDA to boost the actions of antimicrobial
agents.
Therefore, in the current work the ability of CDA to induce
dispersal in established single- and dual-species biofilms
formed by E. coli and K. pneumoniae, and to eradicate their
biofilms when combined with antibiotics, was studied. In
addition, the ability of CDA to prevent biofilm formation
by these pathogens and to increase the inhibitory effects of
antibiotics on the growth of bacterial planktonic cells was
investigated.
METHODS
Bacterial strains, media and growth conditions. The micro-
organisms used in the present study included E. coli (ATCC 25922)
and K. pneumoniae (ATCC 700603). All cultivations were performed
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in artificial urine adopted by Griffith et al. (1976) containing calcium
chloride (0.49 g l21), magnesium chloride hexahydrate (0.65 g l21),
sodium chloride (4.6 g l21), disodium sulphate (2.3 g l21), trisodium
citrate dihydrate (0.65 g l21), disodium oxalate (0.02 g l21),
potassium dihydrogen phosphate (2.8 g l21), potassium chloride
(1.6 g l21), ammonium chloride (1.0 g l21), urea (25 g l21) and
gelatine (5.0 g l21). The pH of the medium was adjusted to 6.1 and
then the medium was sterilized by membrane filtration. Tryptone
soya broth (Merck) was prepared separately, autoclaved and added to
the sterile basal medium to a final concentration of 1.0 g l21. Medium
was supplemented (as required) with antibiotic (chloramphenicol at a
final concentration of 20 mg ml21).
Chemicals and antimicrobial compounds. Three different con-
centrations of CDA (U-Chemo; 100, 310 or 620 nM) were used.
These concentrations were observed previously to have the most effect
on inducing the dispersion of pre-established biofilms and on
inhibiting biofilm development (Davies & Marques, 2009) with no
cytotoxic effects on human cells (Jennings et al., 2012). Ethanol
(10 %) (Merck) was used as a carrier for CDA. This study also
examined the effects of two antibiotics, ciprofloxacin (Sigma) and
ampicillin (Sigma), which are used widely in treatment of urinary
tract infections. Ciprofloxacin was used at a final concentration of
1 mg ml21 and ampicillin was used at a final concentration of 256 mg
ml21. The concentration of the selected antibiotics was established in
our laboratory to be effective against planktonic cells, but have no
inhibitory effect on the biofilm cells of the test micro-organisms.
Single-and dual-species biofilm dispersal bioassays in Petri
dishes. Single-species biofilms of E. coli and K. pneumoniae as well as
their dual-species biofilms were cultivated on the inside surface of
sterile Petri dishes (Barraud et al., 2006). Plates were incubated for
5 days at 37 uC with shaking at 30 r.p.m., in triplicate. Medium in the
plates was replaced every 24 h to reduce the accumulation of native
dispersion-inducing factors and to allow mature biofilms to form.
After the final exchange of medium, the cells were allowed to grow for
~1 h and then dispersion induction was tested by replacing the
growth medium with fresh medium containing one of the indicated
concentrations of CDA (100, 310 or 620 nM) or just the carrier as a
control and the cells were incubated for a further 1 h. Medium
containing dispersed cells was then homogenized for 30 s at
5000 r.p.m. and cell density was determined based on the OD600.
Dispersion bioassays in biofilm tube reactors. Single- and dual-
species biofilms were also grown on the interior surfaces of tubing of a
once-through continuous-flow reactor system at 37 uC. The silicone
tubes were inoculated by syringe injection through a septum 1 cm
upstream from each reactor tube with 3 ml of overnight cultures of
each micro-organism to cultivate single-species biofilms. To grow dualspecies biofilms, 1.5 ml overnight cultures of E. coli and K. pneumoniae
were mixed well and then used as inoculum. Cells were allowed to
attach (static incubation) to the tubing for 1 h, after which the flow was
started at an elution rate of 280 ml min21. After 5 days of biofilm
culture, the influent medium was switched from fresh medium in the
test lines to one of the three indicated concentrations of CDA. Control
lines were switched to new lines containing just the carrier. Samples
were collected in test tubes on ice and were subsequently homogenized,
and cell density was determined as described above.
Combined CDA and antibiotic biofilm microtitre plate assays.
