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Biofouling
The Journal of Bioadhesion and Biofilm Research
ISSN: 0892-7014 (Print) 1029-2454 (Online) Journal homepage: http://www.tandfonline.com/loi/gbif20
Magnetic fields suppress Pseudomonas
aeruginosa biofilms and enhance ciprofloxacin
activity
H.M.H.N. Bandara, D. Nguyen, S. Mogarala, M. Osiñski & H.D.C. Smyth
To cite this article: H.M.H.N. Bandara, D. Nguyen, S. Mogarala, M. Osiñski & H.D.C. Smyth
(2015) Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin
activity, Biofouling, 31:5, 443-457, DOI: 10.1080/08927014.2015.1055326
To link to this article: http://dx.doi.org/10.1080/08927014.2015.1055326
Published online: 23 Jun 2015.
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Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin activity
H.M.H.N. Bandara
a
, D. Nguyen
a
, S. Mogarala
a
, M. Osiñski
b
and H.D.C. Smyth
a
*
a
College of Pharmacy, The University of Texas at Austin, Austin, TX, USA;
b
Department of Electrical & Computer Engineering,
Center for High Technology Materials, The University of New Mexico, Albuquerque, NM, USA
(Received 12 March 2015; accepted 20 May 2015)
Due to the refractory nature of pathogenic microbial biofilms, innovative biofilm eradication strategies are constantly being
sought. Thus, this study addresses a novel approach to eradicate Pseudomonas aeruginosa biofilms. Magnetic nanoparticles
(MNP), ciprofloxacin (Cipro), and magnetic fields were systematically evaluated in vitro for their relative anti-biofilm con-
tributions. Twenty-four-hour biofilms exposed to aerosolized MNPs, Cipro, or a combination of both, were assessed in the
presence or absence of magnetic fields (Static one-sided, Static switched, Oscillating, Static + oscillating) using changes in
bacterial metabolism, biofilm biomass, and biofilm imaging. The biofilms exposed to magnetic fields alone exhibited sig-
nificant metabolic and biomass reductions (p< 0.05). When biofilms were treated with a MNP/Cipro combination, the most
significant metabolic and biomass reductions were observed when exposed to static switched magnetic fields ( p< 0.05).
The exposure of P. aeruginosa biofilms to a static switched magnetic field alone, or co-administration with MNP/Cipro/
MNP + Cipro appears to be a promising approach to eradicate biofilms of this bacterium.
Keywords: Pseudomonas aeruginosa; biofilms; magnetic fields; ciprofloxacin; magnetic nanoparticles; drug delivery
Introduction
In nature, most microorganisms prefer a communal growth
lifestyle rather than surviving as solitary cells or single
species. These microbial communities are termed biofilms
(Harding et al. 2009). Biofilms are defined as a complex
functional community of one or more species of
microbes encased in an extracellular polymeric network
and attached to one another or to a solid surface
(Samaranayake 2006). These communities are hierarchi-
cally arranged and three dimensionally organized in order
to gain ecological advantages for better survival, when
compared to their planktonic counterparts (Hall-Stoodley
et al. 2004). Gram-positive pathogens such as Staphylo-
coccus epidermidis,Staphylococcus aureus and Strepto-
coccus sp., Gram-negative bacteria such as Pseudomonas
aeruginosa and Enterobacteriaceae, and fungi such as
Candida spp. are frequently associated with biofilm
related infections (Lode et al. 2000; Lyczak et al. 2000;
Saltzstein et al. 2007; Romling & Balsalobre 2012). In the
USA, P. aeruginosa is among the 10 most commonly iso-
lated pathogens involved in healthcare-associated infec-
tions and the second most commonly isolated pathogen
associated with ventilator-related pneumonia (Hidron et al.
2008). Moreover, P. aeruginosa biofilms cause lung infec-
tions in 95% of adult cystic fibrosis (CF) patients (Hart &
Winstanley 2002), in addition to chronic wound infections,
catheter associated urinary tract infections, chronic otitis
media, rhinosinusitis and contact lens associated keratitis
(Costerton et al. 1999; Hoiby et al. 2010).
Due to a slow growth rate, altered microbial metabo-
lism, phenotypic changes, oxygen gradients and pH dif-
ferences, extracellular substances and persister cells,
complete eradication of P. aeruginosa biofilms with con-
ventional antibiotics is almost impossible (Romling &
Balsalobre 2012). However, it is generally accepted that
superior biofilm control can be achieved when the
antibiotic is combined with another anti-biofilm agent
(Romling & Balsalobre 2012). For instance, established
P. aeruginosa biofilms in CF lungs are treated with an
intensive course of antibiotics (nebulized tobramycin)
and DNases (to disrupt eDNA/extracellular DNA in bio-
film matrices) (Frederiksen et al. 2006; Lewis 2008;
Hoiby et al. 2011).
Due to the inferior outcomes of existing therapies,
alternative treatment modalities have constantly been
sought to eradicate pathogenic biofilms. Early investiga-
tions explored the possibility of using electromagnetic
fields to eliminate P. aeruginosa (Anwar et al. 1992;
Khoury et al. 1992; Benson et al. 1994). However, these
studies were only conducted on planktonic phases ignor-
ing refractory biofilms, and also failed to meet expected
outcomes (Grosman et al. 1992; Piatti et al. 2002;
Potenza et al. 2004; Gao et al. 2005; Laszlo & Kutasi
2010). Magnetic nanoparticles (MNP) such as iron
oxides have received much attention in anti-tumor thera-
peutic strategies due to greater biocompatibility, low sys-
temic toxicity, and the ability to release thermal energy
in the presence of oscillating magnetic fields (Johannsen
*Corresponding author. Email: hugh.smyth@austin.utexas.edu
© 2015 Taylor & Francis
Biofouling, 2015
Vol. 31, No. 5, 443–457, http://dx.doi.org/10.1080/08927014.2015.1055326
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et al. 2007; Soenen & De Cuyper 2010). However, the
applications of MNPs in biofilm elimination are still in
their infancy (Subbiahdoss et al. 2012; Taylor et al.
