Clinical microbiology

Water dispersible magnetite nanoparticles inuence the efcacy of

antibiotics against planktonic and biolm embedded Enterococcus
faecalis cells
Mariana Carmen Chiriuc
a
, Alexandru Mihai Grumezescu
b,
*
, Ecaterina Andronescu
b
,
Anton Ficai
b
, Ani Ioana Cotar
a
, Valentina Grumezescu
b, c
, Eugenia Bezirtzoglou
d
,
Veronica Lazar
a
, Radu Radulescu
e
a
Department of Microbiology and Immunology, Faculty of Biology, University of Bucharest, Romania
b
Department of Science and Engineering of Oxidic Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, Politehnica University
of Bucharest, Polizu Street, No. 1e7, Bucharest, Romania
c
Laser-Surface-Plasma Interactions Laboratory, Lasers Department National Institute for Lasers, Plasma, and Radiation Physics, Magurele, Romania
d
Democritus University of Thrace Faculty of Agricultural Development, Department of Food Science and Technology Laboratory of Microbiology,
Biotechnology and Hygiene, 68200 Orestiada, Greece
e
Bucharest Universitary Hospital, Department of Orthopedics and Traumatology, 169 Splaiul Independentei, 050098 Bucharest, Romania
a r t i c l e i n f o
Article history:
Received 16 March 2013
Received in revised form
24 April 2013
Accepted 26 April 2013
Available online 7 May 2013
Keywords:
Magnetite nanoparticles
Antibiotics
Enterococcus faecalis
Disk diffusion
Minimal inhibitory concentration
Biolms
a b s t r a c t
The objective of this study was to investigate the potential of magnetic nanoparticles to potentiate, but
also to accomplish a sustained and controlled drug release and subsequently improve the efcacy of
antibiotics against Enterococcus faecalis, one of the most resistant opportunistic pathogens, that poses a
threat to chronically infected or immunocompromised patients and is difcult to eradicate from medical
devices. To our knowledge, this is the rst study trying to investigate the ability of magnetite nano-
particles to improve the anti-bacterial activity of the current antibiotics against planktonic and biolm
growing E. faecalis. Our results are suggesting that the magnetite nanoparticles may be considered an
effective aminoglycoside antibiotics carrier, but a complete understanding of the way in which they
selectively interact with different antibiotics and with the bacterial cell is needed, in order to obtain
improved strategies for elimination of E. faecalis biolms on biomedical devices or human tissues.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Although enterococci are natural inhabitants of the oral cavity,
normal intestinal microora, and female genital tract of both hu-
man and animals, they could infect the urinary tract, bloodstream,
intra-abdominal and pelvic regions, surgical sites and central ner-
vous system [1]. Enterococcus faecalis is responsible for 80e90% of
human enterococcal infections [2], being one of the most important
nosocomial opportunistic pathogens. Hospital-acquired infections
caused by enterococci have increased dramatically since the 1970s,
accounting for approximately 7%e10% of all bloodstream infections
in Europe and respectively in US intensive care units [3,4]. This
Gram positive bacterium often infects root canals during
endodontic dental treatments of patients with persistent apical
periodontitis [5]. Among antibiotic selection pressure, other risk
factors should be considered for the emergence of enterococci in
hospitals, one of them being their ability to form biolms on
medical devices [1]. This putative virulence factor is responsible for
the increasing proportion of enterococcal bacteraemias associated
with central venous catheters [1,6]. Clinical enterococcal strains
have been shown to adhere to biomedical polymers in vitro, but the
intimate mechanisms and the clinical relevance of this nding have
not been determined yet [1]. These bacteria are considered at
present the most antibiotic- and heat-resistant pathogens [7],
which determined the intensication of studies for nding out new
alternatives to antibiotic treatment.
In the recent years nanomaterials gained much attention in
