Immobilized furanone derivatives as inhibitors for adhesion of bacteria on modified poly(styrene- co -maleic anhydride)

Article abstract of DOI:10.1021/bm300932u

The ability of brominated furanones and other furanone compounds with 2(3H) and 2(5H) cores to inhibit bacterial adhesion of surfaces as well deactivate (destroy) them has been previously reported. The furanone derivatives 4-(2-(2-aminoethoxy)-2,5-dimethy

Full text of DOI:10.1021/bm300932u

Article
pubs.acs.org/Biomac
Immobilized Furanone Derivatives as Inhibitors for Adhesion of
Bacteria on Modified Poly(styrene-co-maleic anhydride)
Nonjabulo P. Gule,† Osama Bshena,† Michele de Kwaadsteniet,‡ Thomas E. Cloete,‡
̀
and Bert Klumperman†,*
†Division of Polymer Science, University of Stellenbosch, Stellenbosch, South Africa
‡Department of Microbiology, University of Stellenbosch, Stellenbosch, South Africa
ABSTRACT: The ability of brominated furanones and other furanone
compounds with 2(3H) and 2(5H) cores to inhibit bacterial adhesion of
surfaces as well deactivate (destroy) them has been previously reported. The
furanone derivatives 4-(2-(2-aminoethoxy)-2,5-dimethyl-3(2H)-furanone and
5-(2-(2-aminoethoxy)-ethoxy)methyl)-2(5H)-furanone were synthesized in
our laboratory. These furanone derivatives were then covalently immobilized
onto poly(styrene-co-maleic anhydride) (SMA) and electrospun to fabricate
nonwoven nanofibrous mats with antimicrobial and cell-adhesion inhibition
properties. The electrospun nanofibrous mats were tested for their ability to inhibit cell attachment by strains of bacteria
commonly found in water (Klebsiella pneumoniae Xen 39, Staphylococcus aureus Xen 36, Escherichia coli Xen 14, Pseudomonas
aeruginosa Xen 5, and Salmonella tymphimurium Xen 26). Proton nuclear magnetic resonance spectroscopy (1H NMR),
electrospray mass spectroscopy (ES-MS), and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)
were used to confirm the structures of the synthesized furanones as well as their successful immobilization on SMA. To ascertain
that the immobilized furanone compounds do not leach into filtered water, samples of water, filtered through the nanofibrous
mats were analyzed using gas chromatography coupled with mass spectroscopy (GC-MS). The morphology of the electrospun
nanofibers was characterized using scanning electron microscopy (SEM).
A class of compounds, collectively known as furanones, has
been reported in the literature to possess the ability to inhibit
surface colonization by microorganisms as well as antimicrobial
properties against a wide range of microorganisms.15−22 A good
example of this phenomenon is observed in the marine algae
Delisea pulchra, which does not get colonized by marine
organisms. Furanones do this through suppression of a process
called quorum sensing (QS). QS is a process by which bacteria
communicate, and forms an essential part of biofilm formation.
QS is not necessary for bacterial survival but helps in
coordinating the community-based bacterial behavior, and
even though interference with QS may not bring about a
universally beneficial effect, it makes the bacteria more
susceptible to control/destruction by traditional means.
Biochemical studies to understand different pathways of QS
have been carried out.23,24 Furanone derivatives have been
isolated from nature and have also been synthesized. Specific
QS routes for these furanone derivatives have been
reported.25−27 Research on clinical applications of furanone
compounds is widespread.22,28−30
INTRODUCTION
■
The use of membrane technology in filtration systems is a
promising technology. However, its large-scale industrial
applicability is limited largely because of fouling of the
membranes. Of all the types of fouling, biofouling which is
caused by the attachment of microorganisms on membrane
surfaces, is hardest to control. Chemical biocides such as
chlorine have been used for decades to control biofouling of
membranes.1−5 Most of these chemical biocides are, however,
not very effective at higher pH values and can react to form
harmful byproducts.6 Physical means to clean membranes have
been reported, but only work best as secondary methods to
other biofilm removal methods.7 Other methods such as the
use of bacteriophages, electrical current, and nutrient control
have been explored, but these can take a long time to be
effective and are in most instances very host specific.8−10
The focus is now moving to modification of membrane
surfaces in order to control fouling. The modification of
polymers by incorporating metal elements has been
reported.11,12 The application of these nanofibrous mats for
wound dressing and filter media has been evaluated.13 Munoz-
In a recent article, inhibition of QS by the furanone moiety
was reported to have prevented soft rot caused by Pseudomonas
aeruginosa from attacking onion plants.31 This shows the
potential and wide applicability of furanone-mediated QS
̃
Bonilla and Fernan
́
dez-Garci
́
a reported on a wide range of
polymeric materials with antimicrobial activity.14 In their
review, they highlighted five classes of polymers that have
been found to have antimicrobial properties: polymers with
quaternary nitrogen atoms, guanidine containing polymers,
polymers that mimic natural peptides, halogenated polymers,
and polymers containing phospho and sulpho derivatives.14
Received: June 19, 2012
Revised: August 29, 2012
Published: September 4, 2012
© 2012 American Chemical Society
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Figure 1. Synthesis of furanone 1.
inhibition. Biofilm formation on filtration membranes is one of
the major limitations associated with membrane technology.32
This reduces the quality and quantity of water in water
purification systems and consequently results in higher
treatment costs. To the best of the authors’ knowledge,
however, there have been no reports on the application of
furanone-mediated QS inhibition for filtration applications.
Targeting QS is also advantageous compared to the use of
antibiotics since there is no risk of the bacteria developing
resistance, which causes serious control problems. Many natural
products contain the core 3(2H)-furanone structure classified
as a lactone, which makes this group easily accessible.33 Because
of the high synthetic and biological importance of furanone
compounds, their chemistry has received considerable attention
over the past two decades.34 In this paper, furanone derivatives
were synthesized and immobilized on poly(styrene-co-maleic
anhydride) (SMA). The furanone-modified SMA was then
electrospun into nanofibers and tested for their ability to inhibit
biofilm attachment.
solvents and acids (tetrahydrofuran (THF) 99.9%, diethyl ether
(98%), ethyl acetate (99.8%), triethyl amine (99.5%) dichloro-
methane (DCM, 99.8%), p-toluenesulfonic acid (98%),
phosphoric acid (H3PO4, 95%), and toluene (99.8%)) used
in this study were all purchased from Sigma Aldrich. The
bacterial strains Escherichia coli Xen 14, Salmonella tymphimu-
rium Xen 26, P. aeruginosa Xen 5, Klebsiella pneumoniae Xen 39,
and Staphylococcus aureus Xen 36 were obtained from Caliper
Life Sciences, Hopkinton MA, USA. The bacterial strains
contain a Photorhabdus luminescence luxABCDE operon (lux
gene) to produce the enzyme luciferase, which emits photons
in the presence of ATP and oxygen.