To assess the effect of antibiotics alone or in combination with CDA,
biofilms were grown on the inside surface of sterile polystyrene 96well plates. To cultivate biofilms, plates were inoculated with either
150 ml per well of overnight culture of each test micro-organism (to
grow single-species biofilms) or mixtures of two bacteria overnight
cultures (to cultivate dual-species biofilms) and then were incubated
at 37 uC with shaking at 120 r.p.m. Medium within each well was
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Journal of Medical Microbiology 63
Eradication of catheter-associated biofilms
replaced every 24 h for 5 days. The biofilms were then treated for 1 h
with the indicated concentrations of antibiotics alone or combined
with 310 nM CDA and biofilms were monitored by crystal violet
staining as described by Musk & Hergenrother (2008). All experiments were repeated at least four times.
(100, 310 or 620 nM) on the growth of planktonic cells of the test
micro-organisms. The MICs were determined in triplicate in
Mueller–Hinton broth by using a microdilution assay with bacteria
at a density of 105 c.f.u. ml21. Plates were incubated for 24 h at 37 uC.
The lowest concentration of antibiotics where there was no growth
after 24 h was taken as the MIC (CLSI, 2006).
Biofilm formation on catheters. Biofilm formation was also
assayed under hydrodynamic conditions in silicone catheters. Pieces
of catheter (100 % silicon) with a length of 3.0 cm were cut, sterilized
in 0.5 % sodium hypochlorite for 1 h and then washed extensively
with sterile water. Each piece of catheter was put into a glass tube with
5.0 ml growth medium. The tubes were then inoculated with 50 ml of
a culture of each test micro-organism freshly grown overnight (to
cultivate single-species biofilms) or their mixtures (to grow dualspecies biofilms). The tubes were left at 37 uC in a water bath with
shaking at 110 r.p.m. for 5 days. Medium in each tube was replaced
every 24 h for 5 days. The biofilms were then treated for 1 h with the
indicated concentrations of antibiotics alone or combined with
310 nM CDA. The amount of adhered bacteria on the catheters was
examined by viable counts. The catheter pieces were first washed in
50 ml PBS three times and excess liquid was removed by capillarity on
adsorbent paper to reduce the influence of non-attached or loosely
attached bacteria. The catheter pieces were then placed into plastic
tubes containing 5.0 ml PBS. Adhered bacteria were detached by
sonication for 4 min at 42 Hz at room temperature, followed by
vortexing at maximum velocity for 15 s, and then serial dilution and
plating onto LB agar plates. The sonication procedure did not
adversely affect bacterial viability (this was confirmed by plating
samples of bacterial liquid cultures before and after sonication).
Flow cell (continuous culture) biofilm experiments, antibiotics
sensitivity assays and surface area coverage. To observe the
effect of combined CDA and antibiotic treatments on biofilm surface
area and bacteria viability, single- and dual-species biofilms of GFPexpressing E. coli and K. pneumoniae carrying plasmid pAmr93
(generously provided by E. Peter Greenberg, Department of
Microbiology, University of Washington, School of Medicine,
Seattle WA, USA) were also grown in synthetic urine, supplemented
with chloramphenicol at 37 uC in continuous culture flow cells
(channel dimensions, 164640 mm). After 48 h of biofilm culture,
the influent medium was switched from fresh medium in the test lines
to the antibiotics alone or in combination with 310 nM CDA. After
1 h treatment, biofilms were stained with 30 mM propidium iodide,
which labels dead cells red. Using epifluorescence microscopy (CETI),
15 selected fields of view per flow cell were imaged in the xy plane,
at regular intervals and across the entire channels. Image analysis
(ImageJ software; http://imagej.nih.gov/ij/) was performed, and
results were presented as the percentage of total biofilm surface
reduction in cultures subjected to combined CDA and antibiotic
treatments relative to the total biofilm surface in control cultures that
were not exposed to CDA. Statistical comparison of the percentage of
surface covered by biofilms in the different treatments was performed
using ANOVA. Three replicates per experiment were used and at least
two independent repetition experiments were performed.
Inhibition of biofilm formation on catheters by CDA. Biofilm
formation was assayed in silicone catheters. Each piece of catheter was
put into a glass tube with 5.0 ml growth medium (as described above)
with or without 310 nM CDA. The tubes were then inoculated with
50 ml overnight culture of each test micro-organism (to cultivate singlespecies biofilms) or their mixtures (to grow dual-species biofilms). The
tubes were then left at 37 uC in a water bath with shaking at 110 r.p.m.
for 24 h before analysis. After plating onto LB agar, c.f.u. were counted.
