Journal of Biomaterials and Nanobiotechnology, 2010, 1, 37-41
doi:10.4236/jbnb.2010.11005 Published Online October 2010 (http://www.SciRP.org/journal/jbnb)
37
Effect of Nano - Titanium Dioxide with Different
Antibiotics against Methicillin-Resistant
Staphylococcus Aureus
Aashis S. Roy1, Ameena Parveen2, Anil R. Koppalkar3, M. V. N. Ambika Prasad1*
1Department of Materials Science, Gulbarga University, Gulbarga, Karnataka, India; 2Department of Physics, Gurmithkal, Yadgir,
Karnataka, India; 3Materials Science Lab, S. S. Margol College, Shahabad, Gulbarga, Karnataka, India.
Received June 14th, 2010; revised June 25th, 2010; accepted June 30th, 2010.
ABSTRACT
The different investigation has been carried out on the biological activities of titanium dioxide nanoparticle but the
effect of this nano product on the antibacterial activity of different antibiotics has not been yet demonstrated. In this
study the nano size TiO2 is synthesized using citric acid and alpha dextrose and the enhancement effect of TiO2
nanoparticle on the antibacterial activity of different antibiotics was evaluated against Methicillin-resistant
Staphylococcus aureus (MRSA). During the present study, different concentrations of nano-scale TiO2 were tested to
find out the best concentration that can have the most effective antibacterial property against the MRSA culture. Disk
diffusion method was used to determine the antiba cterial activity of these an tibiotics in th e absence and presence of su b
inhibitory concentration of TiO2 nano particle. A clinical isolate of MRSA, isolated from Intens ive Care Unit (ICU) was
used as test strain. In the presence of sub-inhibitory concentration of TiO2 nanoparticle (20 µg/disc) the antibacterial
activities of all antibio tics have been increased against test strain with minimum 2 mm to maximum 10mm. The highest
increase in inhibitory zone for MRSA was observed against pencillin G and amikacin (each 10 mm). Conversely, in
case of nalidixic acid, TiO2 nanopa rticle showed a Synergic effect on the antibacteria l activity of this antib iotic against
test strain. These results signify that the TiO2 nanoparticle potentate the antimicrobial action of beta lactums,
cephalosporins, aminog lycosides, glyco peptides, ma crolids and lin cosamides, tetra cycline a possible utilizatio n of nano
compound in combination effect against MRSA.
Keywords: Nano - Titanium Oxide, S. Aureus, Drug Resistance, Antibacterial Activity
1. Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is
one of the major nosocomial pathogens responsible for a
wide spectrum of infections, including skin and soft tissue
infections, pneumonia, bacteraemia, surgical site infec-
tions (SSI), catheter related infections [1]. Intensive care
unit characteristically has higher rates of infections and in-
creased transmission rates, high antibiotic use and large
numbers of vulnerable patients [2]. The emergence of bac-
terial resistance to antibiotics and its dissemination, how-
ever, are major health problems, leading to treatment draw-
backs for a large number of drugs [3,4]. Consequently
there has been increasing interest in the use of inhibitors of
antibiotic resistance for combination therapy [5,6].
Nanostructured materials are attracting a great deal of
attention because of their potential of achieving specific
processes and selectivity, especially in biological and
pharmaceutical applications [7-9]. Gold, silver and cop-
per have been used mostly for the synthesis of stable
dispersions of nanoparticles [10,11]. A unique charac-
teristic of these synthesized metal particles is that a
change in the absorbance or wave length gives a measure
of the particle size, shape and interparticle properties [12].
Nanomaterials are called “a wonder of modern medicine”.
It is stated that antibiotics kill perhaps a half dozen
different disease-causing organisms but nanomaterials
can kill some 650 cells [13]. Resistant strains fail to
develop if we apply nanoparticles based formulations in
their culture media.