2012). To illustrate the potential anti-biofilm properties
of MNP, the authors have demonstrated the successful
usage of superparamagnetic iron oxide nanoparticles
(SPION) for enhancing drug transport in CF mucus and
alginate gels. When exposed to magnetic fields, SPIONs
demonstrated enhanced penetration in model alginate
biofilms (McGill et al. 2009; Armijo, Brandt, Matthew
et al. 2012). Therefore, this emerging treatment strategy,
ie usage of polymeric or inorganic nanoparticles, appears
to be a promising approach to eradicate biofilms from
tissues and surrounding surfaces (Francolini & Donelli
2010).
To the authors’knowledge, there are no reports on
the application of various magnetic fields to eliminate
the biofilm phase of pathogenic P. aeruginosa, co-ap-
plication of magnetic fields (oscillating, static, and
combinations), or the use of MNPs as an alternative
treatment modality along with the co-delivery of antibi-
otics with MNPs and magnetic fields as a biofilm elim-
ination strategy. Thus, the aim of this study was to
investigate the efficacy of various magnetic fields in
eliminating in vitro P. aeruginosa biofilms treated with
an aerosolized formulation containing different combina-
tions of MNPs, ciprofloxacin (Cipro) and spray dried
lactose (SDL).
Materials and methods
Ciprofloxacin
Ciprofloxacin hydrochloride USP grade was purchased
from Letco Medical (Decatur, AL, USA, Catalog
No. 690953).
Magnetic nanoparticles
FluidMAG-CMX superparamagentic iron oxide
nanoparticles (SPIONs, 150 nm 40 mg ml
−1
) in distilled
water were purchased from Chemicell
®
(Chemicell
GmBH, Berlin, Germany).
Spray-dried lactose
Spray-dried lactose (Super Tab 11SD, monohydrate
lactose USP) was provided by DFE Pharma (Princeton,
NJ, USA).
Microorganisms
Pseudomonas aeruginosa PAO1 (provided by Prof.
Marvin Whitley, Molecular Biosciences, The University
of Texas at Austin, USA) was used throughout the study.
The identity of the bacterium was confirmed with a
commercially available API 20 E kit (Biomérieux, Mercy
I’Etoile, France). All isolates were stored in multiple ali-
quots at −20°C, after confirming their purity.
Growth media
Blood agar (Sigma Aldrich, St Louis, MO, USA) and
brain heart infusion (BHI, Sigma Aldrich) solution were
used for culturing P. aeruginosa.
Microbial inocula
Prior to each experiment, P. aeruginosa was subcultured
on blood agar for 18 h at 37°C. A loopful of the over-
night bacterial growth was inoculated into BHI medium
and incubated for 18 h in an orbital shaker (80 rpm) at
37°C. Bacteria were harvested, washed twice in phos-
phate buffered saline (PBS, pH 7.2) and resuspended in
PBS. The concentration of P. aeruginosa was adjusted to
1×10
7
cells ml
−1
by ultraviolet-visible spectrophotome-
try and confirmed by hemocytometric counting.
Biofilm formation
P. aeruginosa biofilms were developed as described by
Bandara, Yau et al. (2010) with the following modifica-
tions. Commercially available pre-sterilized, polystyrene,
flat bottom 96-well microtiter plates (BD Biosciences,
San Jose, CA, USA) were used. First, 100 μl of a stan-
dard bacterial suspension (10
7
organisms ml
−1
) were
prepared and transferred into the wells of a microtiter
plate, and the plate was incubated for 1.5 h (37°C,
75 rpm) to promote microbial adherence to the well
surfaces. After this initial adhesion phase, the bacterial
suspensions were aspirated, and each well was washed
twice with PBS to remove loosely adhering bacterial
cells. A total of 200 μl of BHI were added to each well
and the plate was incubated for 24 h (37°C, 75 rpm).
After incubation, the wells were washed twice with PBS
to eliminate traces of the medium. The effects of various
treatments were studied on these 24 h biofilms.
Determination of minimum inhibitory concentration
(MIC)
Planktonic phase
The MIC was determined by a broth microdilution assay
in accordance with the CLSI guidelines (CLSI 2012).
Briefly, bacterial suspensions (5 × 10
5
cells ml
−1
) were
treated with the antibiotic in a concentration gradient
(two-fold dilutions) and incubated in a 96-well microtiter
plate for 24 h at 35°C. At the end of this incubation, the
optical density of the bacterial growth was observed
444 H.M.H.N. Bandara et al.
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by unaided eye. The MIC is defined as the lowest
concentration of antimicrobial agent that completely
inhibits growth of the organism, ie P. aeruginosa grown in
the microtiter wells, compared to the solvent control as
detected by the unaided eye. The assay was performed as
quadruplicates three separate times.
Biofilm phase
P. aeruginosa biofilms were developed in sterile 96-well
plates (BD Biosciences) as described above. Biofilms
were washed twice with PBS and ciprofloxacin was
administered in a concentration gradient (two-fold dilu-
tion). The plates were incubated for 24 h at 37°C and
80 rpm.
At the end of the incubation period, an XTT
reduction assay (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide) was performed to quan-
tify the viability of the biofilms. The lowest concentration
of the antibiotic at which the bacterial cells demonstrate
80% viability (as measured by XTT readings) when
compared to the solvent control, is considered to be the
minimum biofilm inhibitory concentration (MBC) of
the antibiotic against P. aeruginosa. The assay was
performed as quadruplicates three separate times.
Spray drying of formulations
The spray-dried lactose particles loaded with ciprofloxa-
cin, MNP, or both, were prepared using a Buchi
®
B-290
mini spray drier (Buchi, Flawil, Switzerland). The two
formulations with MNPs were prepared in 1 g batches
(ie formulations of SDL + MNP and SDL + MNP +
Cipro) and the other two formulations without MNPs
were prepared in 2.5 g batches (ie formulations of SDL
and SDL + Cipro).
