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Antibiofilm activity of nano sized CuO

Article · November 2011

DOI: 10.1109/ICONSET.2011.6168037

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 Antibiofilm Activity ofNano sized CuO
P. Sriyutha Murthy, V.P. Venugopalan
Biofouling & Biofilm Processes Section, Water & Steam
Chemistry Division,
BARC Facilities, IGCAR Campus,
Kalpakkam 603 102, INDIA,
Abstract- Antibiofilm properties of bulk and nano-sized copper
oxide (CuO) were investigated using four species of bacteria viz:
Staphylococcus aureus, E. coli, Pseudomonas aeuriginosa (PA01)
and Pseudomonas putida. The effect of both commercial and
synthesized nanoparticles on biofilm formation was investigated
using different concentrations 0.02, 0.12, 0.24, 0.5, 1.0, 1.2, 1.4 &
1.8 I1g / mL). Both bulk (91% reduction) and nano-sized (93%
reduction) particles significantly reduced biofilm formation in all
the four species of bacteria. Concentration and size/shape of
nanoparticles seemed to influence biofilm inhibition. Increase in
NP concentration from 0.02 to 1.8 I1g/mL resulted in a marginal
increase in inhibition. While a similar increase in concentration
of bulk particles, resulted in 18% decrease in biofilm inhibition.
Differences in biofilm inhibition between bulk and nano-sized
particles were marginal at low concentrations (0.02, 0.12 & 0.24
I1g/mL), whereas prominent differences were observed from
concentration of 0.5 I1g/mL onwards for the bulk particles. The
decrease in inhibition efficiency of bulk nanoparticles
increase in concentration may be attributed to the differences in
size, shape, aggregation and settling behaviour. In general,
higher efficiency was observed with S. aureus, E. coli and P.
putida biofilms, whereas P. aeuriginosa cells showed
resistance in the presence of CuO nanoparticles. Results of the
present study indicated that size and shape of the nanoparticles,
seem to have an influence on the effective concentration, required
for inhibition of biofilms.
Keywords-component; Copper oxide nanoparticle, S. aureus,
E. coli, P. aeuriginosa, P. putida, Hydrothermal synthesis, Biofilm
inhibition.
I. INTRODUCTION
Bacteria in nature exist as free living I floating (planktonic)
and sessile (biofilms) forms [1]. Biofilms are found in almost
every environment either natural or artificial [2]. Biofilms offer
a protected mode of growth and allow bacteria to withstand
harsh environmental conditions [3]. It has been estimated that
about 65% of infections treated in hospitals are due to bacterial
biofilms [4]. Switch from planktonic to biofilm mode of
growth is accompanied by complex phenotypic changes in
bacteria [5] and changes in gene transcriptionJ6]. One of the
earliest finding related to differing characterestics of planktonic
and biofilm cells was the increased resistance of cells in
biofilms to antimicrobial agents like biocides and antibiotics
[7]. However it is still not clearly understood why and how
bacteria, growing within biofilms develop resistance [8]. A
relatively early concept was that penetration of antimicrobials
into biofilms was hampered by the presence of an
exopolymeric matrix in the biofilms [9] or that altered
978-1-4673-0074-2/11/$26.00 @2011 IEEE
Das, D. Arunya ,So Dhara, R. Pandiyan, A.K. Tyagi,
Surface & Nanoscience Division Material Science Division,
Indira Gandhi Center for Atomic Research,
Kalpakkam 603 102, INDIA,
dasa@igcar.gov.in
microenvironment with different levels of metabolic activity
reduced biofilm susceptibility [10]. A relatively recent
explanation is the existence of persister cells in biofilms, which
offer increased resistance [8]. Apart from this, quorum sensing
systems [11], biosorption [12] and gene expression [13] have
been attributed to antimicrobial resistance of biofilm bacteria.
In this context, the potential of metal oxide nanoparticles to
control formation of biofilms is currently under scrutiny.
Copper oxide nanoparticles (NP) have interesting physical
properties with multifunctional applications in the area of
catalysis, batteries, magnetic storage media, solar energy and
superconductors [14]. Bacteria have been shown to develop
resistance to various heavy metals, including ionic copper [15].
Resistance mechanisms to heavy metals include sequestration
of metals into complexes, reduction of a metal to a less toxic
species and direct efflux of metal out of the cell [16]. However
with
little is known about the effectiveness of CuO nanoparticles on
biofilms and also the changes in biological activity as a
function of the size and shape of the nanoparticles.
more Hence, in the present study the susceptibility of biofilms to
CuO nanoparticles was investigated at very low concentrations.
Effects of two different sizes and shapes of CuO NP were also
investigated.
II. MATERIALS AND METHODS
A. Materials
LB broth (Himedia Laboratories) was used to grow and
maintain bacterial cultures. Copper nitrate, liquid ammonia was
purchased from Mis. E. Merck. Bulk CuO particles were
purchased from Mis. Sigma Aldrich. Microtitre plates were
purchased from Mis. Nunc.
B. Synthesis a/Copper OxideNanoparticles by
hydrothermal method.
Synthesis of CuO is carried out following hydrothermal
process using Cu (N02) as source materials. A typical
concentration of Cu (N02) is O.OIM and pH of the system is
maintained at 8.5 by using 1 M NH40H solution. The mixture
is kept in a sealed teflon coated steel chamber at a fixed
autoclave temperature of 100°e. The reaction is complete in 6
hours with the formation of a black material. This material is
centrifuged and repeatedly cleaned with distilled water and
ethanol to remove residues from the reactants
580
C. Characterization ofNanoparticles at 35.5°, and 38.7° which correspond to (hkl) values of (002),
Morphological and structural studies were perfonned and (111). Other less intense peaks are indexed and also match
using field-emission scanning electron microscope (FESEM; well to bulk copper oxide. Bulk being of large crystalline
Zeiss-SUPRA 40) and micro-Raman spectroscopy (inVia materials shows sharp peak in comparison to hydrothermally
Reinshaw) using 514.5nm excitation of an Ar+ laser prepared nano sized CuO. The average size of the particles
calculated using the Debye-Scherrer's formula for nano CuO is
20 nm.
D. Prepartion and enumeration of test organisms
Staphylococcus aureus, Escherichia coli, Pseudomonas
aeuriginosa and Pseudomonas putida were used as the test ···· llulkCuO
---- CuO IlnnOSlruClure
organisms because these species are known to form biofilms in
medical and industrial settings. Pure cultures were grown in LB
medium and incubated overnight at 37°C with shaking. From
this, one ml of bacteria was sub-cultured in 15 ml of LB broth
to reach a mid log phase observed by ODs7o value of 0.12 to
7
0.16 (with a cell density of 1.5 x 10 CFU mL-)).

