Materials Letters 97 (2013) 67–70

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Materials Letters
journal homepage: www.elsevier.com/locate/matlet

Biocidal effect of copper and zinc oxide nanoparticles on human oral

microbiome and biofilm formation
Shams Tabrez Khan a, Maqusood Ahamed b, Abdulaziz Al-Khedhairy a, Javed Musarrat c,n
a
Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
b
King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia
c
Department of Agricultural Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh 202002, India

a r t i c l e i n f o abstract

Article history: Oral cavity bacteria, specifically those involved in biofilm formation and dental ailments, are major
Received 1 December 2012 health concerns. Thus, the ability of CuO (40 nm) and ZnO (35 nm) nanoparticles (NPs) synthesized in
Accepted 19 January 2013 this study was assessed in vitro for inhibition of culturable oral bacteria and their biofilm formation.
Available online 29 January 2013
Both the NPs at a concentration of 50 mg/ml significantly inhibited the growth of total oral bacteria,
Keywords: extracellular polysaccharide (EPS) production and biofilm formation on the glass, acrylic dentures and
Biofilm cultured human epithelial cells, as models. The results elucidated strong bactericidal action of CuO- and
Nanoparticles ZnO-NPs, which significantly reduces the oral bacterial load and eventually inhibits the growth of oral
Oral bacteria biofilm colonizers.
EPS production
& 2013 Elsevier B.V. All rights reserved.
Dental hygiene

1. Introduction necessitate the investigations on novel alternatives as front-line

Nanotherapeutics has lately evoked tremendous interest in
controlling the development of oral biofilms by the use of biocidal
nanoparticles (NPs). Treatment with conventional antibiotics is
considered inadequate, and often leads to chronic oral infections,
which compel tooth extractions or implant removal, involving
costly restoration or regenerative procedures [1]. Human oral
microbiome consists of a complex polymicrobial community,
which dwells in specific niches within the oral cavity and forms
biofilms (plaques) on teeth, prostheses, and mucosal surfaces.
Dental plaques represent a dynamic oral biofilm ecosystem
comprising of more than 800 bacterial species [2–5]. Initial
colonizers include Streptococcus oralis, Streptococcus sanguinis,
and Streptococcus mitis. The co-aggregating partners comprise of
Eikenella corrodens, Veillonella atypical and Prevotella nucleatum,
and the late colonizers are predominantly Aggregatibacter actino-
mycetemcomitans, Prevotella intermedia, Treponema denticola and
Porphyromonas gingivalis [6]. Oral biofilms are regarded as aetio-
logical agents associated with dental caries and periodontitis
[7,8]. An estimated 65–80% of all infections are contemplated to
be linked to biofilm formation, as a causal factor for failure of
antimicrobial treatments, and thus presents a serious challenge
[9–11]. Lesser susceptibility of biofilms to antimicrobial agents,
and an increasing trend of multiple drug resistance in bacteria
n
Corresponding author. Tel.: þ966 1 4675768.
E-mail address: musarratj1@yahoo.com (J. Musarrat).
antimicrobial agents. In this regard, the metal NPs offer the
possibility of controlling oral biofilm formation, as the bacteria
are less likely to acquire resistance against metal NPs than other
conventional antibiotics [12]. Several NPs have been tested earlier
for their effects on pure cultures of Streptococcus mutans [13].
A recent study has suggested significant inhibition of biofilm
formation of S. oralis (ATCC 35037) and two clinical isolates from
periodontitis patients by the soda-lime-glass-nAg [14]. Never-
theless, the effect of metal NPs on oral cavity microbiome is still
under exploration. Therefore, in this study, we report the effect of
ZnO- and CuO-NPs on oral cavity bacteria and demonstrated
the inhibition of biofilm formation on different matrices including
glass, polystyrene plates, acrylic dentures and human epithelial
cells.
2. Materials and methods
Synthesis and characterization of CuO- and ZnO-NPs: The CuO-
and ZnO-NPs were synthesized by the solvo-thermal method [15]
and sol–gel method [16], respectively. The NPs were character-
ized by X-ray diffraction (XRD) and transmission electron micro-
scopy (TEM). The XRD patterns of CuO- and ZnO-NPs powders
were acquired by the use of a PANalytical X0 Pert X-ray diffract-
ometer (Spectris plc, England) equipped with an Ni filter using Cu
Ka (l ¼ 1.54056 Å) radiations, as an X-ray source. For TEM
analysis, the dried powders of NPs were suspended in deionized
water and sonicated for 15 min at 40 W. A drop of diluted

0167-577X/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.matlet.2013.01.085
68 S. Tabrez Khan et al. / Materials Letters 97 (2013) 67–70

Fig. 1. TEM and XRD analysis of NPs. Panels A and B: representative TEM images of CuO- and ZnO-NPs, respectively. Panels C and D: XRD profiles of CuO- and ZnO-NPs, respectively.