Cipro and MNP were mixed at 5% and 1% (w/w),
respectively, in lactose to prepare dry formulations. SDL,
SDL + Cipro, SDL + MNP, and SDL + MNP + Cipro
formulations were prepared by spray drying. The follow-
ing parameters were used in the spray drying process:
feed solutions consisted of 2.5% (w/v) lactose in distilled
water, inlet temperature 150°C, feed rate 1.8 ml min
−1
,
airflow rate (N
2
/qFlow) 357 l h
−1
,N
2
pressure 80psi, and
aspiration rate 35 m
3
h
−1
. Sixty-one percent, 67% and
77% of SDL + Cipro, SDL + MNP, and SDL + MNP +
Cipro respectively, were recovered after spray drying.
Exposure of biofilms to magnetic fields
Biofilm formation and treatment
P. aeruginosa biofilms were grown for 24 h in sterile
6-well plates and tissue culture treated Petri dishes ( for
subsequent magneTherm (nanoTherics Ltd, Newcastle-
under-Lyme, UK) treatment) as described above.
Twenty-four-hour biofilms were washed twice with sterile
PBS and the physical mixtures of SDL, SDL + Cipro,
SDL + MNP, SDL + MNP + Cipro were applied to the
biofilm using an aerosol dosing method (Figure 1). A dry
powder insufflator (Model DP-4 M, Penn-Century Inc.,
Wyndmoor, PA, USA) was used. The insufflator was
loaded with known weights of spray-dried formulations
and the compound was delivered to a plate. The loaded
formulations were weighed and the amounts of Cipro
delivered were calculated. Subsequently, the initial load-
ing weight of the formulation, and the number of deliver-
ies needed in order to deposit a desired concentration of
Cipro, were calculated. Final Cipro deposition was esti-
mated to be 1 μgml
−1
. Although P. aeruginosa is known
not to metabolize SDL, it is commonly used as an excipi-
ent in inhalational drug formulations (Hugh & Leifson
1953; McGowan 2006; Ungaro et al. 2012); therefore,
SDL was used as the control. Subsequently, the plates
were exposed to one of four different magnetic field treat-
ments. Static magnetic field treatments involved exposing
the 24 h biofilm, with or without spray dried particles, to
magnetic fields from the bottom of a 6-well plate for 6 h
using molybdenum magnets. The strength of the magnetic
field was measured by a Gaussmeter (FW BELL 5180
Gauss/Tesla meter, Stanford Magnets, Irvine, CA, USA).
Figure 1. The method of biofilm treatment with different
particles. A dry powder insufflator (Model DP-4 M) was used
and the final Cipro deposition was estimated to be 1 μgml
−1
.
Biofouling 445
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The well was positioned over the magnet and the probe
was placed on the floor of the well when taking the mea-
surements. The magnetic field strength at the biofilm was
4.44 kG (Figure 2A). Switched static magnetic field treat-
ment involved exposing the 24 h biofilm, with or without
spray dried particles, to magnetic fields from the bottom
of the 6-well plates for 30 min (the magnetic field strength
was 4.44 kG) followed by exposing from the top of the 6-
well plate for 30 min (the magnet was positioned over the
lid of the well and the probe was placed at a similar dis-
tance between the lid and the floor of the well when tak-
ing the measurements; the magnetic field strength was
0.12 kG) for a total exposure period of 6 h (Figure 2B).
The oscillating magnetic field treatment involved expos-
ing the 24 h biofilm, with or without spray-dried particles,
to magnetic fields generated by magneTherm (biofilms in
Petri dishes, 17 turn coil of 474 kHz nominal frequency,
current supply: 20 V, 4.3 A, magneTherm), for 30 min
(Figure 2C). The static plus oscillating (Static + oscillat-
ing) magnetic field treatment involved exposing the 24 h
biofilm, with or without spray-dried particles, to static
magnetic fields as mentioned above for 1 h followed by a
30 min exposure to oscillating magnetic fields (Figure 2D).
After the magnetic field treatments, biofilms were incu-
bated in an 80% humidified incubator for 24 h at 37°C
(Figure 2D). At the end of the incubation period, the bio-
films were washed twice with PBS; XTT reduction assays
were performed to quantify the viability of biofilms by
means of measuring metabolic activity, and crystal violet
assays were used to quantify biofilm biomass.
XTT reduction assay
At the end of the incubation of both test and control
biofilms, a standard XTT reduction assay was per-
formed as described by Bandara, Lam et al. (2010)to
measure the viability of biofilms by means of bacterial
cell metabolic activity. In brief, commercially available
XTT powder (Sigma Aldrich) was dissolved in PBS to
afinal concentration of 1 mg ml
−1
. Then the solution
was filter-sterilized (0.22 μm pore size filter) and stored
at –70°C. Freshly prepared 0.4 mM menadione solution
was used for the XTT reduction assay. XTT solution
was thawed and mixed with menadione solution at 20:1
(v/v) immediately before the assay. Thereafter, PBS:
XTT:menadione in 79:20:1 proportions were added to
each culture dish containing biofilms and incubated in
the dark for 5 h at 37°C. The color changes were mea-
sured with a microtiter plate reader (Infinite M200
microplate reader, TECAN US Inc., Morrisville, NC,
USA) at 492 nm. All assays were carried out in
quadruplicates on three different occasions.