The antibacterial activity of TiO2 has been found to be
due to a reaction of the TiO2 surface with water. On
exposure to ultraviolet (UV) irradiation, TiO2 releases
free radicals such as OH, O2
-, HO2-, and H2O2. This

Effect of Nano - Titanium Dioxide with Different Antibiotics Against Methicillin-Resistant Staphylococcus Aureus
38
potent oxidizing power characteristically results in case
of bacteria and other organic substances [14-16]. The
small nanometer-scale TiO2 particles impose several
effects that govern its antibacterial action we examined
the antimicrobial activity of nanostructured titanium
dioxide with different antibiotics against MRSA. The
different investigation has been carried out on the bio-
logical activities of titanium dioxide nanoparticle but the
combination effects of this product with different anti-
biotics have not been demonstrated. The nanocrystal-
line particles of TiO2 are synthesized using ultrasonic
irradiation, and the particle sizes are controlled using
different solvents during the sonication process.
Objectives of the present study are (i) synthesis of
nano size titanium dioxide using citric acid and alpha
dextrose (ii) analyse the effect of Titanium nanoparticles
on the antibacterial activity of different antibiotics against
MRSA (iii) estimation of MRSA growth in the presence
of TiO2 nanoparticles have been reported having an
extremely good safety profile and no toxicity observed
when taken at different nanosize. Taken together, this
compound as a highly safe compound may be considered
for combination therapy against MRSA, due its potential
synergetic effect with important antibiotics such as beta
lactums, cephalosporins, aminoglycosides.
2. Materials and Methods
Titanium dioxide particles preparation: In the following,
the two step sol-gel preparation method used is described
detail. Nanocrystalline titanium dioxide was prepared by
employing citric acid route were saturated solution of
α-Dextrose used as a surfactant.
Two separate solutions were prepared. In first step:
titanium nitrate and citric acid are taken in 1:3 and are
thoroughly stirred using magnetic stirrer with ammonia
solution at 80˚C about 5-6 hrs. Ammonia solution is used
to maintain the pH 4 of solution. Finally a gel is formed.
In second step, saturated solution of alpha dextrose is
added and stirred for 1hr at 120˚C to the spongy type gel
of nanoscaled TiO2 formed. This spongy gel is ignited at
a temperature of about 300˚C for 1 hr. At this tem-
perature a combustion process takes place in the spongy
gel containing citric acid a result of it we have nano-
structured titanium dioxide.
3. X-Ray Diffraction (XRD)
X-ray diffraction Phase identification was carried out by
X-Ray powder diffraction at ambient temperature. A
Shintag X1 diffractometer with Cu Kα (1.54 A) radiation
in θ – θ configuration was used. The patterns were
recorded in the 2-70 range at 0.05 step size using 3-s
acquisition time per step. The mean particle size was
calculated using the Debye–Scherrer Equation 1 in which
K is a constant equal to 0.9, λ is the wavelength of the Cu
Kα radiation, β is the half peak width of the diffraction
peak in radiant and θ is the Braggs angle of (311) plane.
τ = Kλ/βcosθ (1)
Staphylococcus aureus was isolated from Clinical
specimens collected from ICU of Durgabai Deshmukh
Hospital and Research Center and Osmania Hospital,
Hyderabad, South India. Oxacillin-disc diffusion method
was done for identification of methicillin-resistance. This
MRSA was used as test strain. Antibiotic susceptibility
test was performed for the test strain (MRSA) against 23
antibiotics by disc agar diffusion method (DAD) on
Muller-Hinton agar (Himedia, India), according to the
guidelines recommended by National Committee for
Clinical Laboratory Standards (NCCLS) [17].
4. Disk Diffusion Assay to Evaluate
Combined Effects
To determine combined effects, each standard paper disc
was further impregnated with sub-inhibitory concen-
tration of titanium dioxide nanoparticle (10 µg/disc). A
single colony of test strains were grown overnight in
Muller-Hinton broth medium on a rotary shaker (200 rpm)
at 35˚C. The inoculums were prepared by diluting the
overnight cultures with 0.9% NaCl to a 0.5 McFarland
standard and were applied to the plates along with the
standard and prepared disks containing of titanium dioxide
nanoparticle (10 µg/disc). Clinical isolates of MRSA from
our culture collection were used as test strains. After
incubation at 37˚C for 24 hrs, the zones of inhibition were
measured. The assays were performed in triplicate.