Synthesis and Characterization of Furanone Com-
pounds. In this study, the immobilization of furanone
compounds on SMA copolymer was investigated. The
importance of SMA copolymers is attributed to their usage in
a number of areas for various purposes. Its applications
comprise of additives that are used to upgrade properties of
styrenic polymeric material, coating additives, binder applica-
tion, additives for building materials, microcapsules, blend
compatibilizers, adhesion promoters for polyolefin coatings on
metals, and medical and pharmaceutical applications.35−38 SMA
copolymer is also regarded as a functional or reactive
polymer.39 The functionality is brought about by the maleic
anhydride in the backbone of the copolymer. The maleic
anhydride in the backbone of SMA is reactive toward
nucleophilic reagents (H2O, alcohols, thiols, ammonia, amines,
etc). Introduction of nucleophilic compounds enables the
synthesis of new materials.40,41 SMA was chosen for these
experiments because of its ease to react with amines. Two
furanone compounds with dangling amines (5-(2-(2-
aminoethoxy)ethoxy)methyl)-2(5H)furanone and 4-(2-(2-ami-
EXPERIMENTAL METHODS
■
Materials. A commercial grade of SMA (Mw 110 000)
containing about 28 wt % maleic anhydride as a statistical
copolymer was donated by Polyscope, Geleen, The Nether-
lands (Grade SZ 28110). 2,5-Dimethyl-4-hydroxy-3(2H)-
furanone (DMHF, 98%), butadiene monoxide (98%), acryloyl
chloride, 2,2-(aminoethoxy)ethanol (98%), iodine (I2, 99%),
and sodium hydride (NaH, 95%) were obtained from Sigma
Aldrich, South Africa, and used without further purification.
Boc oxide ((Boc)2O, 98%), Grubbs (II) catalyst (97%), sodium
hydrogen carbonate (NaHCO3, 99.5%), and sodium thiosulfate
(Na2S2O3, 99%) were obtained from Merck chemicals. The
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Figure 2. Synthesis of furanone 2.
noethoxy)-2,5-dimethyl-3(2H)-furanone) referred to as Fur-
anone 1 and Furanone 2, respectively, in this paper, were
synthesized in our laboratories (Figures 1 and 2). These
furanones were then covalently immobilized on SMA forming
furanone-modified SMA (Figure 3).
evaporation to remove any residual solvent resulting in the
desired Boc-protected 2-(2-(aminoethoxy) ethanol (1). While
stirring, 20 mL of THF was gradually added to 6.82 g of
product 1 (0.036 mol) followed by 1.03 g of NaH (1.2 equiv).
The reaction temperature was lowered to −20 °C using a
HAAKE Thermo DC5-K75 cryostat. When the temperature
reached −20 °C and the hydrogen gas had completely evolved,
2.50 g of butadiene monoxide (0.036 mol) was added to the
reaction mixture and stirred for an additional 1 h. The reaction
was then stirred for an additional 16 h at room temperature.
Water (20 mL) was slowly added with stirring until H2
evolution ceased. The organic layer was separated using
CH2Cl2, washed with H2O, and dried using 10 g Na2SO4 to
give 6.16 g (90.32%) of 2.43 To alcohol 2 (6 g, 0.022 mol), 2.33
g (0.26 mol) of acryloyl chloride and 5 mL of ether were added.
The mixture was then poured into water, and the organic layer
was separated. Excess volatiles were removed via vacuum
evaporation, and 5.88 g of product 3 (98%) was obtained.
Product 3 (5.50 g, 0.017 mol) was ring-closed via metathesis by
adding 3.29 g (0.01 mol) of Grubbs’ second-generation catalyst,
giving the Boc-protected 5-(2-(2-aminoethoxy)ethoxy)methyl)-
2(5H)furanone (4), which after deprotection with H3PO4 in
the presence of toluene yielded 84% product (5).44 This
reaction is illustrated in Figure 1. Further purification by
column chromatography on silica was dismissed due to losses
in product yields when these were repeatedly columned.
Synthesis of furanone 2. The amine group in 2-(2-
aminoethoxy)ethanol was Boc-protected as described in the
previous section, and the resultant Boc-protected product (1)
was reacted with DMHF (2.0 g) in a 250 mL round-bottom
flask in the presence of p-toluenesulfonic acid (100 mg) and
anhydrous ethanol (80 mL). A drying tube was attached to the
top of the reflux condenser, and the mixture was refluxed for 6
h. The mixture was then cooled, shaken with solid NaHCO3 (5
g), and filtered through a 2 mm layer of NaHCO3 to remove
the acid catalyst.45 The product contained both the starting
material and the Boc-protected product, possibly because the p-
toluenesulfonic acid deprotected the 2-(2-aminoethoxy)-
Figure 3. Immobilization of furanone derivatives on SMA copolymer
backbone.39,41
.
Synthesis of furanone 1. To a magnetically stirred
mixture of 2-(2-(aminoethoxy) ethanol 20.67 mL (1 mmol)
and (Boc)2O 10.50 g (1 mmol), a catalytic amount of iodine
2.53 g (10 mol %) was added under solvent-free conditions at
room temperature. After stirring the reaction mixture for 3 h,
diethyl ether (10 mL) was added. The reaction mixture was
washed with aqueous Na2S2O3 solution (5%, 5 mL) and
saturated NaHCO3 solution. The organic layer was dried over
Na2SO4, and the solvent was evaporated under reduced
pressure.42 The product was further purified using silica gel
chromatography with ethyl acetate and triethylamine (7:1) as
the solvent system. This step was followed by vacuum
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ethanol. Column chromatography was used to purify the Boc-
protected 4-(2-(2-aminoethoxy)-2,5-dimethyl-3(2H)-furanone.
This was followed by deprotection using 4 equiv of H3PO4 in
the presence of toluene as the solvent, giving a 78% yield
(Figure 2).
0.03% LiCl (w/v), and samples were filtered through a 0.45 μm
GH Polypro (GHP) filter to prevent any impurities from
entering the system. Calibration was performed on poly(methyl
metachrylate) (PMMA) standards (Polymer Laboratories)
ranging from 690 to 1.2 × 106 g/mol. Data acquisition was
done using Millennium32 software, version 4.
Electrospinning. The modified polymers (SMA/5-(2-(2-
aminoethoxy)ethoxy)methyl)-2(5H)furanone (SMA/Furanone
1) and SMA/4-(2-(2-aminoethoxy)-2,5-dimethyl-3(2H)-fura-
none (SMA/Furanone 2)) were dissolved in (1:1) mixtures of
ethanol and methanol to form 10 wt %/vol electrospinning
solutions. Pristine SMA dissolved in THF was used for control
experiments. Bubble electrospinning, which is described in
detail by Gule and co-workers was used for nanofiber
production.46 All the polymer solutions were electrospun at
room temperature. The widget-collector distance used was 20
cm and the relative humidity was maintained between 45 and
60% for continuous fiber formation. The applied voltage used
was between 45 and 50 kV. The nanofibrous mats were cross-
linked by heat treatment at 120 °C for 15 min.