Combined CDA and antibiotic treatment of planktonic cells.
We have also evaluated the probable inhibitory effects of antibiotics
alone or in combination with three different concentrations of CDA
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RESULTS
Very low concentrations of CDA induce biofilm
dispersal
We investigated the effect of exposure to nanomolar
concentrations of CDA on pre-established single- and dualspecies biofilms in Petri dish cultures. CDA treatments
resulted in a significant increase in the populations of
planktonic cells released into the bulk liquid compared with
untreated control samples. The greatest effect was observed
repeatedly with 310 nM CDA with at least a threefold increase
in the number of planktonic cells. At this concentration, the
largest increase in the planktonic population was observed in
the case of single-species biofilms of K. pneumoniae and
also dual-species biofilms (OD600 0.92±0.01, P¡0.05 and
1.10±0.01, P¡0.05, respectively) versus untreated controls
(OD60050.62±0.01, P¡0.05 and 0.78±0.02, P¡0.05,
respectively). The results from these experiments are
summarized in Table 1. We also examined the effect of
exposure to very low concentrations of CDA on preestablished biofilms grown in continuous cultures on the
inner surface of silicone tubing. We again observed an
increase in the population of planktonic cells after treatment
with CDA, indicating the release of biofilm bacteria into the
effluent of cultures treated with CDA. As for semi-batch
biofilm cultures, a threefold increase in the number of
planktonic cells in the effluent runoffs was observed in
cultures treated with 310 nM CDA (Table 2), which was the
largest increase in all cases. At this concentration, CDA caused
a significant increase in the population of planktonic cells
especially in dual-species biofilms (OD600 0.51±0.03,
P¡0.05) compared with the results for untreated controls
(OD600 0.18±0.01, P¡0.05). Therefore, 310 nM CDA was
used for further studies.
The results from these two different dispersal bioassays
demonstrated the ability of CDA to stimulate the release
of cells from established biofilms, formed by catheterassociated bacteria, even within mixed cultures.
Combined CDA and antibiotic treatment killed
and removed pre-established biofilms formed on
different surfaces
We tested the effectiveness of combined CDA treatments
on the removal of pre-established single- and dual-species
biofilms formed on the inside surface of polystyrene
96-well plates. After 120 h growth, biofilms were treated
with two antibiotics (ampicillin or ciprofloxacin) at the
indicated concentrations, alone or in combination with
310 nM CDA. The remaining biofilms were then stained
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A. Rahmani-Badi and others
Table 1. Activity of CDA as an inducer of biofilm dispersion using Petri dish bioassays
Cell density of medium containing dispersed cells was determined by measuring the OD600; values represent the mean±SD calculated from three
replicate experiments (P¡0.05).
Micro-organism
K. pneumoniae
E. coli
E. coli+K. pneumoniae
CDA concentration
Control
100 nM
310 nM
620 nM
0.62±0.01
0.66±0.02
0.78±0.02
0.80±0.02
0.78±0.02
0.86±0.05
0.92±0.01
0.9±0.05
1.10±0.01
0.89±0.02
0.82±0.01
1.00±0.01
with 0.1 % crystal violet. We observed that combined
treatments with both CDA and antibiotics had a significant
effect on removing pre-established biofilms (e.g. in the
absence of CDA, ampicillin caused only 21 % reduction in
dual-species biofilms, whereas addition of 310 nM CDA
resulted in 78 % biofilm removal) (Fig. 1a). However, the
largest biofilm removal was observed when both types of
biofilms were treated with a combination of CDA and
ciprofloxacin, which resulted in 90 % reduction in biofilm
biomass in single-species biofilms and ~80 % biofilm
removal in dual-species biofilms (Fig. 1a).
