85

Document heading
In vitro antibacterial activity of the metal oxide nanoparticles against
urinary tract infectious bacterial pathogens
Sundaram Ravikumar
1*
, Ramasamy Gokulakrishnan
1
, Pandi Boomi
2
1
School of Marine Sciences, Department of Oceanography and Coastal Area Studies, Alagappa University, Thondi Campus, Thondi-623 409,
Ramanathapuram District, Tamil Nadu, India
2
School of Chemistry, Department of Industrial Chemistry, Alagappa University, Karaikudi-630 003, Sivgangai District, Tamil Nadu, India
Asian Pacific Journal of Tropical Disease (2012)85-89
Asian Pacific Journal of Tropical Disease
journal homepage:www.elsevier.com/locate/apjtd
*Corresponding author: Sundaram Ravikumar, School of Marine Sciences, Department
of Oceanography and Coastal Area Studies, Alagappa University, Thondi Campus,
Thondi-623 409, Ramanathapuram District, Tamil Nadu, India.
Tel: 04561-243470, +91-9003306959
E-mail: ravibiotech201321@gmail.com
Foundation Project: This work was financially supported by University Grants
Commission, New Delhi [grant No. F.No. 39-563/2010 (SR)].
1. Introduction
The infectious diseases remain one of the greatest
challenges to global health. Urinary tract infection (UTI) is
the second most common clinical disease and possesses a
significant healthcare burden
[1]
. This infectious disease can
alter the urinary system either structurally (complicated
UTI) or functionally. About 80 to 90 percent of UTIs are
caused by a single type of bacteria. Escherichia coli (E.
coli) is the most common cause of uncomplicated urinary
tracts (anatomically normal urinary tract). The diagnosis
of UTI is very difficult for the elder people because of the
asymptomatic bacteriuria
[2]
. So there is an urgent need
to produce the new antibacterial agents from different
sources. The terrestrial plant such as Phylanthus amarus
and Parquetina nigrescens showed potential antibacterial
activity against UTI pathogens
[3]
. Moreover, the marine
resources such as mangroves, seaweeds, sponges and sea
grasses already showed antibacterial
[4-8]
, antifungal
[6]
,
and antiplasmodial
[9-13]
activities. However, most of the
antibacterial agent entered into clinical practice, resistance
was reported in at least one bacterial pathogen
[14]
. During
the past decades, the nanoparticles are attracting a great
deal in biological and pharmaceutical applications
[15-18]
.
Moreover, the metal oxide nanoparticles have good
antibacterial activity and antimicrobial formulations
comprising nanoparticles could be used as an effective
bactericidal agent
[19-24]
. Nevertheless, studies related with
metal oxide nanoparticles against urinary tract infectious
pathogens are too limited. Hence, the present study has
been made an attempt to find out the potential nanoparticles
against urinary tract infectious pathogens.
ARTI CLE I NFO ABSTRACT
Article history:
Received 15 October 2011
Received in revised form 27 December 2011
Accepted 28 February 2012
Available online 28 April 2012
Keywords:
Antibacterial activity
Metal oxide nanoparticles
Minimum inhibitory concentration
Time kill assay
UTI pathogens
Minimal bactericidal concentration
Well diffusion method
Nanoparticle
Infectious bacterial pathogen
Objective: To investigate the antibacterial properties of the five metal oxide nanoparticles viz.,
Al
2
O
3
, Fe
2
O
3
, CeO
2
, ZrO
2
and MgO against urinary tract infectious pathogens viz., Pseudomonas
sp., Enterobacter sp., Klebsiella sp., Escherichia coli (E. coli), Proteus morganii (P. morganii)
and Staphylococcus aureus (S. aureus). Methods: The antibacterial activity of the five
different nanoparticles was assessed by well diffusion method. Different concentrations of the
nanoparticles were analyzed by minimal inhibitory concentration (MIC) and minimal bactericidal
concentration (MBC) techniques. Finally, the potential nanoparticle Al
2
O
3
which showed maximum
antibacterial sensitivity was also subjected for the time kill assay method. Results: Among the
nanoparticles, Al
2
O
3
nanoparticle showed maximum sensitivity (16.000.21) mm against E. coli.
None of the nanoparticles showed activity against Pseudomonas sp. The MIC results also revealed
that, the Al
2
O
3
nanoparticle showed maximum inhibition at the concentration of 5 g/mL against
E. coli, followed by 10 g/mL against Klebsiella sp. and P. morganii, respectively. Moreover, the
time kill assay revealed that, the bacterial growth was maximum inhibited at the concentration
of 5 g/mL from the 2nd h. Conclusions: It can be concluded from the present findings that,