Scanning Electron Microscopy (SEM). The morphology
of the nanofibers was observed with SEM. Analyses were
performed on a Leo 1430VP coupled with a Cambridge S200
scanning electron microscope after gold sputter coating was
applied. The conditions for image acquisition were an
accelerating voltage of 7 kV and a probe current of 150 pA
for the former and an accelerating voltage of 10 kV and a probe
current of 50 pA for the latter. For determination of fiber
diameters the SEM_Img_Studio software, which runs using the
National Instruments LVRunTime Engine, version 1.1.0, was
used.
Immobilization of Furanone Compounds onto SMA.
In a three-necked round-bottomed flask equipped with a stirrer,
0.5 g of SMA (0.0025 mol MAnh) was placed and N,N-
dimethylformamide (DMF) (20 mL) was added as a solvent.
Then the solution was stirred at 70 °C. After complete
dissolution of SMA, 1 g of 4-(2-(2-aminoethoxy)-2,5-dimethyl-
3(2H)-furanone (0.0051 mol) was added dropwise with
continuous stirring. This resulted in gelation of the reactants;
vigorous stirring as well as increasing the temperature to 150
°C resulted in a clear gold solution. This solution was then
dehydrated using rotary evaporation followed by vacuum
evaporation to remove any residual solvents to give the final
product in a yield of 91% (Figure 3). In the case of 5-(2-(2-
aminoethoxy)ethoxy)methyl)-2(5H)furanone, 1.069 g of SMA
(0.0051 mol MAnh) and 1.026 g of 5-(2-(2-aminoethoxy)-
ethoxy)methyl)-2(5H)furanone (0.0051 mol) were used.
Nuclear Magnetic Resonance (1H NMR). NMR spec-
troscopy was used to elucidate the chemical structures of the
synthesized compounds and to determine their purity. One-
1
dimensional H NMR spectra were acquired with a Varian
Unity Inova 600 MHz NMR spectrometer with 5 mm
broadband probe at 293 K in deuterated chloroform
(CDCl3). Relaxation delays of 1 s and frequencies of 600
1
MHz were used for the H NMR. Spectra were internally
referenced to tetramethylsilane (TMS). All peaks are reported
in parts per million (ppm) downfield of TMS.
Electrospray Mass Spectrometry (ES-MS). The molar
masses of 5-(2-(2-aminoethoxy)ethoxy)methyl)-2(5H)-
furanone and 4-(2-(2-aminoethoxy)-2,5-dimethyl-3(2H)-fura-
none were confirmed by ES-MS. ES-MS was carried out using a
Waters API Q-TOF Ultima equipped with a Waters UPLC.
The sample (3 μL) was injected at a capillary voltage of 3.5 kV,
cone voltage of 35 V, and RFI value of 50. The source
temperature was maintained at 80 °C, and the desolvation
temperature was maintained at 350 °C. The desolvation gas
was set at 350 L/h, and the cone gas was set at 50 L/h.
Attenuated Total Reflectance Fourier Transform
Infrared Spectroscopy (ATR/FTIR). To confirm the
immobilization of the furanone compounds onto SMA, ATR-
FTIR spectroscopy was performed. A Nexus FT-IR provided by
Nicolet Thermo equipped with a FTIR gas analyzer was used
for ATR-FTIR studies. The spectrometer was fitted with a
diamond crystal, and measurements were taken in the 600 cm−1
to 4000 cm−1 infrared-range at a resolution of 6 cm−1. The
spectra were based on a total of 32 scans per sample.
Size Exclusion Chromatography (SEC). Molar mass and
dispersity (Đ) were obtained using SEC. The SEC was carried
out on a dimethylacetamide (DMAc, HPLC grade) solvent
system using a flow rate of 1.0 mL/min. The instrument setup
consisted of a Shimadzu LC-10AD pump, a Waters 717 plus
autosampler, a column system fitted with a 50 × 8 mm guard
column in series with three 300 × 8 mm, 10 μm particle size
GRAM columns (2 × 3000 Å and 100 Å) obtained from PSS, a
Waters 2487 dual wavelength UV detector and a Waters 410
differential refractive index (DRI) detector all in series. 100 μL
injection volumes were used, and the oven temperature of the
column and DRI detector was kept at 40 °C. The solvent was
stabilized with 0.05% butylhydroxytoluene (BHT) (w/v) and
To measure the fiber diameters, the electrospun nanofibrous
mats were viewed under the SEM at a magnification between
1000 and 5000×. This magnification range was chosen such
that up to 50 fibers were included in the image. Each image was
then processed using SEM_Img_Studio Software. A minimum
of 15 measurements were taken, and the highest and lowest
values were discarded prior to calculating the average. Solvent
beaded and clumped fibers were considered outliers and
therefore were not included in the measurements. The porosity
of nanofibrous mats is another very important parameter,
especially since it determines the air and water permeability
through the fiber mat.47 To measure pore sizes, area light
intensity scans were performed using the Scanning Probe
Image Processor (SPIP) Image Metrology A/S, version 5.1.3
software. This software uses SEM images to calculate fiber mat
porosity.
Antimicrobial Inhibition. To determine the antimicrobial
efficiency of the nanofibers, the basic plate counting technique
was adopted. These tests were confirmed with bioluminescent
imaging (BLI) and LIVE/DEAD BacLight to determine the
antimicrobial efficiency against viable but nonculturable
(VBNC) cells. This was done to quantify the cells that enter
a dormant state during contact with the antimicrobial fibers and
to eliminate the chances of overestimating the antimicrobial
efficiency of the nanofibers. The electrospun nanofibers were
supported on 0.22 μm sized Millipore filter membranes for
antimicrobial tests and without a support for cell adhesion-
inhibition tests. For each of the tests, the amount of nanofibers
was 10 mg.
Plate counts. K. pneumoniae Xen 39, S. aureus Xen 36, E.
coli Xen 14, P. aeruginosa Xen 5, and S. typhimurium Xen 26
were used to test the antimicrobial and cell-adhesion inhibition
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properties of the modified polymers. Each pathogen was
cultured in 10 mL brain heart infusion (BHI) broth (Biolab
Diagnostics) along with the appropriate antibiotics overnight
on a rotating wheel at 37 °C. For each strain, cells were pelleted
by 10 min centrifugation at 3000 rpm, and washed three times
with physiological water. The optical densities (ODs) were
measured using a Biorad smart spec plus spectrophotometer.