medicine, particularly in the ght to bacteria resistant to antibiotics
by acting as drug delivery devices [8]. Metallic nanoparticles may
be promising agents for antibacterial applications, due to their
* Corresponding author. Tel.: 40 765349326.
E-mail address: grumezescu@yahoo.com (A.M. Grumezescu).
Contents lists available at SciVerse ScienceDirect
Anaerobe
j ournal homepage: www. el sevi er. com/ l ocat e/ anaerobe
1075-9964/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.anaerobe.2013.04.013
Anaerobe 22 (2013) 14e19
selective mechanism of toxicity, subsequent to binding to bacterial
cell walls and causing membrane disruption through direct in-
teractions or through free radical production [9]. Moreover,
phagocytic cells are able to internalize nanoparticles, and to
degrade them by lysozomal fusion, reducing host tissues toxicity
and free radical damage [10].
Previous studies have demonstrated that ZnO, CuO, and Fe
2
O
3
nanoparticles possess antimicrobial activity against Gram-positive
and Gram-negative bacteria [9,11,12].
The metallic nanoparticles have been studied for the design of
nanotextured surfaces that can reduce microbial adhesion, prolif-
eration, and biolm growth through emergent antimicrobial
properties [4,13e15]. Beside the improvement of tissue-forming
cell functions, that nanofeatured medical device surfaces could
simultaneously prevent bacterial colonization by enhancing surface
energy, increasing selective protein adsorption and promoting
protein bioactivity [4,16,17]. This idea has been already used in
industry, as antimicrobial nanoparticles are incorporated into
numerous paints and other materials that affect our daily lives [4].
Despite the efcacy of nanocoatings to prevent biolm forma-
tion on catheters, there is an important limitation, i.e. the ability of
the material to adsorb always the same concentration of the drug
and also the ability to control their release, which in most cases
results in a non-controlled elution of the drug in the rst hours
subsequent to the insertion [18e20]. Therefore, the objective of this
study was to investigate the potential of magnetic nanoparticles to
incorporate antibiotics in an active form, to potentiate, but also to
accomplish a sustained and controlled drug release and subse-
quently to improve the efcacy of different antibiotics against
planktonic and biolm growing E. faecalis cells.
2. Materials and methods
2.1. Materials
All chemicals were used as received. FeCl
3
, FeSO
4
$7H
2
O, NH
4
OH
(25%), and CH
3
OH were purchased from SigmaeAldrich Chem-
ieGmbh (Munich, Germany).
2.2. Nanostructure fabrication
Magnetic iron oxide nanoparticles were prepared by wet
chemical precipitation fromaqueous iron salt solutions by means of
alkaline media, like NH
3
[21e23]. Briey, 100 mg of each of the
following antibiotics (ATB): vancomycin (VA), penicillin (P), and
streptomycin (S), and 8 mL of NH
4
OH (25%) were added in 200 mL
deionized water under vigorous stirring. Then, 1 g of FeCl
3
and 1.6 g
of FeSO
4
$7H
2
O were dissolved in 200 mL of deionized water and
Fe
3
/Fe
2
solution was dropped into the basic solution of ATB. After
the precipitation of magnetite-antibiotics crystals (Fe
3
O
4
@ATB), it
was repeatedly washed with methanol, separated with a strong
NdFeB permanent magnet. Subsequently, the Fe
3
O
4
@ATB was
added into the 100 mL solution of acetic acid 0.1 N and stirred for
10 min. After this, the Fe
3
O
4
@ATB was separated with a strong
NdFeB permanent magnet, repeatedly washed with deionized
water and nally solubilized in ultrapure water.
2.3. Characterization of the obtained nanostructures
2.3.1. XRD
X-ray diffraction analysis (XRD) was performed on a Shimadzu
XRD 6000 diffractometer at room temperature. XRD is a rapid
analytical technique primarily used for phase identication of a
crystalline material and measurement of sample purity [24]. In all
the cases, Cu Ka radiation from a Cu X-ray tube (run at 15 mA and
30 kV) was used. The samples were scanned in the Bragg angle 2q
range of 10e80