the Al
2
O
3
nanoparticle can be used as an alternative antibacterial agent for the urinary bacterial
diseases after completing successful clinical trials.
Contents lists available at ScienceDirect
Sundaram Ravikumar et al./Asian Paicfic Journal of Tropical Disease (2012)85-89
86
2.1. Isolation of UTI bacterial pathogens
A total of 50 urine samples from 25 male and 25 female
patients admitted in the hospitals as UTI problems were
collected from different hospitals and laboratory localities
along the coastal area of Thondi, Ramanathapuram District,
Tamil Nadu, India (Lat. 944 N and Long. 7910E) in
a separate sterile wide mouth bottle. Before collecting a
sample, the women were instructed to swab the vulvae and
men to retract the foreskin and cleanse the glans penis.
Midstream urine was collected in a sterile wide mouthed
container. For the isolation of UTI bacterial strains, loop
full of urine samples were streaked into the nutrient agar,
Mac Conkey agar, blood agar and chocolate agar plates and
incubated at (372) for 24 h. Next day individual colonies
were selected and identified on the basis of morphological
characteristics, gram staining and biochemical characters
[25,26]
.
2.2. Antibacterial assay
The antibacterial activity of the chosen nanoparticles was
performed by using well diffusion method. About 20 mL of
sterile molten Mueller Hinton agar (HiMedia Laboratories
Pvt. Limited, Mumbai, India) was poured into the sterile
petriplates. Triplicates plates were swabbed with the
overnight culture (10
8
cells/mL) of pathogenic bacteria viz.,
Pseudomonas sp., Enterobacter sp., Klebsiella sp., E. coli,
Proteus morganii (P. morganii) and Staphylococcus aureus
(S. aureus). The solid medium was gently punctured with the
help of cork borer to make a well. Finally, the nanoparticle
samples (50 g/mL) were added from the stock into each
well and incubated for 24 h at (372) . After 24 h, the zone
of inhibition was measured and expressed as millimeter in
diameter.
2.3. Minimum inhibitory concentration (MIC)
About 500 L of different concentrations (2.5, 5, 10, 15 and
20 g) of chosen nanoparticles were prepared with dimethyl
sulphoxide (DMSO) and mixed with 450 L of nutrient broth
and 50 L of 24 h old bacterial inoculum and allowed to
grow overnight at 37 for 48 h. Nutrient broth alone served
as negative control. The MIC was the lowest concentration
of the nanoparticles that did not permit any visible growth
of bacteria during 24 h of incubation on the basis of
turbidity
[27]
.
2.4. Minimum bactericidal concentration (MBC)
To avoid the possibility of misinterpretations due to the
turbidity of insoluble compounds if any, the MBC was
determined by sub-culturing the above (MIC) serial dilutions
after 24 h in nutrient agar plates using 0.01 mL loop and
incubated at 37 for 24 h. MBC was regarded as the lowest
concentration that prevented the growth of bacterial colony
on this solid media
[27]
.
2.5. Time kill assay
The potential nanoparticle (Al
2
O
3
) which showed maximum
antibacterial activity against E. coli was also subjected for time
kill assay. The inoculum of E. coli (50 L) at a concentration
of (10
8
cells/mL) was mixed with 50 L (5 g concentration
of Al
2
O
3
) nanoparticle and the total volume was made
up to 5 mL by using minimal medium (g/L) [sucrose (10);
K
2
HPO
4
(2.5); KH
2
PO
4
(2.5); (NH
4
)
2
HPO
4
(1); MgSO
4
.7H
2
O (0.20);
FeSO
4
.7H
2
O (0.01); MnSO
4
.H
2
O (0.007) and H
2
O (1 000 mL)]. The
negative control was maintained without the nanoparticles.
The growth of the bacterial species was assessed at every 1 h
interval by measuring the optical density at 600 nm by using
spectrophotometer (Shimadzu, Japan)
[6]
.
3. Results
Out of the 60 midstream urine samples, 45 bacterial strains
were isolated and it was identified by using biochemical
tests (Table 2). Of these, Pseudomonas sp. is the predominant
one (38%), followed by Enterobacter sp. (22%), Klebsiella sp.
(18%), E. coli (11%), P. morganii (7%) and S. aureus (4%) (Figure
1). The zone of inhibition of the selected nanoparticles
against UTI pathogens was represented in Table 3. It
revealed that, the Al
2
O
3
nanoparticle showed maximum
sensitivity (16.000.21) mm against E. coli followed by (12.00
0.69) mm and (12.000.72) mm against Klebsiella sp. and
P. morganii, respectively. The MgO nanoparticle showed
maximum sensitivity (13.000.64) mm against E. coli and
showed minimum sensivity (9.000.29) mm against Klebsiella
sp. and P. morgonii (6.000.61) mm. The Fe
2
O
3
, Ceo
2
and ZrO
2