Spiked water samples were prepared by inoculating 1 × 1010
CFU/mL (1 mL) of each strain into 500 mL sterile
physiological water. Several control (cell viability) experiments
(not reported here) were carried out to verify the absence of
osmotic lysing, which often occurs when cells (especially gram
negative) are removed from rich media into physiological
water.48,49 The spiked water was then filtered through pristine
SMA (control) or furanone-modified nanofibrous mats. The
nanofibers were then rinsed several times using 10 mL
physiological water to wash off all remaining bacterial cells
and the wash-off water was plated out on BHI agar plates to
determine living cells. After washings, the membranes were
viewed under the XENOGEN VIVO VISION In Vivo Imaging
Lumina System (IVIS) to ascertain complete removal of the
cells. Other techniques such as vortexing of the membranes in
physiological water to remove all cells that may be trapped
inside the membrane were also investigated and yielded similar
results. This was done after specific contact periods to
determine cell death as a function of time. After incubation
of the plates overnight at 37 °C, colony counting was used to
determine the antimicrobial effect of the filters. The experiment
was performed for each pathogen and each specific contact
period in triplicate. In order to mimic a real-life scenario where
bacteria species coexist, 20 μL of each pathogen from the 106
CFU/mL stock was inoculated into the same tube containing
10 mL BHI broth to make a cocktail of the strains, and
antimicrobial tests were conducted as described previously.
Cell-Adhesion Inhibition Characterization. The cell-
adhesion inhibition capacity of the furanone-modified nano-
fibrous mats was carried out. To do this, a method similar to
the one used by Alvarez and co-workers was modified and used
for our experiments.50 Weighed (10 mg) furanone-modified
nanofibrous mats were immersed in BHI (10 mL) media
containing E. coli Xen 14, S. tymphimurium Xen 26, P.
aeruginosa Xen 5, K. pneumoniae Xen 39, and S. aureus Xen
36. For each of the strains, attachment inhibition was
monitored after culturing at different time intervals (0−36 h)
as illustrated in Figure 4. At each of these time intervals, the
nanofibrous mats were removed from the culture media and
rinsed in physiological water to remove unattached colonies.
These were then either rinsed with physiological buffered saline
(PBS) or vortexed prior to plating out. For imaging, the mats
were rinsed and stained prior to fluorescence imaging. Real
biofilm development and biofilm architecture were not studied,
and future studies on these nanofibers will explore these details.
BLI. BLI is a relatively new development, which uses the
light emitted from genetically modified living organisms as a
tool for molecular imaging in small laboratory animals. It has
been used by other researchers and has been found to offer a
method that is sensitive and innocuous and only allows live or
viable cells to be detected.51−53 This technique measures cell
viability by quantifying total photons emitted by the cells. In
order to apply the BLI system in the antimicrobial evaluation,
engineered bacterial strains obtained from Caliper Life Sciences
(Hopkinton MA, USA) were used for the assessment in this
study. The bacterial strains have a Photorhabdus luminescence
Figure 4. Cell adhesion inhibition characterization setup.
luxABCDE operon (lux gene) to produce the enzyme
luciferase, which emits photons in the presence of ATP and
oxygen. These photons indicate metabolic activity in the
pathogens. Research on BLI corresponded with plate counting
data with a correlation efficiency of about 0.98.54 After filtration
of the spiked samples (as described in the Plate Counts
section) the nanofibrous mats were placed in a XENOGEN
VIVO VISION In Vivo Imaging Lumina System (IVIS)
supplied by Caliper Life Science, and the Living Image 3.1
Software was used to process them. Imaging was performed
immediately after filtering the spiked water through the
furanone-modified nanofibrouos mats and also after 10 min
exposure to monitor the level of bioluminescence emitted by
the strains.
Fluorescence Experiments. Furanone-modified and con-
trol nanofibrous mats (10 mg) were exposed to the pathogen
cocktail (1 × 1010 cells) described in the Plate Counts section
along with the appropriate antibiotics [E. coli Xen 14
(Kanamycin, 30 μg/mL), S. tymphimurium Xen 26 (Kanamycin,
30 μg/mL), P. aeruginosa Xen 5 (Tetracycline, 60 μg/mL), K.
pneumoniae Xen 39 (Kanamycin, 200 μg/mL), and S. aureus
Xen 36 (Kanamycin, 30 μg/mL)] overnight on a rotating wheel
at 37 °C. These mats were then taken out of the media using
forceps and rinsed lightly using physiological water. LIVE/
DEAD BacLight kit with SYTO 9 and propidium iodide
fluorescent dyes purchased from Molecular Probes, Inc. were
used to stain the specimen for imaging purposes. SYTO 9 stains
all cells green, while propidium iodide penetrates cells whose
cell membrane has been damaged and stains them red. Viable
and total counts can be obtained in one staining step. Staining
was done by incubation of samples with 6.5 μM dye at room
temperature for 10 min. These samples were then observed on
an Olympus Cell-R system attached to an IX-81 inverted
fluorescence microscope equipped with a F-view-II cooled
CCD camera (Soft Imaging Systems). Using a Xenon-Arc
burner (Olympus Biosystems GMBH) as light source, images
were excited with the 472 or 572 nm excitation filter. Emission
was collected using a UBG triple-band-pass emission filter cube.
For the image frame acquisition, an Olympus Plan Apo N 60x/
1.4 Oil objective and the Cell-R imaging software were used.
Images were processed and background-subtracted using the
Cell-R software.
Gas Chromatograph-Coupled Mass Spectroscopy
(GC-MS). The leaching of chemical compounds into the
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Figure 5. Overlaid ATR-FTIR of SMA and SMA/Furanone 1 showing the conversion of SMA into SMA/Furanone 1 (A) and SMA/Furanone 2
(B).
Figure 6. Morphology of electrospun neat SMA (A), SMA/Furanone 1 (B), and SMA/Furanone 2 nanofibers.
environment is often accompanied by health and pollution
concerns. To determine whether the furanone derivatives did
not leach into filtered water, water filtered through the
nanofibrous mats was screened for traces of furanones using
GC-MS. As a positive control, distilled water spiked with 0.02
M 5-(2-(2-aminoethoxy)ethoxy)methyl)-2(5H)furanone (fur-
anone 1) was analyzed, and distilled water was used as a
negative control. GC-MS was performed using a Waters GCT
spectrometer equipped with CTC CombiPAL Autosampler,
and a DB XLB column (30 m, 0.25 mm ID, 0.1 μm film
thickness) was utilized. Solid phase microextraction (SPME)
vials were used in the analysis, and the headspace of the
samples were analyzed using a CTC PAL auto sampler.
RESULTS AND DISCUSSION
1H NMR. 1H NMR was used to confirm the structures of the
■
synthesized 5-(2-(2-aminoethoxy)ethoxy)methyl)-2(5H)-
furanone and 4-(2-(2-aminoethoxy)-2,5-dimethyl-3(2H)-fura-
none. All the peaks are reported in ppm downfield of TMS.
