ISSN 0975-2366

DOI:https://doi.org/10.31838/ijpr/2020.12.02.0059
Research Article

Toxicity of Ag & ZnO nanoparticles against MCF – 7 cell

lines whereas not toxic to human peripheral blood
mononuclear cell lines
KANTIPRIYA K1*, DR SARVAMANGALA D2, DR U S N MURTHY3, DR SATYANARAYANA R4, DR
NARSINGARAO B5, RAJKIRAN A6,
1*
Research Scholar, Dept. of Biotechnology, GIT, GITAM (Deemed to be University), Visakhapatnam
2, 4
Associate Professor, Dept. of Biotechnology, GIT, GITAM (Deemed to be University), Visakhapatnam
3
Professor, Dept. of Ophthalmology, GVP Institute of Health Care & Medical Technology, Visakhapatnam
5
Professor & HOD, Dept. of Microbiology, GVP Institute of Health Care & Medical Technology,
Visakhapatnam
6
Delivery Assurance Manager, Trianz Holdings Pvt. LTD., Rasoolpura, Begumpet, Secunderabad
*Corresponding author
Email ID: kantipriya.kondala@gmail.com
Received: 23.12.19, Revised: 08.01.19, Accepted: 04.02.20

ABSTRACT
A leaf extract of Talinum triangulare was used for the synthesis of Ag & ZnO nanoparticles and the synthesized
nanoparticles were evaluated for anticancerous activity against Breast cancer (MCF -7) cell lines and antibacterial
activity against pathogens Staphylococcus aureus, Streptococcus and Pseudomonas aeruginosa. Different concentrations
of Ag and ZnO NP’S were tested against human breast cancer cell lines. Among all the tested concentrations 2
μg/ml for Ag nanoparticles and 6 μg/ml for ZnO nanoparticles were showed significant cytotoxic activity against
MCF -7 cell lines. They were also found to be less toxic and safe to mononuclear cell lines from human peripheral
blood, which IC50 values of both Ag & ZnO nanoparticles represents as 2 μg/ml and 6 μg/ml. The antibacterial
activity on tested organisms was found to be very potential and superior.
Keywords: Talinum triangulare, anticancerous activity, antibacterial activity, MCF – 7 cell lines, Ag & ZnO
nanoparticles, mononuclear cells.

BACKGROUND Pseudomonas aeruginosa is associated with rapid

Cancer is one of the most serious worldwide threats
against human health. Many toxic and adverse side
effects occur due to the use of the synthetic drugs
and it made scientists to look at herbal medicine to
improve the fulfilment of human health needs [1].
Nearly 50% of anticancer agents and 74% of
anticancer compounds are plant products or plant
derived products [2]. There is a lot of increase in
the growth of antibiotic resistant pathogenic
infections and cancers [3]. Scientists showed special
interest towards plant derived natural drugs to
avoid side effects and cost effect from synthetic
drugs [4]. Gram - negative bacteria are the main
cause in nosocomial infections which leads to long
period hospitalization, high mortality and
increasing hospital costs. Among the entire Gram -
negative bacteria, Pseudomonas aeruginosa is
highly susceptible to genetic modifications leading
to bacterial resistance and it has ability to survive in
typical environments also [5,6].
growth and spread of human infections through
health care units such as bacteraemia, pneumonia,
post-operative urinary and gastrointestinal
infections, endocarditis, osteomyelitis and
meningitis [7,8]. Besides Pseudomonas aeruginosa,
other bacteria such as Streptococcus and
Staphylococcus aureus are also can cause
opportunistic infections, especially in the cancer
patients, burn victims and patients with cystic
fibrosis [9]. Decreased sensitivity of broad-spectrum
antibiotics such as carbapenems and
fluoroquinolones such as norfloxacin and
ciprofloxacin have been described in Brazil [10-15].
Nosocomial bacterial infections are frequent due to
hospital environment and cross contamination such
as non-hygienic use of medical equipment [16,17],
which could be reduced by the use of nanoparticle
coated medical equipment, where nanoparticles act
as antimicrobial agents. Studies have been
demonstrated the effectiveness of Ag and ZnO