Crystal violet (CV) assay
At the end of the incubation of both test and control bio-
films, CV assays were performed to quantify biofilm bio-
mass. Biofilms were carefully washed twice with PBS
and stained with a 1% CV solution for 15 min at 25°C
without shaking. Wells were carefully washed three
times with PBS to remove excess stain and air dried at
Figure 2. Methods of exposure of P. aeruginosa biofilm to different magnetic fields. (A) Static one sided magnetic field exposure;
magnets were placed under the 6-well plates for 6 h. (B) Static switched magnetic field exposure; the locations of the magnets were
switched from top to bottom and vice versa every 30 min for 6 h. (C) Oscillating magnetic field exposure; biofilms were exposed to
oscillating magnetic fields for 30 min using magneTherm. (D) Static + oscillating magnetic field exposure; biofilms were exposed to
static magnetic field as shown in (A) for 1 h followed by oscillating magnetic fields for 30 min using magneTherm.
446 H.M.H.N. Bandara et al.
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room temperature. Thirty percent acetic acid was added
to the wells containing stained biofilms before incubation
for 20 min at 25°C. The solution was transferred to a
new well plate and the optical density was measured at
570 nm. All assays were carried out in quadruplicates on
three different occasions.
Confocal laser scanning microscopy (CLSM)
Biofilms were prepared on sterile coverslips placed in
commercially available sterile flat bottom six well plates
(Nunclon, Nunc, Thermo Fisher Scientific, Waltham, MA,
USA) as described above. Pre-formed 24 h biofilms were
exposed to magnetic fields and incubated for another 24 h
at 37°C in a humidified incubator. At the end of incuba-
tion, the prewashed coverslips were stained with Live and
Dead stain (Live/Dead BacLight Bacterial Viability kit,
Invitrogen, Eugene, OR, USA) (Bandara, Yau et al. 2010).
Component A (cyto 9, excitation/emission; 480/500 nm)
and Component B (propidium iodide, excitation/emission;
490/635 nm) were mixed (1:1) and 1 μl of dye mixture
was added to the well containing 1 ml of PBS and the
coverslip. The plate was incubated in the dark at room
temperature for 15 min. The biofilm was then analyzed by
fluorescent microscopy using a confocal laser scanning
microscope (Leica TCS SP5 II upright microscope, Leica,
Wetzlar, Germany). For each coverslip, nine microscope
fields (three vertical fields × three horizontal fields) were
imaged and in the experiment and images were repeated
on two different occasions.
Statistical analysis
Statistical analysis was performed using SPSS software
(version 16.0). A Mann–Whitney U-test was performed to
compare the significance of differences between the corre-
sponding control and test samples of the P. aeruginosa
biofilms and to compare the significance of differences
between test samples of the P. aeruginosa biofilms under
different treatment conditions. A p-value < 0.05 was
considered statistically significant.
Results
The MIC of ciprofloxacin was 0.125 μgml
−1
for plank-
tonic P. aeruginosa and the MBC was 16 μgml
−1
for
P. aeruginosa biofilms formed in 24 h.
The effects of magnetic fields on P. aeruginosa biofilms
treated with control particles
Biofilm metabolism (XTT reduction assay )
When the biofilms were treated with SDL control parti-
cles and with various magnetic fields as mentioned
above, all the test samples exposed to magnetic fields
exhibited a significant reduction in metabolic activity
when compared to the untreated (ie magnetic field free)
biofilm controls (p< 0.05, Figure 3A). Comparing the
different magnetic field treatments to each other, there
were no significant differences in the mean XTT
readings.
Biofilm biomass (CV assay )
When the biofilms were treated with SDL and exposed
to one of the four different magnetic fields, all treatments
showed a significant reduction in biomass compared to
the no magnetic field control (p< 0.05, Figure 3B). Bio-
films exposed to static switched, oscillating and static +
oscillating magnetic fields showed a significantly lower
biomass compared to the biofilms exposed to a static
magnetic field (p< 0.05, Figure 3B). Biofilms exposed
to a static switched magnetic field had a significantly
lower biomass compared with those exposed to the oscil-
lating and static + oscillating magnetic fields (p< 0.05,
Figure 3B).
CLSM
The control biofilm (Figure 3C) that was not exposed to
magnetic fields demonstrated a dense, spatially oriented
and confluent biofilm and a typical live to dead ratio for
a 24 h biofilm. The biofilms exposed to static magnetic
fields exhibited a significantly lower quantity of bacterial
cells and there was no organized structure (Figure 3D).
However, the apparent ratio of live to dead cells did not
show significant differences compared to the control
(Figure 3D). Similar reductions in bacterial count were
observed in the biofilms exposed to static switched mag-
netic fields (Figure 3E). However, remnants of the bio-
film structure were preserved, and isolated islands were
observed. The live/dead cell ratio in the biofilms exposed
to static switched magnetic fields appeared to be similar
to the control (Figure 3E).
Exposure to an oscillating magnetic field caused
complete disruption of the biofilm (Figure 3F). There
was no structured biofilm observed in any of the
CLSM images. Instead, scattered bacterial cells were
visible in the microscope with apparently higher pro-
portions of dead cells compared to the control biofilm
(Figure 3F). The biofilm treated with a static + oscillat-
ing magnetic field also exhibited a significantly dis-
rupted biofilm architecture and haphazardly distributed
bacterial cells (Figure 3G). No comparisons were made
between the XTT and microscope findings due to the
qualitative and definitive nature of the CLSM imaging
assessment.
Biofouling 447
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(A) (B)
(C) (D) (E)
(G)(F)
Figure 3. The effects of various magnetic fields on P. aeruginosa biofilms. (A) The effect of various magnetic fields on SDL-treated
biofilms –XTT reduction assay findings; note the significant reduction in biofilm metabolism (mean XTT values) when exposed to
any of the magnetic fields compared to the unexposed control. (B) The effect of various magnetic fields on SDL-treated biofilms –
CV assay findings; note that the most significant reduction in the biomass (mean OD) was when exposed to static switched magnetic
fields. * Indicates significant changes and p< 0.05 is considered to be statistically significant (n= 12). (C–G) CLSM images of
P. aeruginosa biofilms exposed to different magnetic fields (stained using the LIVE/DEAD BacLight bacterial viability kit; Invitro-
gen); live cells are stained green and dead cells red. (C) Undisturbed control. (D) Exposed to static one sided magnetic fields. (E)
Exposed to static alternating magnetic fields. (F) Exposed to oscillating magnetic fields. (G) Exposed to static and oscillating magnetic
fields. Note the significant reduction in the cellular content, stratified architecture and the lower live: dead cell ratio and lack of extra-
cellular components in the test biofilms (D, E, F and G) compared to the three dimensionally arranged and dense biofilm controls
with substantial extracellular materials.