Fig. 2. Effect of CuO- and ZnO-NPs on bacterial growth, biofilm formation and EPS production. (A) Total CFU counts on NA and MRS media, (B) binding of total oral bacteria
to human epithelial cells, (C) and (D) EPS production. Statistical analysis performed by one-way analysis of variance (ANOVA) using Dunnett’s test (Sigma Plot 11.0, USA).
n
po 0.005.
 S. Tabrez Khan et al. / Materials Letters 97 (2013) 67–70
suspension was placed onto a carbon-coated copper grid, air-
dried and observed with TEM at 40,000  (JEM-2100F, JEOL). The
particle size distribution and Zeta (z) potential were determined by
dynamic light scattering (DLS) using ZetaSizer-HT (Malvern, UK).
Effect of ZnO- and CuO-NPs on total oral bacterial population:
Slurry from the teeth crown surface of a healthy male was
collected using a sterile toothpick and suspended in 1 ml of sterile
1  PBS. The number of bacterial cells in suspension was adjusted
to 5  108/ml in all the experiments. The cells were treated with
ZnO- and CuO-NPs at concentrations of 10, 50, and 100 mg/ml in
NA and MRS broths at 37 1C for 16 h. Untreated and treated cells
were spread on NA and MRS agar plates, and incubated at 37 1C
for 3–5 days. The colony forming units (CFU) were determined
and presented as log10 CFU/ml.
Assessment of biofilm formation: Biofilm formation on glass and
acrylic denture and its inhibition with CuO- and ZnO-NPs were
assessed using 0.2% crystal violet (CV) under a light microscope
NIKON Eclipse 80i equipped with a Nikon DXM1200C digital
camera (Nikon, Japan). Quantitative assessment was performed
on polystyrene plate (Nunc, Denmark), and human epithelial
(WISH) cells, as described by Burton et al. [17] and Rubens et al.
[18], respectively.
Extracellular polysaccharide (EPS) production assay: EPS produc-
tion was determined as described by Packiavathy et al. [19]. For
NPs-induced EPS inhibition, the EPS produced by NPs treated and
untreated bacteria was measured spectrophotometrically at
490 nm, and percentage inhibition was determined considering
EPS production in untreated bacteria as 100%.
Fig. 3. Inhibition of biofilm formation by ZnO- (A1) and CuO-NPs (A2) on the surface of glass (magnification 40  ); and acrylic denture (B).
69
3. Results and discussion
Synthesis and characterization of ZnO- and CuO-NPs, zeta poten-
tial and particle size distribution: Fig. 1A and B shows the typical
TEM images of CuO- and ZnO-NPs, respectively. The CuO-NPs
were mostly spherical, and the ZnO-NPs were polygonal in shape
with smooth surfaces and average sizes of 40 nm and 35 nm,
respectively. The XRD pattern of CuO-NPs in Fig. 1C indicates the
peaks of CuO-NPs, indexed to the monoclinic crystal system CuO
(C2/c space group, JCPDS Card no. 45-0937). Similarly, the peaks
of ZnO-NPs at 2y ¼32.061, 34.661, 36.541, 47.821, 56.891 and
63.161 were assigned to (100), (002), (101), (102), (110) and
(103), respectively and suggested a polycrystalline wurtzite
structure (Zincite, JCPDS 5-0664). The crystallite sizes based on
Scherrer’s equation [20] were determined to be 39.87 nm and
35.23 nm for CuO- and ZnO-NPs, respectively. The XRD based
particle sizes were found in agreement with the TEM observa-
tions. DLS analysis revealed the hydrodynamic particle sizes of
CuO- and ZnO-NPs in ultrapure water as 198 nm and 491 nm,
respectively, with the zeta (z) potential of 20.8 for Cu-NPs and
16.4 for ZnO-NPs (Supplementary Fig. 1S).
Biocidal effect of CuO- and ZnO-NPs on bacterial viability:
Treatment of oral bacterial population with CuO- and ZnO-NPs
resulted in significant decrease in total bacterial counts on
both the NA and MRS agar plates (Fig. 2A). At a concentration of
10 mg/ml of ZnO- and CuO-NPs, the CFU counts on NA plates were