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 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles
nanoparticles such as incorporated in to clothing
[18-21], bandage cloth and dressing for burn
victims and used to treat nappy rash in children
[22].
The importance of medicinal plants in
pharmacology is very crucial because they contain
active constituents (that are used in the
management of various disease conditions) such
as; quinine for malaria, opioid analgesics for
cancer pain, NSAIDS for pyrexia, laxatives for
constipation, etc which have side effects both on
acute and chronic administration. Hence, their
study is important to the development of new and
safer drugs [23].
Plant mediated nanoparticles are safe and effective
to use as antimicrobial and anticancerous agents.
Talinum triangulare (jacq.) wild. is an herbaceous
perennial plant widely grown in tropical regions as
a leaf vegetable. It is probably native to tropical
America and the crop is grown in West Africa,
Southeast Asia and warmer parts of North America
and South America. Along with celosia species, it is
one of the most important leaf vegetables of
Nigeria. Common names include; water leaf, leaf
Fig.1: Ceylon spinach plant with pink coloured flowers
Preparation of 1M AgNO3 and 1M ZnSO4
solutions: Silver nitrate (AgNO3) was purchased
from Hi Media laboratories Pvt Limited, Mumbai,
India. Zinc sulphate (ZnSO4) was purchased from
Thermo Fisher Scientific India Pvt Limited, Delhi,
586| International Journal of Pharmaceutical Research | Apr - Jun 2020 | Vol 12 | Issue 2
ginseng, American ginseng, Surinam purslane,
Surinam spinach, Philippine spinach, Ceylon
spinach, Florida spinach, potherb fame flower,
Lagos bologi, Sweetheart [24]. Talinum triangulare
is an herbaceous perennial plant belongs to the
family Portulaceae. Plant grows erect, bears small,
pink flowers and broad, fleshy leaves. Talinum
triangulare is rich in vitamins, including vitamins A
and C, minerals such as iron and calcium and also
high in oxalic acid [25]. In the present study
Talinum triangulare leaf extract mediated Ag and
ZnO nanoparticles were used to evaluate anti-
microbial and anti-cancerous activity.
METHODS
Plant material: Talinum triangulare commonly
called as Ceylon spinach which belongs to the
Portulaceae family, the fresh plants were collected
from Srikakulam (Fig.1)
Plant extract preparation: 100 gms of fresh leaves
were taken and washed thoroughly and ground
with 200 ml of distilled water and filtered. The leaf
extract was used for reduction of silver nitrate and
zinc sulphate and helps in nanoparticle formation.
India. 1M concentration of AgNO3 solution was
prepared by dissolving 169 gms of AgNO3 in 1000
ml of distilled water and stored in amber coloured
bottle to avoid oxidation of silver. 1M concentration
 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles
of ZnSO4 solution was prepared by dissolving 287
gms of ZnSO4 in 1000 ml of distilled water.
Synthesis of Ag and ZnO Nanoparticles: Synthesis
of silver nanoparticles was done using 10ml of leaf
extract was added with 90ml of 1M silver nitrate
(AgNO3) aqueous solution. Synthesis of Zinc oxide
nanoparticles were done using 30 ml of leaf extract
was added with 70 ml of 1M Zinc sulphate (ZnSO4)
aqueous solution.
Characterization
UV-VIS-NIR Spectrophotometer: UV-VIS-NIR
Spectrophotometer Ocean Optics DH-2000-BAL
was used to characterize optical absorption
properties and formation of Ag and ZnO
nanoparticles.
FTIR: FTIR BRUKER instrument by KBr pelleting
method was used to know the possible functional
groups involved in the synthesis of Ag and ZnO
nanoparticles [26].
FESEM: The morphology and distribution of Ag and
ZnO nanoparticles were examined using Field
Emission Scanning Electron Microscopy (FESEM,
Caril Zeiss FEsem Marline compact) and the images
operated at 30 keV.
EDAX: The EDAX spectrum was recorded using
attachment to FESEM and showed strong signal of
Ag and Zn [27].
TEM: Transmission Electron Microscopy (TEM)
analysis was done using TEM instrument JEOL
1200 EX operating at 120kv voltage to analyse size
and shape of the nanoparticle.
Antibacterial activity: To assess the antimicrobial
activity different concentrations of (1µl, 2µl) Ag and
ZnO nanoparticles were used to inoculate on to MH
agar plates with organisms Streptococcus,
Staphylococcus aureus and Pseudomonas
aeruginosa. Zone of inhibition was measured after
24hrs and 48 hrs.
Minimum Inhibitory concentration (MIC)
MIC was determined by serial dilution method [28].
To each test tube, 105 CFU/ml of active bacterial
cultures was inoculated. The culture tubes were
incubated at 370c for 24 hours. After the
incubation, the bacterial growth in tubes was
checked and MIC was determined and expressed in
µg/ml. The results are tabulated in Table 1.
Determination of invitro anticancer (Cytotoxic)
Activity:
Cell lines and cell culture: The human cancer cell
lines were maintained in Dulbecco’s modified
essential medium (DMEM), 10 % fetal bovine serum
(FBS) (growth medium) at 370 C in CO 2 incubator
587| International Journal of Pharmaceutical Research | Apr - Jun 2020 | Vol 12 | Issue 2
[29]. MCF-7 cells (Fig.2) were procured from
National Center for Cell Sciences, Pune, India.
Fig.2: MCF-7 (Breast cancer) cell lines
MTT assay
The MTT assay developed by Mosmann (1983) was
modified and used to determine the inhibitory
effects of test compounds on cell growth in vitro.
The trypsinized cancer cells (MCF-7) from T-25
flask were incubated in a 96 well plate and allowed
to adhere to the wells overnight in 5% CO2
incubator. After reaching confluency, the cell lines
are treated with different concentrations of drug (Ag
and ZnO nanoparticles) and incubated for 24 and
48 hours. After 24 hrs of incubation add 20
microliters of MTT. Incubated until purple color was
obtained. Supernatant solution was removed and
DMSO was added. OD was measured at 492 nm
in microplate reader. IC 50 values of the test
samples were calculated and tabulated in Table 2.
Calculation
% of cell viability = Test/control * 100
% of cell Inhibition = 100-% of cell viability
Isolation of Human blood lymphocytes:
5 mL of peripheral blood human volunteers was
taken. Blood was added carefully to the layer of
Hisep 1077. Gradient centrifugation at 7000RPM
for 30 minutes at 370c. mononuclear cell (MNC)
layer was removed carefully and suspended in
medium. Centrifuged at 2000 rpm for 5minutes.
MNCs obtained were cultured in 96 well plates.
They are incubated for 24 hrs in 5% CO2
incubator. After reaching confluency, the cell lines
are treated with different concentrations of drug
and incubated for 24, 42 and 72 hrs. After 24, 48
and 72 hrs. of incubation add 20 microliters of MTT
(5mg/ml in PBS). Incubated until purple color was
obtained. Supernatant solution was removed and
 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles

DMSO was added. OD was measured at 492 nm MCF-7 Breast Cancer cells of Silver & Zinc oxide
in microplate reader. nanoparticles.

STATISTICAL ANALYSIS RESULTS AND DISCUSSION

All measurements and data were expressed as
mean ± Standard deviation and analyzed using
Minitab 17.0 statistical analysis tool. One Sample
T-tests were carried out to test the significance of
measurements (p<0.05). One Sample T-tests using
t-statistic (T-value) is used to compute the
confidence intervals and perform the hypothesis
test. It is the ratio of the departure of the estimated
value of a parameter from its hypothesized value to
its standard error has been used to examine the
effect of Silver and Zinc Oxide Nanoparticles on
mononuclear cells and the cell viability results on
The present work reports the formation of Ag and
ZnO nanoparticles using the plant Talinum
triangulare leaf extract. These biosynthesized
nanoparticles were characterised using UV-VIS-NIR
Spectrophotometer, FTIR, FESEM, EDAX.
Antibacterial and anti-cancerous activities were
studied and discussed below.
UV-VIS-NIR Spectroscopy of Talinum triangulare
plant leaf extract shown a peak at 267 nm, where
the formation of Ag and ZnO nanoparticles was
shown in the spectra. The peak at 242 nm
represents the formation of ZnO nanoparticles and
peak at 428 nm represents the formation of Ag
nanoparticles (Fig 3, 4, 5).