448 H.M.H.N. Bandara et al.
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The effects of various magnetic fields on P. aeruginosa
biofilms co-treated with magnetic nanoparticles
Biofilm metabolism (XTT reduction assay )
When SDL + MNP-treated biofilms were exposed to the
aforementioned different magnetic fields, all test treat-
ments (except those exposed to oscillating magnetic
fields) showed a significant suppression of biofilm meta-
bolism (p< 0.05) compared to an untreated biofilm con-
trol (Figure 4A). Biofilms that were exposed to static
switched magnetic fields demonstrated the lowest meta-
bolic activity, and had a significantly lowered metabolic
activity compared to the biofilms exposed to the other
magnetic field treatments (p< 0.05, Figure 4A).
Biofilm biomass (CV assay )
Biofilms treated with SDL + MNP and exposed to static
switched magnetic fields and oscillating magnetic fields
showed a significant reduction in their biomass compared
to the controls and compared to the other magnetic field
treatments (p< 0.05, Figure 4B). Conversely, biofilms
exposed to static magnetic fields showed a significant
increase in biomass compared to the SDL + MNP-treated
controls (p< 0.05, Figure 4B).
The effects of various magnetic fields on P. aeruginosa
biofilms co-treated with ciprofloxacin
Biofilm metabolism (XTT reduction assay )
When P. aeruginosa biofilms were treated with SDL +
Cipro and exposed to the aforementioned different mag-
netic fields, all the test biofilms except the one exposed
to the static + oscillating magnetic fields exhibited a
significant reduction in metabolism compared to the
untreated biofilm controls (p< 0.05, Figure 5A).
Biofilm biomass (CV assay )
Biofilms that were co-treated with Cipro and exposed to
static switched, oscillating, and static + oscillating mag-
netic fields exhibited a significantly lower biomass when
compared to the controls and to the static field treatment
(p< 0.05, Figure 5B).
The effects of various magnetic fields on P. aeruginosa
biofilms co-treated with both magnetic nanoparticles
and ciprofloxacin
Biofilm metabolism (XTT reduction assay )
When P. aeruginosa biofilms were treated with SDL +
MNP + Cipro and exposed to static switched magnetic
fields, a significant decrease in metabolism was observed
compared to the untreated biofilm controls and the other
magnetic field treatments (p< 0.05, Figure 6A). In con-
trast, biofilms treated with SDL + MNP + Cipro, and
exposed to other magnetic fields, did not significantly
differ from the controls.
Biofilm biomass (CV assay )
The outcome of the exposure of SDL + MNP + Cipro-
treated biofilms to different magnetic fields was com-
pared. All the biofilms exposed to magnetic fields showed
a significantly reduced biomass compared to the unex-
posed sample (p< 0.05, Figure 6B). When compared
Figure 4. The effects of various magnetic fields on P. aeruginosa biofilms treated with magnetic nanoparticles. (A) The effect of
various magnetic fields on SDL + MNP-treated biofilms –XTT reduction assay findings; note the most significant reduction of bio-
film metabolism (mean XTT values) when exposed to static switched the magnetic fields compared to the unexposed control and
other exposed biofilms. (B) The effect of various magnetic fields on SDL + MNP-treated biofilms –CV assay findings; note that the
most significant reduction in the biomass (mean OD) was when exposed to static switched magnetic fields. * Indicates significant
changes and p< 0.05 is considered statistically significant (n= 12).
Biofouling 449
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with the biofilm exposed to a static magnetic field, the
remaining three biofilms exposed to different magnetic
fields showed a significant reduction in overall biomass
(p< 0.05, Figure 6B). There were no significant changes
among other comparisons.
The effects of various particle treatments on P.
aeruginosa biofilms
Biofilm metabolism (XTT reduction assay )
When considering the different particle treatments alone
(without magnetic exposure), only SDL + MNP + Cipro-
treated biofilms showed a significantly lower metabolic
activity compared to the SDL-treated control (p< 0.05,
Figure 7A).
In contrast, when different particle treatments were
exposed to one of the magnetic fields, there were no
significant changes observed in any tested biofilms
compared to their respective controls.
Biofilm biomass (CV assay )
A significant reduction in the biomass of all three test P.
aeruginosa biofilms (treated with SDL + MNP, SDL +
Cipro and SDL + MNP + Cipro) that were not exposed
to test magnetic fields was observed compared to biofilm
treated with SDL (p< 0.05, Figure 7B).
Figure 5. The effects of various magnetic fields on P. aeruginosa biofilms treated with ciprofloxacin. (A) The effect of various mag-
netic fields on SDL + Cipro-treated biofilms –XTT reduction assay findings; note the most significant reduction of biofilm metabolism
(mean XTT values) when exposed to static switched the magnetic fields compared to the unexposed control and other exposed bio-
films. (B) The effect of various magnetic fields on SDL + Cipro-treated biofilms –CV assay findings; note that the most significant
reduction of the biomass (mean OD) was when exposed to static switched magnetic fields. * Indicates significant changes and p< 0.05
is considered statistically significant (n= 12).
Figure 6. The effects of various magnetic fields on P. aeruginosa biofilms treated with magnetic nanoparticles and ciprofloxacin.
(A) The effect of various magnetic fields on SDL + MNP + Cipro-treated biofilms –XTT reduction assay findings; note the most sig-
nificant reduction of biofilm metabolism (mean XTT values) when exposed to static switched magnetic fields compared to the unex-
posed control and other exposed biofilms. (B) The effect of various magnetic fields on SDL + MNP + Cipro-treated biofilms –CV
assay findings; note that the most significant reduction in the biomass (mean OD) was when exposed to static switched magnetic
fields. * Indicates significant changes and p< 0.05 is considered statistically significant (n= 12).