.
2.3.2. TGA
The thermogravimetric (TG) analysis of the Fe
3
O
4
@ATB and
Fe
3
O
4
was assessed with a Shimadzu DTG-TA-50H instrument.
TG analysis records the weight changes in a sample with respect to
the temperature [25]. In this purpose, samples were screened to
200 mesh prior to analysis, were placed in alumina crucible, and
heated with 10 K min
1
from room temperature to 800

C, under
the ow of 20 mL min
1
dried synthetic air (80% N
2
and 20% O
2
).
2.3.3. FT-IR
A Nicolet 6700 FT-IR spectrometer (Thermo Nicolet, Madison,
WI) connected to software of the OMNIC operating system (Version
8.0 Thermo Nicolet) was used to obtain FT-IR spectra of hybrid ma-
terials. The samples were placed in contact with attenuated total
reectance(ATR) ona multibounceplate of ZnSecrystal at controlled
ambient temperature (25

C). FT-IR spectra were collected in the
frequency range of 4000e650 cm
1
by co-adding 32 scans and at a
resolution of 4 cm
1
with strong apodization. The spectra were
recorded as absorbance values at each data point in triplicate.
2.3.4. Particle size analysis
Particles size were determined by using dynamic light scattering
technique (Zetasizer Nano ZS, Malvern Instruments Ltd., U.K.), at a
scattering angle of 90

and 25

C. The particle size analysis data was

evaluated using intensity distribution. The average diameters
(based on StokeseEinstein equation) were calculated from the
three individual measurements.
2.4. Qualitative and quantitative assessment of the inuence of
Fe
3
O
4
@ATB on planktonic cells growth
The study of antimicrobial activity of Fe
3
O
4
@ATB against
E. faecalis ATCC 29212 reference strain was performed by a qualita-
tive method for antimicrobial susceptibility testing based on disk
diffusion following CLSI 2012 recommendations. In brief, the inoc-
ulum represented by a bacterial suspension from a 16e18 h culture
developed on solid medium, and adjusted according to McFarland
0.5 standard was seeded on a Muller-Hinton Agar (MHA) medium
plate. After inoculationplates were left standing for 10 min to let the
culture get absorbed. Afterwards, the antibiotic disks, and antibiotic
disks plus 10 mL of Fe
3
O
4
@ATB, respectively were placed at corre-
sponding distance on MHA plates, and plates were incubated la
35 2

C. The results reading were performed by comparatively
measuring of inhibition zone diameter generated by different anti-
biotics, and antibiotic disks plus 10 mL of Fe
3
O
4
@ATB, respectively,
according with their dimensions from CLSI, 2012. For these experi-
ments there were used penicillin, vancomycin and streptomycin.
The quantitative method for the minimal inhibitory concentra-
tion (MIC) assay of each naonosystem (Fe
3
O
4
@ATB) consisted of
two-fold microdilutions of nanoparticules stock solutions prepared
in sterile saline were performed in liquid culture medium (nutrient
broth) distributed in 96 multi-well plates. The concentration of
Table 1
ATB concentration in the initial solution of Fe
3
O
4
@ATB and ATB concentration from
Fe
3
O
4
@ATB determined by TG analysis.
Fe
3
O
4
@ATB The ATB concentration in
the initial solution of
Fe
3
O
4
@ATB (mg/mL)
ATB concentration (%)
from Fe
3
O
4
@ATB determined
by TGA at 600

C
Penicillin (P) 4.5 1.9
Streptomycin (S) 7.8 2.4
Vancomycin (VA) 453 5.6
M.C. Chiriuc et al. / Anaerobe 22 (2013) 14e19 15
each antibiotic adsorbed on magnetite surface is presented in
Table 1. Each well was inoculated with 5 mL of microbial suspen-
sions corresponding to a 0.5 McFarland density. Sterility, negative
control wells (nutrient broth) and microbial growth, positive con-
trols (inoculated nutrient broth) were used. The plates were incu-
bated for 24 h at 37

C, and the inuence of nanoparticles on the
planktonic cells growth in liquid medium was appreciated by
measuring the A 600 nm of the obtained cultures. The MIC was
considered as the last dilution of the tested compound which
inhibited the microbial growth.
2.5. Assessment of the inuence of Fe
3
O
4
@ATB on E. faecalis
biolms
After performing the MIC assay, the 96 well plates were
emptied, washed 3 times with phosphate buffered saline, xed
with cold methanol and stained with violet crystal solution 1% for
30 min. The biolm formed onto the plastic wells was resuspended
in 30% acetic acid and the intensity of the stained suspension was
assayed by measuring the absorbance at 490 nm [26]. The results
interpretation was performed by comparing the obtained value of
absorbance at 490 nm for strains treated with nanoparticles with
those obtained for control strains (bacteria grown in standard/
normal conditions).
3. Results and discussion
Magnetic nanoparticles consisting of magnetite (Fe
3
O
4
) possess
unique characteristics that make them promising as carriers in
biomedical applications. In terms of diagnosis they can be used
both for in vitro and in vivo applications, such as: immobilization
and detection of biomolecules [27e29], cell separation [30], puri-
cation [31] and gene transfer [32], and could serve as contrast
agents in magnetic resonance imaging [33e35]. They can also be
applied for drug delivery in target therapy [36e41], inhibition of
microbial biolm development [42,43] and for hyperthermia
treatment [44], due to the heat they produce in an alternating
magnetic eld [45].
The size distribution for Fe
3
O
4
@ATB samples are plotted in Fig. 1.
The size of the fabricated nanostructures based magnetite and
antibiotics ranged between 30 and 70 nm.
The crystalline properties of the Fe
3
O
4
@ATB and Fe
3
O
4
was
investigated by XRD (Fig. 2). The Fe
3
O
4
@ATB samples have the
characteristics of bulk magnetite crystallite phase (Fe
3
O
4
), and the
broad peaks suggest the nanocrystallite nature of magnetite par-
ticles. The patterns of Fe
3
O
4
@ATB and Fe
3
O
4
have characteristic
peaks at 30.5