We also examined the effect of combined CDA and
antibiotic treatment on the killing and eradication of preestablished biofilms grown on urinary catheters. When
120 h biofilms were treated in the absence of CDA, both
antibiotics caused a roughly twofold decrease in c.f.u.
counts compared with untreated controls, whilst combined
exposure of cultures to either 310 nM CDA and only
256 mg ampicillin ml21 or 310 nM CDA and 1 mg
ciprofloxacin ml21 resulted in at least a fourfold decrease
in c.f.u. counts (Fig. 1b). This demonstrated that CDA
removed biofilms not only from the polystyrene surfaces,
as shown in microtitre plates, but also from silicone
surfaces of urinary catheters.
continuous culture flow cells. When 48 h biofilms were
treated in the absence of CDA, none of the antibiotics
exerted a significant effect on the percentage of surface area
covered by the biofilms (Fig. 2). In contrast, after
combined treatment, the biofilm cells remaining on the
surface were easily removed and killed by antibiotics when
examined by using live/dead staining (Fig. 2). As for
microtitre assays, the largest reduction in biofilm biomass
was observed when pre-established single- and dual-species
biofilms were treated with a combination of CDA and
ciprofloxacin, giving ~90 and 80 % biofilm removal,
respectively (Fig. 2a).
Therefore, combined treatments with both CDA and
antibiotics caused almost complete removal of preestablished single- and dual-species biofilms formed by
catheter-associated micro-organisms.
CDA prevented biofilm formation on urinary
catheters
Combined CDA and antibiotic treatment
significantly reduced biofilm surface coverage
Test micro-organisms were also used to investigate the
inhibitory role of additional CDA in biofilm formation by
both single- and mixed-cultures on silicone catheters.
Determinations of c.f.u. (mm catheter)–1 revealed a
twofold reduction in biofilms by only 310 nM CDA (Fig.
3), indicating the ability of CDA to inhibit biofilm
formation by other types of bacteria in addition to
Pseudomonas aeruginosa.
To further examine the effect of CDA on biofilm
eradication, we also tested combined CDA and antibiotic
treatment against pre-established biofilms cultivated in
To further investigate the effect of CDA on the sensitivity of
test micro-organisms towards antibiotics, we also evaluated
very low concentrations of CDA for inhibitory effects on the
Table 2. Activity of CDA as an inducer of biofilm dispersal using tube reactor bioassays
Cell density of medium containing dispersed cells was determined by measuring the OD600; values represent the mean±SD calculated from three
replicate experiments (P¡0.05).
Micro-organism
K. pneumoniae
E. coli
E. coli+K. pneumoniae
1512
CDA concentration
Control
100 nM
310 nM
620 nM
0.15±0.01
0.17±0.01
0.18±0.01
0.35±0.01
0.3±0.01
0.34±0.02
0.44±0.01
0.36±0.02
0.51±0.03
0.39±0.02
0.31±0.01
0.46±0.01
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Eradication of catheter-associated biofilms
–CDA
(a)
+CDA
100
60
E. coli
50
Biofilm remaining
(% of control)
Biofilm remaining
(% of control)
+CDA
40
30
20
10
0
–CDA
E. coli+K. pneumoniae
+CDA
80
K. pneumoniae
50
Biofilm remaining
(% of control)
–CDA
60
40
30
20
60
40
20
10
0
Amp 256 mg ml–1 Cip 1 mg ml–1
Amp 256 mg ml–1
Cip 1 mg ml–1
E. coli
K. pneumoniae
Dual-species biofilm
(b) 3.0
Biofilm formation [c.f.u. (mm catheter)–1] (×106)
0
Amp 256 mg ml–1 Cip 1 mg ml–1
2.5
2.0
1.5
1.0
0.5
0
Amp (–CDA) Amp+CDA Cip (–CDA)
Cip+CDA
Control
Fig. 1. Effect of CDA in combination with antibiotics on pre-established single- and dual-species biofilm removal. (a) The
amount of biofilm remaining after treatment with the tested concentrations of antibiotics [ampicillin (Amp) and ciprofloxacin
(Cip)] alone (–CDA) or in combination with 310 nM CDA (+CDA) for 1 h was determined by the measurement of A590 of
crystal violet by staining the 120 h biofilms in a microtitre plate assay. All readings are corrected to reflect 0 and 100 % controls
(blank well, 0 %; biofilms without any treatments, 100 %). Bar, SD (n54). (b) After treatment of the biofilms with antibiotics alone
or combined with 310 nM CDA, c.f.u. plate counts were determined to assess the viability of the bacteria (n59).
growth of planktonic cells. Compared with antibiotics alone,
the combination of nanomolar concentrations of CDA with
antibiotics had no additional inhibitory effects on the
growth of planktonic cells (Table 3).
DISCUSSION
Microbial biofilms are an increasing concern in the medical
field where the use of artificial medical devices for
therapeutic and restorative purposes is on the rise.