450 H.M.H.N. Bandara et al.
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Discussion
Due to the refractory nature of pathogenic biofilms,
many different biofilm eradication strategies and new
therapeutic agents have been investigated. Some strate-
gies have used chemical approaches such as anti-
adhesins/polymeric coatings (Cassinelli et al. 2000; van
de Belt et al. 2001; von Bismarck et al. 2001; John
et al. 2007; Johansson et al. 2008), quorum sensing
inhibitors (Francolini et al. 2004; Cirioni et al. 2006;
Antoniani et al. 2010), biosynthesis inhibitors (Ueda
et al. 2009), biofilm dispersal agents (Raad et al. 2007;
Kim et al. 2008; Barraud et al. 2009; Cai et al. 2009;
Davies & Marques 2009) and natural products
(Bjarnsholt et al. 2007; Kalishwaralal et al. 2010;
Kostenko et al. 2010; Wu et al. 2011), among others.
Physical methods have also been investigated, including
ultrasound waves (Ensing et al. 2005), low frequency
vibrations (Bandara et al. 2014) and other methods (Di
Poto et al. 2009).
Unlike previous reports, the current study sought to
evaluate the potential for eradication of biofilms using
a combination of chemical and physical approaches.
Nanoparticles, a test antibacterial compound, and mag-
netic fields were systematically evaluated to assess the
relative anti-biofilm contributions of each approach and
combinations thereof. The following discussion is orga-
nized in increasing order of complexity with the initial
focus on the separate treatments followed by discus-
sion of the treatment combinations. Interestingly, the
most effective treatment conditions observed in the
present study were obtained using magnetic fields
alone.
Magnetic fields alone disrupt P. aeruginosa biofilms
Significant reductions in biofilm metabolic activity and
biomass were observed when different magnetic fields
were applied in the absence of nanoparticle treatments.
The highest suppression of viability and biomass was
observed in the biofilm exposed to a static switched
field. The reduction in cell viability must be attributed to
bacterial cell death and the reduction in biomass must be
related to both bacterial death and loss of the extracellu-
lar matrix of the biofilm. Furthermore, ultrastructural
views confirmed the severe destructive effects of mag-
netic fields on the architecture of P. aeruginosa biofilms.
Thus, the magnetic fields appear to negatively affect the
structure, cell viability and extracellular materials (either
their synthesis or direct disruption) of mature P. aerugi-
nosa biofilms.
There are several early reports on the effects of mag-
netic fields on bacterial growth (Kohno et al. 2000; Piatti
et al. 2002; Potenza et al. 2004; Gao et al. 2005; Laszlo
& Kutasi 2010). Most previous studies were conducted
using static magnetic fields ranging from 30 mT to 14.1
T, and exposing from 30 min to six days, using a variety
of microbes and growth conditions. After the exposure
to static magnetic fields, E. coli,Staphylococcus aureus,
S. mutans,Rubus fruticosus,Shewanella oneidensis,Sac-
charomyces cerevisiae,Bacillus circulans,Micrococcus
luteus,Pseudomonas fluorescens,Salmonella enteritidis,
and Serratia marcescens did not exhibit any significant
changes in their growth (Grosman et al. 1992; Piatti
et al. 2002; Potenza et al. 2004; Gao et al. 2005; Laszlo
& Kutasi 2010). The present results, in comparison,
demonstrated that static magnetic fields had a significant
Figure 7. The effects of various particle treatments on P. aeruginosa biofilms. (A) Particle treatment of biofilms with no magnetic
exposure biofilms –XTT reduction assay findings; note the significantly low metabolic activity of the biofilm treated with SDL +
MNP + Cipro compared to the SDL-treated biofilm. (B) Particle treatment of biofilms with no magnetic exposure –CV assay find-
ings; note the significantly low biomass of the biofilm treated with all particle combinations compared to the SDL-treated biofilm. *
Indicates significant changes and p< 0.05 is considered statistically significant (n= 12).
Biofouling 451
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inhibitory effect on P. aeruginosa biofilm viability and
biomass. In line with the present results, Piatti et al.
(2002) also noted that there was a significant growth
inhibition of S. marcescens and Hordeum vulgare when
exposed to static magnetic fields for 24 h. It should be
noted that previously reported studies were conducted in
planktonic bacterial cultures, thus the extrapolation of
these findings to a biofilm environment must be made
with caution.
As yet, few mechanisms have been identified by
which magnetic fields may influence microorganism via-
bility and growth. However, magnetic field effects are
likely dependent upon many variables including species,
strain, exposure time, the strength of the magnetic field
and the growth environment (Bajpai & Basu 2013). For
instance, to elicit growth inhibition using static magnetic
fields in S. aureus,S. mutans and E. coli they must be
grown under anaerobic conditions (Kohno et al. 2000).
The present authors did not study the effects of the vari-
ous factors that influence the effects of magnetic fields
on biofilms, such as magnetic field strength or time of
field application. Thus, the discrepancies between the
present study and previous reports may be due to differ-
ences in any of the aforementioned conditions. In addi-
tion, magnetic fields may interact with iron related
cellular processes (Schwartz et al. 1989; Stepanian et al.
2000; Zhang et al. 2002). P. aeruginosa also depends on
iron for virulence, alginate production, mucoid pheno-
type change and biofilm formation (Banin et al. 2005;
Wiens et al. 2014). Although the exact mechanism is yet
to be elucidated, the reduction in the biofilm biomass
and the viability of P. aeruginosa in the current study
may also be related to the blockage of iron acquisition
enzymes, as shown previously in E. coli K-12 and
Mycobacterium tuberculosis (Schwartz et al. 1989;
Stepanian et al. 2000; Zhang et al. 2002).