5. Estimation of MRSA Growth in the
Presence of Nanocrystalline TiO2
The 2 mL of the overnight-cultured MRSA was added to
100 mL nutrient broth, containing 0.12% glucose with
and without 0.01, 0.5 and 1% nano-TiO2 and incubate at
30˚C for 24 hrs. Optical density measurements were
taken at 600 nm to monitor the bacterial concentration.
6. Results and Discussions
The nano size titanium dioxide is synthesized using citric
acid and alpha dextrose. The small nanometer scale TiO2
particles as seen in the Figure 1 will impose several
effects that govern its antibacterial action. The X-ray
diffraction pattern shows cubic peaks of TiO2, which
indicates the nanocrystalline nature of pure
nanostructured titanium dioxide and is shown in the
Figure 2. By comparing the XRD pattern standard JCPS
data (432-161) of TiO2, indicating the prominent peaks
corresponding to 2θ = 27˚, 39˚, 48˚, 55˚ and 63˚ are due to
(110), (200), (112),

Effect of Nano - Titanium Dioxide with Different Antibiotics Against Methicillin-Resistant Staphylococcus Aureus
39
Figure 1. Showing TEM image of TiO2 nanoparticles.
Figure 2. XRD pattern of TiO2.
(220) and (310) planes which indicates formation of
single phase titanium dioxide. The crystalline size of the
Titanium dioxide is calculated by using debye-scharrer
equation and it was found to be around 20 nm. Titanium
dioxide has a very good potential to move into the clinic
[18]. In this investigation the effect of TiO2 nanoparticle
on the antibacteria of different antibiotics was invest-
tigated against MRSA using disk diffusion method. The
antimicrobial resistance of MRSA against various anti-
biotics is increased without nano-TiO2 and decreases
with nanoscaled TiO2. The diameter of inhibition zones
(mm) around the different antibiotic discs with TiO2 and
without titanium dioxide nanopatrticles against test strain
are shown in [Table 1].
The antibacterial activities of all antibiotics have been
increased in the presence of nanosize titanium dioxide
against test strain. The highest antibacterial activities in-
creases in area were observed for penicillin and amikacin
(10 mm) followed by ampicillin and Gentamycin (in
each 09 mm), oxacillin, cloxacillin (08 mm), amoxycillin,
cephalexin, cefotoxime, ceftazidime, vancomycin, strep-
tomycin (in each 07 mm) erythromycin, clindamycin (06
mm) and tetracyclin (05 mm). The moderate increases in
inhibition zone areas for ciprofloxacin, rifampicin, sul-
phazidime and cotrimoxazole (04 mm).The lowest in-
crease in inhibition zone area against the Chloramphe-
nicol (03 mm) followed by norfloxacin and clarithromy-
cin (02 mm).
Conversely, for nalidixic acid, titanium dioxide nano-
particle shows no effect on the antibacterial activity of
this antibiotic against the test strain. It should be pointed
out that the titanium dioxide nanoparticle content of 10
µg/disc was chosen to guarantee that the effect produced
was due to the combination and not to the effect of the
TiO2 nanoparticle itself. So the effect observed in this
condition could be due to the antibiotic-titanium dioxide
nanoparticle combination. At the concentration tested,
TiO2 nanoparticle significantly improved antibiotic effi-
cacy against S. aureus when combined with beta lac-
tums, cephalosporins, aminoglycosides.
The optical density of the medium was investigated as the
number of bacteria after contact with the nano- particles.
Figure 3 shows the growth of MRSA at diff- erent
concentrations and the effect of different 0.01, 0.5 and 1%
nano-TiO2 on the in growth and killing of MRSA. As
demonstrated by the figure, 0.01% nano-TiO2 did not have
antibacterial efficiency on MRSA but the concentrations of
0.5 and 0.1% nano-TiO2 inhibited the bacterial growth. Also
shows that 0.5% nano-TiO2 showed 1.9 times decrease the
optical density of bacterial cultures as compared to the
control. While, in the presence of 1% nano-TiO2, the optical
density of MRSA cultures decreased 4.5 times as compared
to the control experiment.
A study states the nano-TiO2 as a strong and effective
bactericidal agent [18]. During the present study,
different concentrations of nanosized TiO2 were tested to
find out the best concentration that can have the most
effective antibacterial property against the MRSA culture.