CAUTIs are notable examples of this, and illustrate the
significant economic and medical problems associated
with the use of such devices (Stamm, 1991; Tambyah et al.,
2002).
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In the long quest aiming to prevent or reduce the
accumulation of microbial biofilms, many different
strategies have been proposed with different degrees of
success (Yang et al., 2012). In various industrial settings, a
range of biocides and toxic metals (e.g. tin and copper)
have been used for antifouling coatings and sanitizing
purposes (Cloete et al., 1998; Chambers et al., 2006). These
substances are, however, not appropriate for use in medical
implants and therefore other strategies have been sought.
For example, in urology, a range of urinary catheters has
been developed which incorporate and/or release antimicrobial compounds, e.g. metals (such as silver salts) and
antibiotics (Darouiche et al., 1999; Johnson et al., 2006).
Nevertheless, the general risk of antibiotic resistance
development associated with these compounds has resulted
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A. Rahmani-Badi and others
(a)
K. pneumoniae
E. coli
Dual-species biofilm
Untreated control
Untreated control
Untreated control
Amp (–CDA)
Amp+CDA
Amp (–CDA)
Amp+CDA
Amp (–CDA)
Amp+CDA
Cip (–CDA)
Cip+CDA
Cip (–CDA)
Cip+CDA
Cip (–CDA)
Cip+CDA
60
50
40
30
20
10
0
Control
–CDA
80
Surface coverage (%)
Surface coverage (%)
+CDA
E. coli
Amp 256 µg ml–1 Cip 1 µg ml–1
+CDA
70
+CDA
E. coli+K. pneumoniae
100
60
50
40
30
20
10
0
–CDA
K. pneumoniae
Surface coverage (%)
–CDA
(b) 80
70
Control
Amp 256 µg ml–1 Cip 1 µg ml–1
80
60
40
20
0
Control
Amp 256 µg ml–1 Cip 1 µg ml–1
Fig. 2. Biofilms were grown using GFP-labelled E. coli and K. pneumoniae for 48 h. Following dispersion of biofilms by CDA,
cells remaining on the surface were easily removed by antibiotics [ampicillin (Amp) and ciprofloxacin (Cip)]. After treatments,
biofilms were stained with 30 mM propidium iodide, which labels dead cells red, and quantified (per cent surface coverage)
using digital image analysis. (a) Microscopic images of the biofilms on the surface of coverslips after combinatorial treatments.
Images are top-down views (xy plane). Bars, 50 mm. (b) The columns show the levels of biofilm biomass after antimicrobial
treatments when used alone (–CDA) or combined with CDA (+CDA). Bar, SD. Results are representative of three separate
experiments.
in limited use of such strategies (Ramos et al., 2011).
Therefore, additional strategies are being developed in
order to reduce catheter-associated biofilms. In a previous
study, Cobrado et al. (2012) suggested that cerium nitrate,
low-molecular-mass chitosan and hamamelitannin are
biocompatible compounds that could be used to coat
medical devices. However, their in vivo studies revealed
that amongst these compounds, only hamamelitannin
could inhibit biofilm formation by all the bacteria tested.
According to Cobrado et al. (2013), a more durable coating
strategy and further in vivo testing for these compounds are
needed in order to develop a cost-effective and biocompatible indwelling catheter that could reduce medical deviceassociated biofilms more effectively than the present
available options.
The main feature of biofilms is the production of EPSs to
build a matrix for embedding the cells, and protecting
them from shearing forces and undesirable conditions,
1514
including the presence of most antimicrobial agents
(Flemming & Wingender, 2010). In a previous study,
Davies & Marques (2009) reported that CDA, a signalling
molecule synthesized by Pseudomonas aeruginosa, induces
dispersion of pre-established biofilms in this bacterium, as
well as many other strains of micro-organisms. Induction
of biofilm dispersal can potentially use the microorganisms’ own energy to remove EPSs and disrupt preestablished biofilms, revert cells to a planktonic phenotype,
and restore their susceptibility to antimicrobial agents.
Therefore, in this study, we first examined the impacts of
nanomolar concentrations of CDA (as an inducer of
biofilm dispersal) on dispersion of single- and mixedspecies biofilms formed by the top two species in CAUTIs,
E. coli and K. pneumoniae, which are also excellent biofilm
formers (Dalgaard, 1995; Hancock et al., 2010). Our data
showed that treatment of pre-established biofilms with
only 310 nM CDA resulted in at least a threefold increase
in the number of planktonic cells in all the cultures tested.