Fig.3: UV of plant extract

Fig.4: UV of ZnO nanoparticles Fig.5: UV of Ag nanoparticles

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 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles

FTIR peaks for the plant extract were changed after represents the involvement of components like
the formation of Ag and ZnO nanoparticles which
100 proteins, phenols and flavonoids (Fig 6, 7, 8).
80
Transmittance [%]

60
40
20
0

3889.20

3732.02
3664.39
3596.87
3550.41
3489.98

3068.40

2928.48

2312.31

1625.26

1381.68
1320.55

1105.45
1060.62

828.24
780.49

670.50
4000 3500 3000 2500 2000 1500 1000
Wavenumber cm-1

Fig.6: FTIR of plant extract

D:\FTIR DATA\2018 JULY\ABM.3 BPE SOLID 7/28/2018

3068.40 peak resembles =C-H stretch (alkene), 1625.26 peak resembles C=C stretch (alkene),
2928.48 peak resembles C-H stretch (alkane),Page1320.55
1/1 peak resembles C-F stretch (alkyl halide)
100
80
Transmittance [%]

60
40
20
0

3871.63
3778.46

3420.68

2924.76
2855.65

2373.40
2339.17

1599.50

1454.33
1384.85
1309.44

1022.79

770.23

616.02

4000 3500 3000 2500 2000 1500 1000

Wavenumber cm-1

Fig.7: FTIR of Ag nanoparticle

C:\Program Files\Bruker\OPUS_7.5.18\SITARAM.4 B2 SOLID 3/11/2017

3420.68 peak resembles N-H stretch (amine), 1022.79 peak resembles C-F stretch (alkyl halide),
1599.50 peak resembles C=C stretch (aromatic),Page770.23
1/1 peak resembles C-Cl stretch (alkyl halide)
1309.44 peak resembles C-N stretch (amine),

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 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles

100
80
Transmittance [%]

60
40
20
0

3910.84
3880.54
3862.89
3780.27
3704.10
3658.84

3425.88

2924.57
2854.88

2314.35

2006.58

1883.86
1853.68
1813.47
1786.50
1757.07
1731.72
1717.70
1631.90
1524.77
1490.65
1472.93
1441.83
1411.48
1381.37
1323.73
1116.56
1023.64
4000 3500 3000 2500 2000 1500 1000
Wavenumber cm-1

Fig.8: FTIR of ZnO nanoparticles

C:\Program Files\Bruker\OPUS_7.5.18\SITARAM.6 B1 SOLID 3/11/2017

3425.88 peak resembles O-H stretch (alcohol), FESEM of Ag and ZnO nanoparticles represents the
2924.57 peak resembles C-H stretch (alkane), shape and size of the particles and EDAX analysis
Page 1/1
1631.90 peak resembles C=C stretch (alkene), represents presence of elemental compounds (Ag
1023.64 peak resembles C-F stretch (alkyl halide) and Zn). (Fig 9, 10).

Fig 9: FESEM of Ag nanoparticles Fig 10: FESEM of ZnO nanoparticles

EDAX analysis shows the presence of elemental Ag and Zn (Fig 11, 12).

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 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles

Quantitative results
L,
80

60

Weight%
40

20

0
C O Ag

Fig 11: EDAX of Ag nanoparticles

Quantitative results

60

50

Weight%
40

30

20

10

0
O Zn

Fig 12: EDAX of ZnO nanoparticles

TEM ANALYSIS
Size and shape of the silver and zinc oxide nanoparticles represented in the TEM analysis [Fig 13].