(220), 35.9

(311), 43.5

(400), 57.3

(511) and
63.1

(440), well matching the standard pattern of Fe

3
O
4
.
The amount of antibiotic fromFe
3
O
4
@ATB was established by TG
analysis (Fig. 3).
The weight loss of the ATB amount from Fe
3
O
4
@ATB varies
between 1 and 8% (Table 1).
FT-IR analysis was used to show that the extreme conditions,
including acid and base treatment steps, encountered in the
fabrication process do not affect the integrity of the antibiotic
functional groups. In all samples, FT-IR recorded spectra show ab-
sorption bands arising at w530 cm
1
fromcharacteristic vibrations
of magnetite (FeeO) [22]. There are many well-dened peaks in the
Fig. 1. Size distribution of Fe
3
O
4
@ATB evaluated by DLS technique: (a) Fe
3
O
4
@P (63 nm); (b) Fe
3
O
4
@VA (58 nm); (c) Fe
3
O
4
@S (78 nm).
Fig. 2. XRD pattern of Fe
3
O
4
and Fe
3
O
4
@ATB e samples have the characteristics of bulk
magnetite crystallite phase (Fe
3
O
4
).
Fig. 3. TG analysis of Fe
3
O
4
and Fe
3
O
4
@ATB (the TGA records allow to establish
the amount of the ATB entrapped on the Fe
3
O
4
@S, Fe
3
O
4
@VA and Fe
3
O
4
@P, being
proportional with the weight changes at high temperature).
M.C. Chiriuc et al. / Anaerobe 22 (2013) 14e19 16
ngerprint region between 1800 and 1000 cm
1
. The nger-
print region of the spectra of V, S, P and and Fe
3
O
4
@V, Fe
3
O
4
@S,
Fe
3
O
4
@P regions shows no differences after synthesis.
V and Fe
3
O
4
@V show the absorption bands at 3380 cm
1
(OeH
bands), 1665 and 1710 cm
1
(C]O band), 1229 cm
1
(COeC band)
and several bands detected at 1128 cm
1
and 1063 cm
1
which
testify the presence of CeN band.
S and Fe
3
O
4
@S show the absorption bands at 33352 cm
1
(OeH
stretch overlapped with NeH stretch) and for CeOeC bonds at
1091 cm
1
; 2917 and 2859 cm
1
(CeH stretch); 1459e1377 cm
1
(asymmetric CeH bending of CH
2
group).
P and Fe
3
O
4
@P show the absorption bands at 2919 and
2858 cm
1
(CeH stretch), 1775 cm
1
(b-lactam C]O stretching),
1670 cm
1
(Amide I), 1611 cm
1
(asymetric COO