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Journal of Medical Microbiology 63
Eradication of catheter-associated biofilms
Biofilm formation [c.f.u. (mm catheter)–1]
14000
Table 3. Susceptibility profiles (MIC; mg ml”1) of E. coli and K.
pneumoniae to the examined antibiotics in the presence and
absence of three different concentrations of CDA
No CDA
12000
310 nM CDA
10000
Treatment
8000
6000
4000
2000
0
K. pneumoniae
E. coli
Dual-species
biofilm
Fig. 3. Addition of CDA inhibits biofilm formation of E. coli and K.
pneumoniae on urinary catheters. Biofilm formation by test bacteria
in synthetic urine in the presence and absence of 310 nM CDA
was examined on urinary catheters (100 % silicone) by viable
counts. Catheters were incubated for 24 h, washed and sonicated,
and serial dilutions were plated for determination of c.f.u. Results
displayed are mean±SD of three replicates. Bacteria formed
significantly less biofilm on catheters in medium with CDA
(‘310 nM CDA’) than in medium without additional CDA (‘No
CDA’) (P,0.01 by t-test).
Then, we successfully removed both types of biofilm
culture by using a combination of CDA and common
antibiotics at concentrations that had not been significantly
effective against biofilms in previous studies (Barraud et al.,
2006). The results presented here demonstrated that
following exposure to low concentrations of CDA, cells
remaining on the surface were easily killed by antibiotics
even within mixed cultures. We confirmed that the
combination of 310 nM CDA with ciprofloxacin and
ampicillin, which are used widely in the treatment of
CAUTIs, resulted in up to 90 % reduction in biofilm
biomass in all cases. Moreover, live/dead staining experiments showed that most of the biofilm cells remaining on
the surface were killed by antibiotics. We also showed that,
in addition to dispersal of pre-established biofilms, CDA
inhibited biofilm formation by bacterial species other than
Pseudomonas aeruginosa. This anti-biofilm activity could
be useful in prophylactic measures for preventing biofilmassociated infections such as CAUTIs. CDA-based strategies to induce biofilm dispersal or to prevent biofilm
formation contain very low concentrations of CDA, only in
the nanomolar range, which should be safe to humans and
to the environment. Moreover, in a previous study,
Jennings et al. (2012) have shown that CDA has no
cytotoxic or stimulatory effect on human cells even at high
concentrations (up to 250 mg ml21). Some free fatty acids
have been reported to have antimicrobial properties
(Desbois et al., 2008; Desbois & Smith, 2010) and play
an important role in maintaining the microbial flora of the
http://jmm.sgmjournals.org
Ampicillin (2 CDA)
Ampicillin+100 nM CDA
Ampicillin+310 nM CDA
Ampicillin+620 nM CDA
Ciprofloxacin (2 CDA)
Ciprofloxacin+100 nM CDA
Ciprofloxacin+310 nM CDA
Ciprofloxacin+620 nM CDA
Micro-organism
E. coli
K. pneumoniae
128
128
128
128
0.06
0.06
0.06
0.06
128
128
128
128
0.125
0.125
0.125
0.125
skin (Kenny et al., 2009; Takigawa et al., 2005). However,
we demonstrated that CDA did not inhibit bacterial growth
at the nanomolar range that induces biofilm dispersal.
These results were highly consistent with results obtained
from Jennings et al. (2012) showing that CDA inhibited
bacterial growth only at micro- to millimolar concentrations. This lack of growth inhibition at lower concentrations was not surprising as bacteria produce this fatty acid
and use it as a signalling molecule (Davies & Marques,
2009). CDA mediates the transition from a biofilm to a
planktonic phenotype via a signalling mechanism (because
it acts at nanomolar concentrations, consistent with all
known cell–cell signalling molecules) rather than a toxic
effect. Therefore, CDA-based biofilm control strategies
would not be expected to select for resistant strains, as seen
with antibiotics. The application of a dispersion inducer
prior to, or in combination with, treatment by antimicrobial agents provides a promising mechanism for enhancing
the activity of these treatments through the disruption of
existing biofilms.
ACKNOWLEDGEMENTS
We sincerely thank Dr David G. Davies (Department of Biological
Sciences, State University of New York, Binghamton, USA) for
valuable information about CDA and also for providing protocols for
biofilm dispersal bioassays.
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