Furanone 1: (1H NMR: 600 MHz, CDCl3 δ = 3.42 (m, 2H),
3.58 (m, 5H), 3.81 (m,2H), 4.42 (s,2H), 4.98 (m,1H), 5.35 (m
= 1H), 6.32 (s = 2H)). Furanone 2: (1H NMR: 600 MHz,
CDCl3 δ =1.22 (s,3H), 2.16 (s,3H), 2.98 (m,2H), 3.32 (m,
4H), 3.62 (m,2H), 3.74 (m.1H), 6.67(s,2H)). These results
confirmed the structures of the furanone compounds.
ES-MS. ES-MS spectra confirmed the molar masses of 5-(2-
(2-aminoethoxy)ethoxy)methyl)-2(5H)furanone and 4-(2-(2-
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Figure 7. Nanofiber diameters of SMA/Furanone 1 (A) and of SMA/Furanone 2 (B) and pore sizes of SMA/Furanone 1 (C) and of SMA/
Furanone 2 (D) nanofiber mats.
Table 1. Summary of Antimicrobial and Cell Adhesion-Inhibition Achieved by the Synthesized Furanones
P. aeruginosa
Xen 5
E. coli
Xen 14
S. typhimurium
S. aureus
Xen 36
K. pneumoniae
Xen 26
Xen 39
antimicrobial efficacy after 30 min
SMA nanofibers
0
0
0
0
0
(log CFU/mL reduction)
neat furanone 1
2.68
3.45
1.12
3.78
0
2.79
3.62
0.79
2.62
0
2.01
3.09
0.8
1.35
3.76
0.83
3.02
0
2.23
3.66
0.6
neat furanone 2
SMA/Furanone 1 nanofibers
SMA/Furanone 2 nanofibers
SMA nanofibers
3.26
0
2.66
0
cell adhesion-inhibition after 36 h
(log CFU/mL reduction )
neat furanone 1
2.38
4.68
1.88
3.22
2.49
4.67
1.21
4.22
1.51
3.79
2.03
1.79
1.75
4.66
1.35
3.66
2.02
3.66
2.23
3.66
neat furanone 2
SMA/Furanone 1 nanofibers
SMA/Furanone 2 nanofibers
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Figure 8. Antimicrobial (A) and antifouling (B) potential of neat furanone 1 over 30 min and 36 h respectively.
Figure 9. Antimicrobial (A) and antifouling (B) potential of neat furanone 2 over 30 min and 36 h, respectively.
Figure 10. Antimicrobial (A) and cell-adhesion inhibition (B) potential of SMA/Furanone 1 nanofibrous mats over 30 min and 36 h, respectively.
aminoethoxy)-2,5-dimethyl-3(2H)-furanone to be m/z 215.01
and m/z 201.1, respectively. This corresponds to calculated
molar masses of 215.101 g for 5-(2-(2-aminoethoxy)ethoxy)-
methyl)-2(5H)furanone and 201.110 g for 4-(2-(2-amino-
ethoxy)-2,5-dimethyl-3(2H)-furanone.
ATR/FTIR Spectroscopy. ATR/FTIR spectroscopy was
used to confirm successful immobilization of the furanone
moiety on SMA. Figure 5A,B compares the ATR/FTIR spectra
of pristine SMA with that of the synthesized furanones and
SMA modified with furanones. The absorption peak at 1185
cm−1 is characteristic of the amide stretch.55 The imide groups
around 1694 cm−1 are characteristic of the asymmetric and
symmetric CO stretching vibrations. The signals at 1576
cm−1 and around 1500 cm−1 are caused by (CC) stretching
of the aromatic ring and (C−H) bending vibration of the
aromatic ring, respectively. The band at 916 cm−1 is due to
cyclic anhydride groups. From the highlighted similarities on
the spectra (Figure 5A and 5B), the incorporation of the
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Figure 11. Antimicrobial (A) and cell-adhesion inhibition (B) potential of SMA/Furanone 2 nanofibrous mats over 30 min and 36 h, respectively.
Figure 12. Antimicrobial (A) and attachment-inhibition (B) potential of nanofibrous mats on mixed strains.
precipitate out any residual SMA. There was no precipitate,
which indicated that there was no excess SMA present in the
furanone-modified SMA. This gave a good indication that
complete conversion of SMA to SMA/Furanone had taken
place. The furanone containing SMA was rotary evaporated and
dried in a vacuum pump to remove the methanol, and the
yields remained consistent to further confirm that none of the
SMA was precipitated. SEC of the polymer was consistent with
the polymer, with a molar mass (Mn) of 112 000 and dispersity
(Đ) of 2.7 for SMA/Furanone 1. For SMA furanone 2, Mn was
111 900, and the dispersity (Đ) was 2.3.
Morphology of the nanofibers. The morphology of both
SMA/Furanone 1 and SMA/Furanone 2 nanofibers did not
differ much from that of neat SMA (Figure 6A−C). The fibers
exhibited smooth morphology with no solvent beading.
Individual fibers in these mats had diameters averaging between
120 and 220 nm (Figure 7A,B). Although there is no specific
diameter range for nanofibrous materials in filter applications,
according to the filtration theory, smaller fiber diameters give
better filtration efficiency.56 This is because thinner fibers result
in high surface-to-volume ratios, which are advantageous for
filtration application. This was a good property since it
increased the surface area for water filtration. These nano-
fibrous mats also had average pore sizes of less than 120 nm2
(Figure 7C,D). The sizes of the mat pores are very important
Figure 13. In vivo images illustrating the antimicrobial efficacy of the
furanone-modified nanofibrous mats after 10 min of exposure of a
mixed culture.
furanone moiety onto the SMA backbone is clearly
demonstrated.
Conversion Efficiency of SMA to SMA/Furanone. After
immobilization of the furanone compounds on SMA, both
furanone-containing polymers were dissolved in methanol to
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antimicrobial and cell adhesion-inhibition properties. In both
cases (neat and immobilized), furanone 2 showed greater
activity than furanone 1. To fully understand this phenomenon
would require further insight into the biochemical QS pathway
taken by these furanone compounds specifically. Neat furanone
1 reduced populations of P. aeruginosa Xen 5, E. coli Xen 14, S.
typhimurium Xen 26, S. aureus Xen 36, and K. pneumoniae Xen
39 by an average of 1.5 log after 30 min (Figure 8A). P.
aeruginosa Xen 5 was reduced more than all the other strains by
2.7 log, and S. aureus Xen 36 was the least reduced by 1.4 log.
The free furanone also inhibited attachment by more than 1.5
log for all of the studied strains after 36 h (Figure 8B). SMA/
Furanone 1 nanofibers on the other hand exhibited lower
antimicrobial efficiency (up to 1.1 log reduction of P. aeruginosa
Xen 5) after 30 min exposure (Figure 10 A). Adhesion-
inhibition of more than 1.2 log was achieved for all strains after
36 h (Figure 10 B).