E. Preparation of experimental suspensions and Microtitre

Assays
The working suspension was prepared as follows.
30 40 50 60 70 80
Known quantity of nanoparticles was dissolved in sterile 20 (degree)

autoclaved ultra pure water and sonicated to ensure that the

particles did not agglomerate. The aliquot was serially diluted
to get double the concentrations of 0.02, 0.l2, 0.24, 0.5, 1.0,
1.2, 1.4 & 1.8 Ilg / mL in 100 III of water. To this aliquot 80111
of sterile LB broth and 20 III of the above mentioned mid-log
phase bacterial suspension were added to make a final volume
Figure I. Powder XRD pattern of (a) CuO NP and (b)Bulk CuO
Fig. 2 is the FESEM images presenting morphology of the
CuO. Fig. 2a displays large particles of CuO and the average
particle size is 100 to 200 nm. This particle shows sharp peak
�

of 200 Ill. The suspensions were then transferred to microtitre
plates and incubated in a rocking platform for 24 hours. For
estimating planktonic growth, ODs7o was recorded after 1, 2, 3,
4, 19, 21 and 24 h incubation. OD value after 24 hours of
incubation was taken for comparison.
in XRD pattern similar to the fully grown crystallites
resembling 'bulk' nature of the CuO powder. Completely
different morphology is obtained for hydrothermally grown
CuO with an average size 20 - 30 nm. These results closely
support the broad XRD features for nano materials.

F. Biojilm formation Assay and Biojilm Quantification

Biofilm formation assay was performed in microtitre plates
[17]. After 24 h of incubation of multiwell plates, with and
without nanoparticles, the culture medium was removed from
the wells. The wells were rinsed five times with sterile distilled
water to remove loosely attached bacteria. Plates were air dried
for 45 minutes and each well was stained with 150 III of 1%
crystal violet solution in water for 45 min. After staining the
plates were washed with sterile distilled water five times.
Biofilms were visible as purple rings formed on the side of
each well. The quantitative analysis of biofilm production was
done by adding 200 III of 95% ethanol to destain the wells. One
hundred microliters from each well was transferred to a new
microtitre plate and the level (OD) of the crystal violet present
in the destaining solution was measured at 570 nm. Each
concentration of nanoparticles was tested in triplicate. Parallel
controls with plain LB medium without bacteria were also
carried out.

III. RESULTS

A. Copper oxide nanoparticle characterization

Fig. 1 shows the XRD patterns of CuO synthesized by

FIGURE 2. FESEM images of a) Copper oxide Bulk b) Copper
hydrothennal method [18]. This pattern reveals intense peaks
oxide nanoparticles.