Ag ZnO

Fig 13: TEM images of Ag and ZnO nanoparticles

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 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles

ANTIBACTERIAL ACTIVITY
Antibacterial activity of silver and zinc oxide
nanoparticles evaluated against the
microorganisms are tabulated in the Table. 1. The
maximum zone of inhibition was recorded and
observed that the zone increased as the
concentration of silver and zinc oxide nanoparticles
increases Fig. 13. The exact mechanism for the
green synthesis of nanoparticles were still unknown.
However, some recent literature has shown that
biomolecules such as proteins, phenols and
flavonoids present in the bio-systems play an
important role in the reduction of metal ions and
capping of the resulting nanoparticles [30].
Nanoparticles involved in the antibacterial activity
first attach to the surface of cell wall and gradually
disturbs its proper functioning such as permeability
and respiration results to breakage of outer
membrane. Then they are able to penetrate inside
the bacteria and inhibits the protein and nucleic
acid synthesis and cause further damage by
interacting with sulphur- and phosphorous
containing compounds such as DNA. Then they
release silver ions which exhibits bactericidal effect
[31]. Based on this plant leaf extract used for the
synthesis of Ag & ZnO nanoparticles contains
biomolecules such as phenols, flavonoids and
proteins were not only responsible for reduction of
Ag+ and Zn+ ions into nanoparticles but also acts
as stabilizing agents by capping. The inhibition of
bacterial growth depends on its size and
concentration of Ag and ZnO nanoparticles. Silver
nanoparticles shown a great zone of inhibition as
previous literature says, simultaneously the zinc
oxide nanoparticles also exhibit great bactericidal
effect and they are less toxic and cost effective too.

Table 1: Determination of MIC of Ag and ZnO nanoparticles

Organisms Ag Nanoparticles ZnO Standard Standard Plant
Nanoparticles Ag Zn extract
1 2 1 2
µg/ml µg/ml µg/ml µg/ml
Staphylococcus aureus 26 28 26 28 18 15 -
Streptococcus 27 28 26 27 17 13 -
Pseudomonas 25 27 25 26 15 12 -
aeriginosa

1µl 2µl 1µl

2µl

ZnO NP’S Ag NP’S

Fig.13: Zone of inhibition of Ag and ZnO Nanoparticles on Streptococcus plates(1µl and 2µl)

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 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles

ANTI CANCEROUS (CYTOTOXIC) ACTIVITY
Cytotoxicity of AgNP’S and ZnONP’S were
evaluated invitro against MCF-7 (Breast cancer) cell
lines. The cytotoxic activity was carried out by MTT
assay and results were summarized in the Table. 2.
The sample revealed a considerable cytotoxicity
against MCF-Cell line [32]. Interaction of
biomolecules with noble metals may be helpful in
cellbiology and medicine [33], biogenic
nanoparticles could be used as alternative or
complimentary agents in cancer treatment. In this
study nontoxic dose of AgNP’S and ZnONP’S on
PBMC (Peripheral Blood Mononuclear Cells)
reached 2µg/ml and 6µg/ml Fig. 14,15. The dose
recorded 62.37% of cell viability for silver
nanoparticles and 65.69% of cell viability for zinc
oxide nanoparticles on MCF-7 cell proliferation
Fig.16, 17. As they are near to 50% of cell death ,
the IC 50 values of silver nanoparticles and zinc
oxide nanoparticles were recorded as 37.63% and
34.31% and they are considered to be safe dose to
PBMC’S.