The antimicrobial activity of neat furanone 2 was more than
3 log for all the strains tested (Figure 9A). The furanone
compound also showed more than 4.5 log cell adhesion-
inhibition of P. aeruginosa Xen 5, E. coli Xen 14, and S. aureus
Xen 36. S. typhimurium Xen 26 and K. pneumoniae Xen 39 were
also inhibited by more than 3.6 log after 36 h (Figure 9B).
Nanofibrous mats made from SMA/Furanone 2 showed up to
3.8 log reductions in populations of P. aeruginosa Xen 5, closely
followed by S. typhimurium Xen 26, which was reduced by up to
3.3 log after 30 min of exposure (Figure 11A). E. coli Xen 14, S.
aureus Xen 36, and K. pneumoniae Xen 39 were all reduced by
at least 2.6 log after 30 min of exposure. The SMA/Furanone 2
nanofibers also inhibited cell-adhesion by more than 3 log for
all the strains except S. typhimurium Xen 26 over 36 h of
exposure (Figure 11B).
The cultured mixed strains (Figure 12 A) were also
significantly reduced by the furanone-modified nanofibers,
and a similar trend was observed where the furanone 2-
modified polymer nanofibrous mats showed higher potency
than the furanone 1-modified polymer nanofibrous mats. The
slightly higher activity in the mixed strains could be due to
competition stresses for nutrients, which were already being
experienced by the strains especially after 36 h of incubation.
This is, however, not significant enough to draw any concrete
conclusions.
BLI. In vivo imaging of nanofibrous mats after exposure to a
mixed strain culture of P. aeruginosa Xen 5, E. coli Xen 14, S.
typhimurium Xen 26, S. aureus Xen 36, and K. pneumoniae Xen
39 was carried out to confirm the results obtained from plate
counting. Metabolically active (viable) bacterial cells show
bioluminescence as indicated in Figure 13. The degree of
bioluminescence changes with increase in concentration as
illustrated by the scale in Figure 13. From these results, the
antimicrobial nature of the furanone compounds is clearly
demonstrated. The mats made from SMA/Furanone 2 still
demonstrated high antimicrobial potential compared to those
made from SMA/Furanone 1.
Fluorescence Microscopy. In Figure 14, the fluorescence
microscopy results after staining for live/dead cells are shown.
The green dye (SYTO 9) stains all bacteria colonies on the
mats and the red dye (propidium iodide) stains cells with
ruptured membranes (dead cells). The fibers also absorbed
some of the dyes, and bacteria colonies can be differentiated
from the fibers because they appear as distinct dots on the
nanofiber surfaces. The control SMA images in Figure 14A did
not have antimicrobial activity. This is indicated by the fact that
Figure 14. Fluorescence microscopy images showing cell-adhesion
inhibition and antimicrobial efficacy of the furanone-modified
nanofibrous mats.
for this study since, for accuracy in measuring antimicrobial
efficacy, the pores have to be smaller than the sizes of the
studied bacteria strains. The recorded pore sizes were all less
than 250 nm2 and therefore appropriate for further tests.
Antimicrobial and Cell-Adhesion Inhibition Charac-
terization. A lot of research has been done on furanone
compounds with both 2(5H) and 3(2H) cores. Free furanone
derivatives with 2(5H) cores have demonstrated antimicrobial
activity against bacterial strains and even fungi.22,57−62 In zone
inhibition tests, 4-amino-5-hydroxy-2(5H)-furanones demon-
strated an average of 10 mm inhibition of E. coli, S. aureus, P.
aeruginosa, K. pneumoniae and Enterobacter spp.57 Work done by
Lonn-Stenstud and co-workers also showed that this class of
̈
furanone compounds significantly reduced P. aeruginosa lung
infection in mice.58 Even though these studies did not use
similar techniques to quantify antimicrobial and cell-adhesion
inhibition efficiencies, they showed convincingly that furanone
derivatives with 2(5H) cores had antimicrobial properties.
The antimicrobial efficiency of synthetic and natural
furanone derivatives with the 3(2H) core has also received
vast interest among researchers.46,63−65 One of the most
interesting reports was published by Kataoka, where he
investigated the composition of Japanese soy sauce, which
among other properties has been found to be antimicrobial and
anticarcinogenic. This sauce was found to contain 4-hydroxy-
2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF),
DMHF, and 4-hydroxy-5-methyl-3(2H)-furanone (HMF).
This sauce has demonstrated up to 3 log reduction of strains
S. aureus, Shigella spp., Vibrio cholera, Salmonella spp., and E. coli
within 4−6 h of contact.66 A similar study also demonstrated up
to 4.5 log reductions in populations of K. pneumoniae Xen 39, S.
aureus Xen 36, E. coli Xen 14, P. aeruginosa Xen 5, and S.
typhimurium Xen 26.46 Sung and co-workers also reported
exceptional antimicrobial efficacy of DMHF toward strains of P.
aeruginosa, S. aureus, E. coli, and Enterococci.63
In the present study, as summarized in Table 1, the neat
furanones as well as SMA-immobilized furanones showed good
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Figure 15. GC-MS spectra indicating that furanone compounds did not leach from nanofibrous mats into the filtered water.
none of the available (Figure 14A1) bacteria colonies were dead
(they did not absorb propidium iodide; Figure 14A2). These
nanofibers also did not inhibit attachment as indicated by the
attached colonies (Figure 14A1). Furanone-modified nano-
fibers, on the other hand, demonstrated inhibition of cell
adhesion in that no colonization was visible (Figure 14B,C).
These results also confirmed cell deactivation (antimicrobial
activity), and this is indicated by the colony, which managed to
attach to the nanofibers, but was deactivated (absorbed the red
dye indicating cell death) (Figure 14 B2).
Leaching. GC-MS experiments were carried out to
investigate the leaching of the furanone derivatives from the
fibers. As expected for covalently bound compounds, the GC-
MS results indicated that the furanone compounds did not
leach into the filtered water. The characteristic signal observed
at 11.59 min in the positive control (spiked with the furanone-
derivative) was absent in all the leaching experiments (Figure
15).
CONCLUSIONS
■
The syntheses of 5-(2-(2-aminoethoxy)ethoxy)methyl)-2(5H)-
furanone and 4-(2-(2-aminoethoxy)-2,5-dimethyl-3(2H)-fura-
none were successfully carried out. The structures and molar
masses were confirmed using 1H NMR and ES mass
spectrometry. Both furanone derivatives were successfully
immobilized onto the SMA copolymer. The furanone-modified
SMA was electrospun to obtain nanofibers. The nanofibrous
mats demonstrated good antimicrobial and cell-adhesion
inhibition efficiency against P. aeruginosa Xen 5, E. coli Xen
14, S. typhimurium Xen 26, S. aureus Xen 36, and K. pneumoniae
Xen 39 individually and in mixed cell culture form. The 3(2H)
furanone had high activity compared to the 2(5H) furanone. It
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Ramasamy, K.; Cuihua, X.; Manijeh, P.; Gholam, H. Bioorg. Med.