showed maximum sensitivity (10.000.35) mm against E. coli,
P. morganii (11.000.51) mm and Enterobacter sp. (12.00
0.26) mm, respectively. None of the nanoparticles showed
sensitivity against Pseudomonas sp. The MIC and MBC
revealed that, the Al
2
O
3
nanoparticle showed sensitivity at
the concentration of 5 g/mL against E. coli and Klebsiella
sp. and P. morganii 10 g/mL, respectively. Moreover,
MgO and ZrO
2
nanoparticles showed maximum sensitivity
against E. coli at the concentration of 10 g/mL, respectively
(Table 4). The effect of Al
2
O
3
nanoparticle against E. coli was
performed with time kill assay. It revealed that, the growth
2. Materials and methods
Commercial nanoparticles of Al
2
O
3
, Fe
3
O
4
, CeO
2
, ZrO
2
and
MgO were procured from Sigma Aldrich Company, India. The
characteristics of the nanoparticles are presented in Table 1.
Table 1
Properties of nanoparticles.
Formula Molecular weight Form Particle size (nm) (transmission electron microscope)
Al
2
O
3
101.96 Powder <50
Fe
3
O
4
231.53 Powder 9-11
CeO
2
172.11 Powder <25
ZrO
2
123.22 Powder <100
MgO 40.30 Powder <30
Sundaram Ravikumar et al./Asian Paicfic Journal of Tropical Disease (2012)85-89
87
of the pathogen was inhibited from the 2
nd
h when compared
with control (Figure 2).
11%
7%
4%
38%
18%
22%
Pseudomonas sp. Enterobacter sp. Klebsiella sp.
E. coli P. morganii S. aureus
Figure 1. Percentage occurrence and distribution of bacterial
pathogens in UTIs among the patients (n=45).
O
D