Fig.14: Cell Viability Results on MCF-7 (Breast cancer) cell lines with Ag nanoparticles

Statistical Inference
A one sample T-Test of Silver (Ag) Cell Viability Results on MCF-7 Breast cancer cells showed that all the
concentration values are less than the standard (74.21) at T=-2.52, P=0.043

One Sample T-test

Variable N Mean St Dev SE Mean 95% U-Bound T-Value P-Value

Silver (Ag) 4 67.94 4.97 2.49 73.79 -2.52 0.043

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 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles

Fig.15: Cell Viability Results on MCF-7 (Breast cancer) cell lines with ZnO nanoparticles

Statistical Inference
A one sample T-Test of Zinc (Zn) Cell Viability Results on MCF-7 (Breast cancer) cells showed that all
the concentration values are less than the standard (93.79) at T=-10.64, P=0.001

One Sample T-test

Variable N Mean St Dev SE Mean 95% U-Bound T-Value P-Value

Zinc (Zn) 4 71.57 4.18 2.09 76.49 -10.64 0.001

Fig.16: Effect of Silver Nanoparticle on Mononuclear cells at different concentrations

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 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles

Statistical Inference
A one sample T-Test of Silver (Ag) nanoparticle showed that all the concentration values are less
on mononuclear cells at different concentrations, than the standard (93.75) at T=-12.45, P=0.001

One Sample T-test

Variable N Mean St Dev SE Mean 95% U-Bound T-Value P-Value

Silver (Ag) 4 94.940 0.451 0.226 91.471 -12.45 0.001

Fig.17: Effect of Zinc oxide Nanoparticle on Mononuclear cells at different concentrations

Statistical Inference
A one sample T-Test of Zinc (Zn) nanoparticle on showed that all the concentration values are less
mononuclear cells at different concentrations, than the standard (92.16) at T=-5.55, P=0.006.

One Sample T-test

Variable N Mean St Dev SE Mean 95% U-Bound T-Value P-Value

Zinc (Zn) 4 90.720 0.519 0.259 91.331 -5.55 0.006

Table 2: Anticancer effect of Ag and ZnO nanoparticles as IC 50 in µg/ml

Cell line Ag Nanoparticles Std. Silver ZnO Nanoparticles Std. Zinc

MCF - 7 62.37% cell viability 74.21% 65.69% cell viability 93.79%
MNC 90.57% cell viability 93.75% 90.69% cell viability 92.16%
IC 50 Value At 2 µg/ml - At 6 µg/ml -

CONCLUSION characterized. Both obtained silver and zinc oxide

This study reports that Talinum triangulare plant nanoparticles showed good antibacterial and
leaf extract based biogenic AgNPs and ZnONPs anticancerous activity. Silver nanoparticles are
have been successfully synthesized and reported to possess anti-fungal, anti-bacterial,

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 Kantipriya K et al / Anti-bacterial and Anti-cancerous activity of Ag & ZnO nanoparticles

anti-viral and anti-inflammatory [35]. According 10. Iversen BG, Jacobsen T, Eriksen HM, et al. An
to the above silver nanoparticles showed great outbreak of Pseudomonas aeruginosa infection
antibacterial activity and cytotoxic activity, caused by contaminated mouth swabs. Clin
surprisingly zinc oxide nanoparticles also showed Infect Dis. 2007;44(6):794–801.
similar good results in both antibacterial and 11. Lanini S, D’Arezzo S, Puro V, et al. Molecular
anticancerous studies. As ZnONPs are less cost epidemiology of a Pseudomonas aeruginosa
and less toxic, along with single step and eco- hospital outbreak driven by a contaminated
friendly synthesized AgNPs could be a disinfectant-soap dispenser. PLoS One.
promisingagent in the field of drug delivery and 2011;6(2): e17064.
cancer therapy. 12. Kiffer C, Hsiung A, Oplustil C, et al; MYSTIC
Brazil Group. Antimicrobial susceptibility of
ACKNOWLEDGEMENTS gram-negative bacteria in Brazilian hospitals: the
My sincere thanks to Miss Animisha and Mr MYSTYC Program Brazil 2003. Braz J Infect Dis.
Radha Krishna for giving helping hand in the 2005;9(3): 216–224.
13. Andrade SS, Jones RN, Gales AC, Sader HS.
laboratory work and my special thanks to Dr.
Increasing prevalence of antimicrobial resistance
Lakshmi.
among Pseudomonas aeruginosa isolates in Latin
No conflicts of interest
American medical centres: 5-year report of the
SENTRY Antimicrobial Surveillance Program
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