581
 Figure. 3 shows the Raman spectra of bulk and
hydrothennally synthesized CuO using Cu (N03)2 as precursor.
The observed peaks are assigned to Ag, and Bg of CuO [Xl The Figure 5. Multiple graphs showing biofilm quantity in controls and at
values of Ag, and Bg of bulk CuO are 296, and 344 cm-1, different nanoparticle concentration a) S. aureus,b) E. coli,c) P. aeurigionsa
respectively. There is an observable shift to lower wave and d) P. putida.
nwnber with a broadening effect that emanates from the nano
materials of CuO grown hydrothermally.
0.5 0.5
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SA EC PA01 PP iii SA EC PA01 PP
Bacterial Strains Bacterial Strains
sized CuO. Treatments using nano-sized CuO showed 90% _Bulk _Nano sized _Bulk _Nano sized
inhibition of biofilm formation_at the lowest (0.02 f.1g1mL)
concentration which increased to 92% at the highest (l.8
f.1g/mL) concentration tested for S. aureus (Fig. 4a). In
comparison an increase in percentage reduction from 89 to
93% was observed between the low and high concentrations Figure 5. Multiple graphs showing biofilm quantity between bulk and
nanosized particles at different nanoparticle concentration a) 0.02,
for E. coli (Fig. 4b). Both Pseudomonas aeuriginosa (Fig. 4c)
b) 0.12, c) 0.24, d) 0.5, e)1.0, t)1.2, g)1.4 and h)1.8 J.!g!mL.
and P. putida (Fig. 4d) showed an increase in percentage

gj �
reduction from 89 to 92% at low and high concentrations
In comparison to nano-sized CuO, inhibition of biofilms
respectively. decreased with increase in concentration of bulk CuO particles.
D'35 2.5�
Percentage inhibition of biofilm formation reduced from 88 to
E D.36 2.6�
�E 0.30 78% with increase in concentration of bulk particles (Fig. 5) in
e
!; 0.30 (a) 2.0 �tD (b)
2.0 :;;
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tD 0
S. aureus. Similarly inhibition of biofilms reduced from 90 to
tD 0

� 0.20 8 0.20 1.5 �

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� 0.15 1.0 � �� 0.15 1� � 80% in E. coli, 90-72% in P. aeuriginosa (90-72%) and 91 to
'j 0.10 ctI � 0.10 m
0.5 � 0.05 0.5
78% in P. putida respectively.

� �
0.05
0.0 'Ei
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iii ... ....'} ...." ••:" iii ... ...." ...'" ,," :I
��'"<;:0":-'"�..,. <::.�
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NP Concentration (....g/mLI NP Concentration (l-IglmL) It was observed that a clear distincition in biofilm
�Untreated __ Bulk _ Nano sized
inhibiton between bulk and nano-sized particles was observed
EI: D'5D
0.45
2.5� E D'4D 2.5� above 0.5 f.1g1mL concentration.
0.40 (c) C>
2.0 � � 0.35 (d) T 2.0 e:g
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a.. 0.25 § 0.20 §
1.0 � � 0.15 1� IV. DISCUSSION
i �:�� 0.5 � 0.10 5
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Size and shape of metallic nanoparticles play an
� 0.00 � 0.00
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()�(;:"'.()-t ()t;. ......" ..." ....'"
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NP Concentration (f,lg/mL) NP Concentration (f,lglmL)

C=:IUntreated __ Bulk ____ Nanosized �Untreated __ Sulk ____ Nanosized

582
characterization of novel nano scaled materials with new
physiochemical properties is of great interest in the formulation
of bactericidal materials. Literature on antibiofilm properties of
CuO nanoparticles is very scarce and hence direct comparison
of inhibition is difficult. Studies by Heinlann [19] on
planktonic cells demonstrated that nano CuO exhibited a 48
fold increase in toxicity on bacteria compared to bulk
counterpart.
In the present study about 90% inhibition of biofilm
formation was observed for S. aureus at the lowest
concentration of 0.02 Ilg/mL which were several folds higher
compared to inhibition (55% inhibition) of biofilms by ZnO
polymer composites [20].
Copper is an important co-factor for many enzymes,
however, high levels of copper are toxic. Therefore, bacteria
must strike a balance between sufficient availability of copper
as co-factor and to limit intracellular levels to prevent toxicity
(Baker et aI., 2010). Results of the present study show that
nano and bulk sized CuO particles exhibit higher toxicity on
biofilms compared to low toxicity of 35% observed for copper
ions on planktonic cells [15]. Concentration of copper ions
required for complete inhibition of growth was found to be 250
ppm, whereas in the present study 0.02 ppb was able to
suppress 90% of biofilm growth.
V. CONCLUSIONS
In summary, results of our study showed that both nano and
bulk particles of CuO were very effective in preventing biofilm
formation in four different species of bacteria at ppb levels.
Changes in size and shape of particles seem to have an effect
on microbial toxicity in a concentration dependent manner.
(
ACKNOWLEDGMENT HEADING 5)
We thank Dr. Kalavathi, Scientist of MSG, IGCAR for
helping in XRD analysis.
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