Chem. Lett. 2003, 11, 4303−4313.
is, however, not clear why the 3(2H) furanone has a higher
activity compared to the 2(5H) furanone; structural differences
between the two furanone compounds may be attributed to
this, because the same number of moles of the furanone
compounds was used in the SMA modifications. Further
biochemical studies need to be done for proper understanding
of the QS pathways taken by the furanone compounds to
inhibit cell attachment-inhibition and antimicrobial properties
of these furanones.
(22) Wu, H.; Song, Z.; Hentzer, M.; Andersen, J. B.; Molin, S.;
Givskov, M.; Høiby, N. J. Antimicrob. Chemother. 2004, 53, 1054−
1061.
(23) Manefield, M.; Rasmussen, T.,B.; Henzter, M.; Andersen, J.,B.;
Steinberg, P.; Kjelleberg, S.; Givskov, M. Microbiology 2002, 148,
1119−1127.
(24) Rasmussen, T. B.; Givskov, M. Int. J. Med. Microbiol. 2006, 296
(2−3), 149−169.
(25) Kim, C.; Kim, J.; Park, H. -Y.; Park, H. -J.; Lee, J. H.; Kim, C. K.;
Yoon, J. Appl. Microbiol. Biotechnol. 2008, 80, 37−47.
AUTHOR INFORMATION
■
̀
(26) Estephane, J.; Dauvergne, J.; Soulere, L.; Reverchon, S.;
Notes
Queneau, Y.; Doutheau, A. Bioorg. Med. Chem. Lett. 2008, 18,
4321−4324.
The authors declare no competing financial interest.
(27) Defoirdt, T.; Miyamoto, C. M.; Wood, T. K.; Meighen, E. A.;
Sorgeloos, P.; Verstraete, W.; Bossier, P. Environ. Microbiol. 2007, 9,
2486−2495.
ACKNOWLEDGMENTS
■
The authors would like to acknowledge the University of
Stellenbosch, the Andrew Mellon Foundation, Eskom and the
South African Research Chairs Initiative (SARChI) from the
Department of Science and Technology (DST), and the
National Research Foundation (NRF) for funding this project.
(28) Defoirdt, T.; Crab, R.; Wood, T. K.; Sorgeloos, P.; Verstraete,
W.; Bossier, P. Appl. Environ. Microbiol. 2006, 72 (9), 6419−6423.
(29) Morohoshi, T.; Shiono, T.; Takidouchi, K.; Kato, M.; Kato, N.;
Kato, J.; Ikeda, T. Appl. Environ. Microbiol. 2007, 73, 6339−6342.
(30) Manefield, M.; Harris, L.; Rice, S. A.; De Nys, R.; Kjelleberg, S.
Appl. Environ. Microbiol. 2000, 66, 2079−2084.
REFERENCES
(31) Abd-Alla, M. H.; Bashandy, S. R. Int. Schol. Res. Net. (ISRN)
Microbiol. 2012, 2012 (ArticleID 161890), 1−7.
■
(1) Chen, X.; Stewart, P. S. Environ. Sci. Technol. 1996, 30, 2078−
2083.
(32) Ponnusamy, K.; Paul, D.; Kim, Y. S.; Kweon, J. H. Braz. J.
Microbiol. 2010, 41, 227−234.
(2) Kramer, J. F. Biofilm control with bromo-chloro-dimethylhydantoin;
Paper number 1277; NACE International: Houston, TX, 2001.
(3) Vidella, H. A. Int. Biodeterior. Biodegrad. 2002, 49, 259−270.
(4) Stewart, P.; Roe, F.; Rayner, J.; Elkins, F. G.; Lewandowski, Z.;
Ochsner, U. A.; Hassett, D. J. Appl. Environ. Microbiol. 2000, 66, 836−
838.
(5) Presterl, E.; Suchomel, M.; Eder, M.; Reichmann, S.; Lassnigg, A.;
Graninger, W.; Rotter, M. J. Antimicrob. Chemother. 2007, 60, 417−
420.
(6) Richards, M. Self immobilization of single and combinations of
enzymes in spherical particles and evaluation of their anti-biofouling
potential. Masters Dissertation, University of Pretoria, Pretoria, South
Africa, 2010.
̀
(33) Perez, A. G.; Olías, R.; Olías, J. M.; Sanz, C. J. Agric. Food Chem.
1999, 47, 655−658.
(34) Mahajan, V. A.; Borate, H. B.; Wakharkar, R. D. Tetrahedron
2006, 62, 1258−1272.
(35) Brouwer, H. D.; Schellekens, M. A.,J.; Klumperman, B.;
Monteiro, M.,J.; German, A.,L. J. Polym. Sci., Part A 2000, 38,
3596−3603.
(36) Guha, S. K. U.S. Patent 5488075, 1996.
(37) Edgren, D.; Wong, P. S. L.; Theeuwes, F. U.S. Patent 4587117,
1986.
(38) Bellettini, A. G.; Bellettini, R. J. U.S. Patent 6210653, 2001.
(39) Zschoche, S.; David, R.; Tavana, H.; Poschel, K.; Petong, N.;
̈
(7) Meyer, B. Int. Biodeterior. Biodegrad. 2003, 51, 249−253.
(8) Allison, D. G.; Ruiz, B.; SanJose, C.; Jaspe, A.; Gilbert, P. FEMS
Microbiol. Lett. 1998, 167, 179−184.
Dutschk, V.; Grundke, K.; Neumannb, A. W. Colloids Surf. A 2007,
307, 53−61.
(40) Saad, G. R.; Morsi, R. E.; Mohammady, S. Z.; Elsabee, M. Z. J.
Polym. Res. 2008, 15, 115−123.
(9) Van der Borden, A. J.; Van der Mei, H. C.; Busscher, H. J. J.
Biomed .Mater. Res. 2004, 68B, 160−164.
(41) Mpitso, K. Synthesis and characterization of styrene−maleic
anhydride copolymer derivatives. M.Sc. Thesis, Stellenbosch Uni-
versity, Stellenbosch, South Africa, 2009.
(10) Curtin, J. J.; Donlan, R. M. Antimicrob. Agents Chemother. 2006,
50, 1268−1275.
(11) Al-Sha’alan, N. Molecules 2007, 12, 1080−1091.
(12) Revanasiddappa, H. D.; Vijaya, B.; Kumar, S.,L.; Prasad, K.,S.
World J. Chem. 2010, 5, 18−25.
(42) Varala, R.; Nuvula, S.; Adapa, S. R. J. Org. Chem. 2006, 71,
8283−8286.
(43) Claessens, S.; Naidoo, D.; Mulholland, D.; Verschaeve, L.; van
Staden, J.; De Kimpe, N. Synlett 2006, 4, 621−623.