stretching) and
1395 cm
1
(symetric COO

stretching).
Antibiotic multi-resistance and strong biolm production abil-
ities, together with a high phenotypic expression of gelatinase are
an important equipment of E. faecalis to colonize prosthesis tissues
and to spread out as causative agents of medical devices associated
infections [46]. The alarming increase of E. faecalis biolm associ-
ated infections urged the search for the design and development of
new and effective strategies to ght against this important
pathogen.
In this study we have investigated the inuence of magnetic
nanoparticles functionalized with antibiotics on the planktonic
growth and biolm formation by E. faecalis.
Previous studies have shown the ability of E. faecalis to form
biolms on biomaterials, and this feature varied on different sub-
strata [47].
The results of the qualitative assay of the antimicrobial activity
of the obtained nanosystems showed that the magnetic nano-
particles clearly slightly improved the activity of the tested anti-
biotics, as revealed by the increase of the growth zone inhibition
diameter for all tested antibiotics, as compared with the antibiotic
disks charged with the same antibiotic concentration (Fig. 4).
In the quantitative assay, the nanoparticles improved the anti-
microbial activity of streptomycin, as revealed by the signicant
decrease of the MIC value of this antibiotic (Fig. 5).
The magnetic nanoparticles didnot improve the efcacyof any of
the testedantibiotics toprevent the E. faecalis biolms development.
Fig. 6 present the degree of E. faecalis biolmdevelopment in the
presence of the rst two higher concentrations of the tested
nanostructures, as compared with the antibiotic control and
magnetite control.
It is to be noticed that magnetite itself exhibited a superior anti-
biolm activity, as compared with the antibiotic loaded into the
Fig. 4. The growth inhibition diameter values of Fe
3
O
4
@ATB versus control antibiotics
on E. faecalis ATCC 29212.
Fig. 5. The MIC values of Fe
3
O
4
@ATB versus MIC values of control antibiotics on
E. faecalis ATCC 29212.
Fig. 6. The effect of Fe
3
O
4
@S (2925 mg/mL), Fe
3
O
4
@VA (16,988 mg/mL) and Fe
3
O
4
@P (1688 mg/mL) on E. faecalis biolm development.
M.C. Chiriuc et al. / Anaerobe 22 (2013) 14e19 17
nanocarrier, being closer or even better, in case of streptomycin,
than the antibiotic solution, demonstrating the utility of these
metallic nanoparticles for the design on anti-biolm surfaces.
Taken together, the obtained results are clearly demonstrating
that magnetic oxide nanoparticles are improving the antimicrobial
activity of streptomycin, both against planktonic, as well as
adherent E. faecalis cells. The fact that the Fe
3
O
4
@S was much more
efcient than the antibiotic alone against planktonic E. faecalis cells,
is demonstrating that the potentiating effect of the magnetic
nanoparticles consists in binding to bacterial cell walls causing
membrane disruption [11] and favouring the aminoglycoside drug
streptomycin activity.
The fact that in case of the adherent bacterial cells, the hybrid
antimicrobial nanosystemFe
3
O
4
@S efciency was inferior to that of
Fe
3
O
4
itself or antibiotic solution is demonstrating that magnetite
nanoparticles could carry the antibiotic, but do not release it in an
active form, that could diffuse and reach the biolm cells.
This could probably be due to the binding of antibiotics func-
tional groups to the surface of the nanoparticles. The rest of the
molecule is solvated in dispersion medium or in a uid. The pro-
cess, known to be entropic or steric, refers to the inhibition of
particles aggregation by an entropic force, which appears when the
particles are close to each other [48].
On the other side, the obtained results could be explained by the
nding that enteroccocal biolms reach a few tens of micrometres
thick, offering additional degrees of resistance to treatment, due to
their multilayer structure, cemented by the extracellular polymeric
substance [49,50].
4. Conclusion
To our knowledge, this is the rst study trying to investigate the
ability of magnetic nanoparticles to improve the anti-bacterial
activity of the current antibiotics against E. faecalis, one of the
most resistant opportunistic pathogens, both in planktonic and
adherent state. Our results are suggesting that the magnetic
nanoparticles may be considered an effective carrier for amino-
glycoside antibiotics, but a complete understanding of the way in
which they selectively interact with different antibiotics and the
bacterial cell is needed in order to obtain improved strategies for
elimination of E. faecalis biolms on biomedical devices or human
tissues.
Acknowledgements
The results presented in this work were supported by the stra-
tegic grant POSDRU/89/1.5/S/58852, Project Postdoctoral program
for training scientic researchers conanced by the European
Social Fund within the Sectorial Operational Program Human Re-
sources Development 2007e2013 and by the PN-II-PT-PCCA-2011-
3.2-0284: Novel nanostructured prosthetic tubular devices with
antibacterial and antibiolm properties induced by physicochem-
ical and morphological changes funded by the National University
Research Council in Romania.
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