v
a
l
u
e

a
t

6
0
0

n
m
1.2
1
0.8
0.6
0.4
0.2
0
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (h)
Figure 2. Time dependent assay of nanoparticle (Al
2
O
3
) against
chosen UTI pathogen E. coli.
Pathogen alone
Pathogen + Al
2
O
3
nanoparticle
4. Discussion
Nanotechnology is an emerging field and it has been
applied in science and technology for the purpose of
manufacturing new materials at the nanoscale level
[28]
. In
Table 2
Biochemical characterization of isolated bacteria from UTI patients.
Characteristics Pseudomonas sp. E. coli Klebsiella sp. Enterobacter sp. P. morganii S. aureus
Gram staining - - - - - +
TSI Slant K A A A K A
Butt K A A A A A
GAS - G G G G -
H
2
S - - - - - -
Mannitol Acid Acid Acid Acid - -
Motility Motile Motile Non-motile Motile Motile Motile
Indole test - - - - + -
Methyl red test + + - - + -
V.P. test - - + + - -
Citrate test - - + - - -
Urease test + - + - + -
Oxidase test + - - - - -
Catalase test + - - - + +
+: positive; -: negative; K: alkaline; A: acid; G: gas.
Table 3
Antibacterial activity of chosen 5 nanoparticles against UTI pathogens (meanSD) (mm) .

Name of
the nanoparticles
Pseudomonas sp. (n=17) Enterobacter sp. (n=10) Klebsiella sp. (n=8) E. coli (n=5) P. morganii (n=3) S. aureus (n=2)
Al
2
O
3
- - 12.000.69 16.000.21 12.000.72 9.000.61
Fe
2
O
3
- 7.000.23 - 10.000.35 - 7.000.67
Ceo
2
- 6.000.12 6.000.74 9.000.39 11.000.51 8.000.24
ZrO
2
- 12.000.26 7.000.45 10.000.59 - -
MgO - - 9.000.29 13.000.64 6.000.61 -
n: number of isolates; -: no sensitivity.
Table 4
MIC and MBC (

g/mL) of chosen 5 nanoparticles against UTI pathogens.

Name of the
nanoparticles
Pseudomonas
sp.
Enterobacter
sp.
Klebsiella sp. E. coli P. morganii S. aureus
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
Al
2
O
3
- - - - 10 10 5 5 10 20 - -
Fe
2
O
3
- - - - - - 15 20 - - - -
Ceo
2
- - - - - - 20 20 20 20 - -
ZrO
2
- - 20 20 - - 10 20 - - - -
MgO - - - - 15 15 10 10 - - - -
-: no activity.
Sundaram Ravikumar et al./Asian Paicfic Journal of Tropical Disease (2012)85-89
88
the present scenario, the nanoparticles are being emerged as
novel antimicrobial agents with unique biological, chemical
and physical properties
[29,30]
. Moreover, the advantages of
the metal nanoparticles are less toxicity, heat resistance
and suitable for biological application
[19,31,32]
. All the
nanoparticles showed sensitivity against all the pathogens
except Pseudomonas sp. Of the selected nanoparticles, the
Al
2
O
3
nanoparticle showed maximum sensitivity against
E. coli. The MIC result reveals that, the Al
2
O
3
nanoparticle
showed maximum sensitivity at a concentration of 5 g/
mL against E. coli, 10 g/mL against Klebsiella sp. and P.
morganii, respectively and this activity might be due to the
size, surface morphology, particle morphology and structure
of the nanoparticles
[33]
and the possible mechanism for the
cell lyses is, the nanoparticles release ions which react with
the thiol (-SH) groups of protein present in the cell wall,
inactivate the protein and decrease the cell permeability
which leads to cellular death
[34]
. Earlier investigations
reveal that, the silver and gold nanoparticles showed various
biological properties
[35-54]
. Moreover, the TiO
2
and CdO,
Fe
3
O
4
and ZnO nanoparticles showed antibacterial activity
against E. coli
[34,55-57]
. Generally, the toxic effects of the
Al
2
O
3
nanoparticles are time dependent. This oxidative stress
in the cell wall might increase the production of lactate
dehydrogenase, which is an indicator of cell membrane
damage
[58]
. It is concluded from the present findings that,
the Al
2
O
3
nanoparticle could be used as an alternative
antibacterial agent for the urinary bacterial diseases after
completing successful clinical trials.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
The authors are thankful to the authorities of Alagappa
University for providing required facilities and also to
University Grants Commission, New Delhi for financial
assistance with the grant number: F.No.39-563/2010 (SR).
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