(44) Li, B.; Berliner, M.; Buzon, R.; Chiu, C. K.-F.; Colgan, S. T.;
Kaneko, T.; Keene, N.; Kissel, W.; Le, T.; Leeman, K. R.; Marquez, B.;
Morris, R.; Newell, L.; Wunderwald, S.; Witt, M.; Weaver, J.; Zhang,
Z.; Zhang, Z. J. Org. Chem. 2006, 71, 9045−9050.
(45) Buttery, R. G.; Ling, L. C. J. Agric. Food Chem. 1996, 44 (6),
1512−1514.
(13) Zhuang, X.; Cheng, B.; Kanga, W.; Xua, X. Carbohydr. Polym.
2010, 82, 524−527.
(14) Munoz-Bonilla, A.; Fernan
2012, 37, 281−339.
́
dez- Garci
́
a, M. Prog. Polym. Sci.
̃
(15) Gottesdiener, K.; Mehlisch, D. R.; Huntington, M; Yuan, W.;
Brouwn, P.; Gertz, B.; Mills, S. Clin. Ther. 1999, 21, 1301−1312.
(16) Baveja, J. K.; Li, G.; Nordon, R. E.; Hume, E. B. H.; Kumar, N.;
Willcox, M. D. P.; Poole-Warren, L. A. Biomaterials 2004, 25, 5013−
5021.
(46) Gule, N. P.; De Kwaadsteniet, M.; Cloete, T. E.; Klumperman,
B. Fabrication and characterization of antibiofouling furanone
modified nanofibers from poly(vinyl alcohol) via bubble electro-
spinning. In preparation.
(47) Aydilek, A. J. Comput. Civil Eng. (ASCE) 2002, 16 (4), 280−290.
(48) Suwannakul, S.; Stafford, G. P.; Whawell, S. A.; Douglas, C. W.
I. Microbiology 2010, 156, 3052−3064.
(49) Rojas-Chapana, J. A.; Correa-Duarte, M. A.; Ren, Z.; Kempa, K.;
Giersig, M. Nano Lett. 2004, 4 (5), 985−988.
(50) Zodrow, K.; Brunet, L.; Mahendra, S.; Li, D.; Zhang, A.; Li, Q.;
Alvarez, P. J. J. Water Res. 2009, 43, 715−723.
(17) Lattmann, E.; Ayuko, W.,O.; Kinchinaton, D.; Langley, C. A.;
Singh, H.; Tisdale, M.,J. J. Pharm. Pharmacol. 2010, 55, 1259−1265.
(18) Khan, M. S.; Husain, A. Pharmazie 2002, 57, 448−452.
(19) Leite, L.; Jansone, M.; Veveris, M.; Cirule, H.; Popelis, Y.;
Melikyan, G.; Avetisyan, A.; Lukevics, E. Eur. J. Med. Chem. 1999, 34,
859−865.
(20) Klunk, W. E.; Covey, D. F.; Ferrendelli, J. A. Mol. Pharmacol.
1982, 22, 438−443.
(21) Ali, A. M.; Shahram, H.; Jamshid, C.; Ghadam Ali, K.; Farshid,
H.; Fen-Tair, L.; Tai, W. L.; Kak-Shan, S.; Chi-Feng, Y.; Moti, L. J.;
3149
dx.doi.org/10.1021/bm300932u | Biomacromolecules 2012, 13, 3138−3150

Biomacromolecules
Article
(51) Close, D. M.; Xu., T.; Sayler., G. S.; Ripp., S. Sensors 2011, 11,
180−206.
(52) Cronin, M.; Akin, A. R.; Collins, S. A.; Meganck, J.; Kim, J.-B.;
Baban, C. K.; Joyce, S. A.; van Dam, G. M.; Zhang, N.; van Sinderen,
D.; O’Sullivan, G. C.; Kasahara, N.; Gahan, C. G.; Francis, K .P.;
Tangney, M. PLoS One 2012, 7 (1), 1−11.
(53) O’Neill, K.; Lyons, S. K.; Gallagher, W. M.; Curran, K. M.;
Byrne, A. T. J. Pathol. 2010, 220 (3), 317−327.
(54) Francis, D. L.; Visvikis, D.; Costa, D. C. Eur. J. Nucl. Med. Mol.
Imag. 2003, 30, 988−994.
(55) Bshena, O. E. S. Synthesis of permanent non-leaching
antimicrobial polymer nanofibers. Ph.D. Thesis, University of
Stellenbosch, Stellenbosch, South Africa, 2012.
(56) Yun, K. M.; Suryamas, A. B.; Iskandara, F.; Bao, F. L.; Niinuma,
H.; Okuyama, K. Sep. Purif. Technol. 2010, 75, 340−345.
(57) Lattmann, E.; Dunn, S.; Niamsanit, S.; Sattayasai, N. Bioorg.
Med. Chem. Lett. 2005, 15, 919−921.
(58) Lonn-Stensrud, J.; Landin, M. A.; Benneche, T.; Petersen, F. C.;
Scheie, A. A. J. Antimicrob. Chemother. 2009, 63, 309−316.
(59) Sindhu Ramachandran, C. V.; Sreekumar, P. K. Int. J. Pharm.
Pharm. Sci. 2011, 3, 225−228.
̀
(60) Weber, V.; Coudert, P.; Rubat, C.; Duroux, E.; Vallee-Goyet, D.;
Gardette, D.; Bria, M.; Albuisson, E.; Leal, F.; Gramain, J.-C.;
Couqueleta, J.; Madesclaire, M. Bioorg. Med. Chem. 2002, 10, 1647−
1658.
(61) Paulitz, T.; Nowak-Thompson, B.; Gamard, P.; Tsang, E.;
Loper, J. J. Chem. Ecol. 2000, 26 (6), 1515−1524.
(62) Hume, E. B. H.; Baveja, J. K.; Muirf, B.; Schubert, T. L.; Kumar,
N.; Kjelleberg, S.; Griesser, H. J.; Thissen, H.; Read, R.; Poole-Warren,
L. A.; Schindhelm, K.; Willcox, M. D. P. Biomaterials 2004, 25, 5023−
5030.
(63) Sung, W. S.; Jung, H. J.; Park, K.; Kim, H. S.; Lee, I. S.; Lee, D.
G. Life Sci. 2007, 80, 586−591.
(64) Kozminykh, V. O.; Igidov, N. M.; Kozminykh, E. N.; Semenova,
Z. N.; Andreichikov, Y. S. Pharmazie 1992, 47, 261−263.
(65) Winzer, K.; Hardie, K. R.; Burgess, N.; Doherty, N.; Kirke, D.;
Holden, M. T. G.; Linforth, R.; Cornell, K. A.; Taylor, A. J.; Hill, P. J.;
Williams, P. Microbiology 2002, 148, 909−922.
(66) Kataoka, S. J. Biosci. Bioeng. 2005, 100 (3), 227−234.
3150
dx.doi.org/10.1021/bm300932u | Biomacromolecules 2012, 13, 3138−3150