Although there are no reports of the usage of oscillat-
ing magnetic fields in controlling animal pathogens,
these have been suggested as a disinfection method in
agricultural products and food due to their significant
inhibitory effects on plant bacterial pathogens (Lipiec
et al. 2004). Interestingly, the current study also clearly
indicates that P. aeruginosa biofilms can be significantly
disrupted through exposure to oscillating magnetic fields,
as well as by a combination of static and oscillating
magnetic fields. The aforementioned agricultural studies
were not conducted in biofilm environments, thus direct
comparisons between these studies cannot be made.
There are no reports to the authors’knowledge that
describe the potential mechanism by which magnetic
fields control P. aeruginosa biofilms. According to an
investigation of global gene expression conducted by
Sandvik (2013), extremely low-frequency magnetic fields
up-regulated transposase activity, membrane transport
processes, and signal transduction systems in a frequency-
dependent manner, suggesting that magnetic fields
induced changes in ion transport in P. aeruginosa.
MNPs alone do not affect the viability of P. aeruginosa
biofilms
The major advantage of using nanoparticles is that the
target area can be precisely located and the subsequent
release of the drug can be pre-planned (Francolini &
Donelli 2010). MNPs can be precisely controlled by a
magnetic field and driven to the specific location in the
body for the desired action (Corchero & Villaverde
2009, Xie et al. 2009). Previously, MNPs have been suc-
cessfully used in targeting cancer cells, imaging and drug
delivery (Gindy & Prud’homme 2009).
Recent studies have suggested that MNP may possess
potential antimicrobial properties depending on the
nanoparticle type, dose and the pathogen (Taylor & Webster
2009). For instance, iron oxide with silver nanoparticles
showed good antimicrobial properties against E. coli,S. epi-
dermidis,Bacillus subtilis and MRSA (methicillin resistant
S. aureus)biofilms (Kvitek & Soukupova 2009; Durmus &
Webste r 2013) but not against genetically engineered P.
aeruginosa PTSOX4 (Kafayati et al. 2012). The antimicro-
bial activity of nanoparticles in previous studies was sug-
gested to be due to their high surface-area-to-volume ratio,
which may be accompanied by the enhanced synthesis of
reactive oxygen species (ROS) and free radicals (Nel et al.
2009;Allaker2010;Mahmoudietal.2011). Thus,
nanoparticles may interact with microbial membranes and
kill them by damaging the microbial structure (Nel et al.
2009;Allaker2010;Mahmoudietal.2011). The disruption
of cell membrane and cytoplasmic leakage in response to
chitosan nanoparticles has been previously observed by
atomic force microscopy (Qi et al. 2004)
Consistent with the aforementioned previous reports
on P. aeruginosa PTSOX4, it is also reported here that
MNP alone had no significant effects on the metabolism
of P. aeruginosa biofilms. However, a significant
reduction in the biomass of the biofilm was observed
(Figure 4). The differential effect of MNP on biofilm
viability and biomass suggests that the dose of MNP
needed to induce bacterial lysis may be higher than that
needed to disrupt the extra cellular matrix. In addition,
there are concerns about the use of MNP in biofilm elim-
ination, as the exposure of P. aeruginosa biofilms to
SPIONs at concentrations up to 200 μgml
−1
resulted in
an increase in biofilm biomass and cell density (Haney
et al. 2012). Similar positive effects were not observed
in the present study, most likely because MNP concen-
trations that were estimated to be several-fold lower than
200 μgml
−1
were used. Nevertheless, as described in the
current literature, the usage of MNPs has been mainly
452 H.M.H.N. Bandara et al.
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investigated in planktonic microorganisms and the possi-
ble prevention of biofilm formation (Taylor & Webster
2009; Anghel et al. 2013; Wang et al. 2013). Thus, more
studies are necessary to explore the properties of MNPs
for use in the eradication of existing biofilms.
MNPs significantly disrupt P. aeruginosa biofilms in
the presence of static switched magnetic fields
The uptake of nanoparticles by biofilms is usually low
due to their poor diffusion and penetration properties
(Fabrega et al. 2009; Liu et al. 2009; Gunawan et al.
2011; Raghupathi et al. 2011, Cui et al. 2012). How-
ever, nanoparticles that possess magnetic properties, eg
SPIONs, chitosan coated SPIONs and silver nanoparti-
cles, have been shown to overcome this limitation. In the
presence of magnetic fields, these nanoparticles can be
targeted to the infection site, and disrupt and eradicate
pathogenic biofilms (Lee et al. 2005; McGill et al. 2009;
Armijo, Brandt, Matthew et al. 2012; Armijo, Brandt,
Rivera et al. 2012; Chen et al. 2012). For instance,
Durmus and Webster (2013) reported that the exposure
of SPIONs to static magnetic fields further increased
their antibacterial properties against MRSA compared to
unexposed controls (Durmus & Webster 2013). Similarly,
the exposure of P. aeruginosa biofilms treated with
SPIONs to static switched magnetic fields caused a sig-
nificant reduction in biofilm metabolism and biomass.
The use of static one sided magnetic exposure failed to
elicit similar results, and displayed an increase in the
biomass possibly due to the additive effect of MNP mass
to the existing biofilm mass. This suggests that static
switched magnetic fields may be more effective in direct-
ing MNPs to a target site and in destroying established
biofilms when compared to static magnetic fields. The
latter finding further confirms the results obtained by
Park et al. (2011). They also reported > 4 log inactiva-
tion of P. aeruginosa PA01 biofilm after treatment for
8 min with SPIONs and a magnetic field. The treatment
caused a substantial disintegration of the bacterial cell
membrane in the biofilm and was suggested to be purely
due to the thermal effect generated by MNPs and the
magnetic field (Park et al. 2011).