This is the first report of combination effect of TiO2
nanoparticles with different antibiotics. Today, TiO2

Effect of Nano - Titanium Dioxide with Different Antibiotics Against Methicillin-Resistant Staphylococcus Aureus
40
Table 1. the comparative activities of various antibiotics and antibiotic with NanosizedTiO2 against MRSA.
Sl.
No. Antibiotics Symbol
Inhibition Zone of Antibiotic
(mm)
Inhibition Zone of Antibiotic with
TiO2 (20nm) in (mm) Increased zone size (mm)
1 B-lactams
01. Penicillin G P (10U) 34 44 10
02. Oxacillin Wx (1 μg) 11 19 08
03. Cloxacillin Cx (30 μg) 19 27 08
04. Ampicillin A (10 μg) 29 38 09
05. Amoxycillin Am (25 μg) 20 26 07
2 Cephalosporins
06. Cephalexin Cp (30 μg) 25 32 07
07. Cefotoxime CX (30 μg) 24 33 07
08. Ceftazidime Ca (30 μg) 16 23 07
3 Glycopeptides
09. Vancomycin V (30 μg) 15 22 07
4 Aminoglycosides
10. Amikacin Ak (10 μg) 15 25 10
11. Gentamycin G (50 μg) 14 24 09
12. Streptomycin S (25 μg) 13 19 07
5 Flouroquinolones
13. Ciprofloxacin Cf (5 μg) 20 24 04
14. Norfloxacin No (10 μg) 15 17 02
6 Azlides
15. Clarithromycin Cw (15 μg) 17 19 02
7 Macrolides
16. Erythromycin E (15 μg) 15 21 06
8 Lincosamides
17. Clindamycin Cl (10 μg) 20 26 06
9 Sulphonamides
18. Cotrimoxazole Co (25 μg) 17 21 04
19. Nalidixicacid Na (30 μg) 16 16 00
20. Rifampicin R (15 μg) 25 29 04
21. Tetracyclin T (30 μg) 21 26 05
22. Sulphazidime Sz (25 μg) 12 17 04
23. Chloramphenicol C (30 μg) 18 21 03
Figure 3. MRSA growth at different concentrations of TiO2.
nanoparticle are cosmetic ingredient has drawn the atten-
tion of scientists because of its extensive pharmaceutical
properties. In different phases, clinical trials, no toxicity
except mild dehydration was observed when taken at
doses as high as g/day and it is reported as an attractive
choice for many disease therapies.
Recently some metal nanoparticles have been eva-
luated for increasing the antibacterial activities of dif-
ferent antibiotics. Several investigations have suggested
the possible mechanisms involving the interaction of na-
nomaterials with the biological molecules. It is believed
that microorganisms carry a negative charge while metal
oxides carry a positive charge. This creates an “elec-
tromagnetic” attraction between the microbe and treated
surface. Once the contact is made, the microbe is oxi-
dized and dead instantly. Generally, it is believed that
nanomaterials release ions, which react with the thiol
group (-SH) of the proteins present on the bacterial sur-
face. Such proteins protrude through the bacterial cell
membrane, allowing the transport of nutrients through
the cell wall. Nanomaterials inactivate the proteins, de-
creasing the membrane permeability and eventually caus-
ing the cellular death [19]. In this study using disk diffu-
sion assay we showed that the antibacterial activity of

Effect of Nano - Titanium Dioxide with Different Antibiotics Against Methicillin-Resistant Staphylococcus Aureus
41
beta lactums, cephalosporins, aminoglycosides, glycol-
peptides, erythromycin, clindamycin and tetracycline can
be increased by TiO2 nanoparticles. Therefore, this com-
pound or its future derivatives have a good potential for
combination effect against MRSA.
7. Conclusions
The synthesis of nanosize titanium dioxide of 20nm was
carried out successfully using citric acid and alpha dex-
trose as double surfactants. The small nanometer scale
TiO2 particles which impose several effects that govern
its antibacterial action. The antibacterial activities at
different concentrations of nano-TiO2 were investigated.
The antimicrobial resistance of MRSA against various
antibiotics is increased without nano-TiO2 and decreases
with nano-TiO2. Need the further work to find out the
exact reason to for enhancement of activity of antibiotics
in presence of TiO2 nanoparticles.
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