decreased by 30% and 66%, while on MRS agar, the reduction was
35% and 59%, respectively. Exposure at a higher concentration
70 S. Tabrez Khan et al. / Materials Letters 97 (2013) 67–70
of 50 mg/ml significantly affected the cell viability, resulting in
82–92% reduction in the survival of treated bacteria as compared
to the untreated control. Thus, the data in Fig. 2A suggested that
the reduction of planktonic cells was significant only for NPs
concentrations 450 mg/ml. Also, the biocide activity of CuO-NPs
was significantly higher than that of the ZnO-NPs, which is
consistent with the results obtained with the biofilm formation
(Fig. 2C). The EC50 values of CuO- and ZnO-NPs were estimated to
be 22.5 mg/ml and 70.5 mg/ml on NA plates vis-a -vis 25 mg/ml and
66 mg/ml on MRS agar, respectively. [Hall-Stoodley et al. [10]
reported the inhibition of oral bacteria Actinomyces viscosus and S.
mutans with ZnO whisker in the range between 78 and 312.5 mg/
ml.] The EC50 value of nano-Cu has been reported to be 78 mg/ml
on Vibrio fischeri [21]. Indeed, the susceptibility to conventional
antimicrobial agents varies greatly from organism to organism;
however, our results demonstrated the strong biocidal effects of
ZnO- and CuO-NPs on oral bacterial community.
NPs-induced inhibition of biofilm formation: Effects of CuO- and
ZnO-NPs on binding of oral bacteria to a monolayer of human
epithelial cells are shown in Fig. 2B. The binding was not affected at
lower concentration range up to 10 mg/ml of NPs. However, it was
significantly decreased (po0.001) with CuO-NPs at concentrations
above 50 mg/ml. Also, the biofilm formation on polystyrene surface
was significantly inhibited at both 50 and 100 mg/ml of CuO- and
ZnO-NPs (Fig. 2C). A recent study demonstrated ZnO- and CuO-NPs-
induced inhibition of S. mutans on artificial teeth and glass surface
[22]. Our results also exhibited complete inhibition of biofilm
formation at 100 mg/ml of ZnO- and CuO-NPs on the glass
(Fig. 3A1 and A2) and acrylic denture (Fig. 3B) surfaces. Over all,
the results suggest greater toxic effects of CuO-NPs vis-a -vis ZnO-
NPs, which could be attributed to (i) differences in shape of CuO-NPs
(spherical) and ZnO-NPs (polyhedral), and (ii) higher z-potential of
20.8 for Cu-NPs and 16.4 for ZnO-NPs. It is suggested that more
cationic NPs exhibit greater toxicity associated with cell wall
disruption and higher membrane permeability.
NPs-induced inhibition of EPS production: Fig. 2D shows the NPs
concentration dependent reduction in EPS production. About 27%
and 18% inhibition occurred at 10 mg/ml of ZnO- and CuO-NPs,
respectively. The extent of EPS inhibition increased remarkably to
59% and 61% at concentration of 50 mg/ml NPs. Thus, NPs-induced
biofilm inhibition is attributed to repressed EPS synthesis. Earlier
studies suggested the role of glucosyltransferase in the synthesis of
polysaccharide and demonstrated its inhibition by metal ions [23].
4. Conclusions
In the study, the CuO- and ZnO-NPs exhibited significant
inhibitory activity on oral bacteria and biofilm formation. The
results substantiated the potential of these NPs as antimicrobial
agents, and elucidated their effective control on oral biofilm
formation, as a novel approach in preventing dental infections.
Further studies are warranted to ascertain the sensitivity of
specific dental plaque colonizers to different types of biocidal
NPs and understand the mechanism of biofilm inhibition, for
developing effective strategies for dental hygiene.
Appendix A. Supporting information
Supplementary data associated with this article can be found
in the online version at http://dx.doi.org/10.1016/j.matlet.2013.
01.085.
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