Exposure of Pseudomonas fluorescens to iron oxide
magnetic nanoparticles and an oscillating magnetic field
of 873 kHz/100 Oe (Oersteds) resulted in a complete
eradication of planktonic bacteria and a significant
reduction in biofilm viability at 8 min exposure, at 55°C
(Rodrigues et al. 2012). However, the complete eradica-
tion of a biofilm was not seen even after 17 min expo-
sure at 60°C (Rodrigues et al. 2012). In contrast, when
P. aeruginosa biofilms were treated with MNP and
exposed to oscillating magnetic fields, no significant
changes in biofilm viability were observed. However,
biomass was significantly reduced. The difference in the
outcome could be due to a lower dose of MNP and/or
magnetic field and/or the temperature used. Nevertheless,
the present findings again suggest that the dose of MNP
needed for bacterial cell lysis may be higher than that
needed for matrix disruption.
Ciprofloxacin significantly eliminates P. aeruginosa
biofilms in the presence of static switched magnetic
fields
Early research conducted in the 1990s suggested the
possibility of using magnetic fields as a treatment option
to improve the efficiency of antimicrobials on biofilms
formed on various medical devices (Khoury et al. 1992;
Benson et al. 1994). Benson et al. (1994) reported that
the application of static magnetic fields (5–10 G),
together with gentamycin, significantly reduced the
P. aeruginosa counts on various biomaterial surfaces.
Thus they suggested that static magnetic fields may
enhance the activity of gentamycin against P. aeruginosa
biofilms by forming radical intermediates and the
up-regulation of the bacterial membrane protein, porins
(Benson et al. 1994). In another study, cephalothin-
resistant P. aeruginosa exhibited re-sensitization to
cephalothin after exposure to a static electromagnetic
field (0.35 Tesla of electromagnetic field for 15 min)
(Samarbaf-Zadeh et al. 2006). Similarly, in the present
study, ciprofloxacin-treated P. aeruginosa biofilms
exposed to static switched magnetic fields exhibited the
most significant inhibitory effects on biofilm metabolism
and biomass. Static one sided and oscillating magnetic
field treatment regimens also exhibited less-significant
findings. It can be speculated that static switched mag-
netic fields disrupt the biofilm matrix more efficiently
than static one sided magnetic fields, providing cipro-
floxacin with better access to deeper layers of the bio-
film. Further investigations are necessary to explain the
mechanism of bacterial killing and biofilm reduction
mediated by ciprofloxacin with assistance from static
switched magnetic fields.
Magnetic nanoparticles assist ciprofloxacin in biofilm
killing
In the latter part of the study P. aeruginosa biofilms were
treated with MNP, ciprofloxacin and magnetic fields. To
the authors’knowledge, there are no reports in the litera-
ture of simultaneous application of all three treatment
modalities to bacterial biofilms. However, several studies
have reported that co-administration of antibiotics with
nanoparticles resulted in better inhibitory effects on bio-
films. For instance, Cotar et al. (2013) reported that
MNPs conjugated to polymixin B, streptomycin and cefo-
taxime exhibited a dose-dependent significant suppression
of biofilm formation by P. aeruginosa and E. coli as
Biofouling 453
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estimated by the total biofilm biomass. Armijo, Brandt,
Rivera et al. (2012) suggested that when administered
together, SPIONs capped with PEG (polyethylene glycol)
may contribute to a better distribution of antibiotics in
P. aeruginosa biofilms in cystic fibrosis lungs, due to
their superior penetrating properties. Furthermore, Au and
Ag nanoparticles functionalized with ampicillin were also
shown to be effective broad-spectrum bactericides against
both Gram-negative and Gram-positive bacteria (Brown
et al. 2012). The present results also confirm the supe-
rior inhibitory effects of MNP and antibiotics on
P. aeruginosa biofilms. SDL + MNP + Cipro-treated
P. aeruginosa biofilms, even without exposure to mag-
netic fields, showed significantly lower metabolism and
biomass compared to SDL-treated control biofilms.
However, when biofilms were treated with MNP and
ciprofloxacin individually (SDL + MNP and SDL +
Cipro), no significant results were observed (despite a
significant reduction in their biomass). This clearly indi-
cates that the combined treatment of MNP and ciproflox-
acin is more effective for eradicating P. aeruginosa
biofilms than their individual effects.
Interestingly, when the combined treatment of SDL +
MNP + Cipro was exposed to magnetic fields, a static
switched magnetic field was the only magnetic exposure
that could exert a significant reduction in biofilm meta-
bolism compared to the unexposed controls, despite the
significant reduction in biomass in all biofilms except the
one exposed to static magnetic fields. Hence, when all
different treatment situations are considered, the best bio-
film suppression was observed with a static switched
magnetic field exposure.
Conclusions
In summary, it is reported here, for the first time, that
mere exposure of biofilms to magnetic fields resulted in
the significant destruction and killing of P. aeruginosa
biofilms. Static switched magnetic fields demonstrated
superior anti-biofilm properties when applied alone or
when co-applied with MNP, Cipro or MNP + Cipro.
Thus, upon further optimization, a combined therapy of
antibiotics with MNPs and exposure to magnetic fields
can be used as a promising novel therapeutic approach/
treatment strategy for removal of pathological biofilms.
Further studies are necessary to evaluate the efficacy of
these magnetic fields in the management of biofilm-as-
sociated infections in soft tissues as well as on medical
devices.
The results also indicate that the combination ther-
apy of MNP and ciprofloxacin demonstrates superior
properties in eliminating biofilms compared to their indi-
vidual effects. In the spectrum of the dosage used,
although MNP itself did not elicit significant effects in
respect of bacterial killing, it did reduce the biofilm
mass. Exposure to magnetic fields did not show any
added advantage when treated with MNP. Hence, a
longer exposure period with stronger magnetic fields
and a superior safety margin may be required to trigger
a significant effect.
Acknowledgements
The authors would like to thank Dr Kristin Fathe from the
University of Texas at Austin for editorial assistance.
Conflict of interest disclosure statement
No potential conflict of interest was reported by the authors.
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