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AUTHOR'S PROOF!

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Article Title

Antimicrobial potentials of Helicteres isora silver nanoparticles against

extensively drug-resistant (XDR) clinical isolates of Pseudomonas
aeruginosa

Article Sub- Title

Article Copyright Year

Springer-Verlag Berlin Heidelberg 2015

(This will be the copyright line in the final PDF)

Journal Name

Applied Microbiology and Biotechnology

Family Name

Particle

Given Name

8
9

Corresponding
Author

Kumar
Vinay

Suffix
Organization

S. P. Pune University

10

Division

Department of Biotechnology, Modern College of Arts,

Science and Commerce

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Address

Ganeshkhind, Pune 411 016, India

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e-mail

vinaymalik123@gmail.com

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Family Name

Mapara

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Particle

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Given Name

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Nikunj

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Author

Organization

S. P. Pune University

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Division

Department of Biotechnology, Modern College of Arts,

Science and Commerce

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Address

Ganeshkhind, Pune 411 016, India

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e-mail

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Given Name

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Sharma
Mansi

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Author

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S. P. Pune University

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Department of Biotechnology, Modern College of Arts,

Science and Commerce

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Address

Ganeshkhind, Pune 411 016, India

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e-mail

AUTHOR'S PROOF!
29

Family Name

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Particle

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Given Name

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Suffix

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Author

Shriram
Varsha

Organization

S. P. Pune University

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Division

Department of Botany, Prof. Ramkrishna More Arts,

Commerce and Science College

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Address

Akurdi, Pune 411044, India

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e-mail

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Author

Bharadwaj
Renu

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Organization

B. J. Government Medical College

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Department of Microbiology

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Address

Pune 411001, India

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Author

Mohite
K. C.

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Savitribai Phule Pune University

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School of Energy Studies, Department of Physics

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Address

Ganeshkhind, Pune 411007, India

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e-mail

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Received

8 June 2015

Revised

6 August 2015

Accepted

11 August 2015

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Schedule

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Abstract

Pseudomonas aeruginosa is a leading opportunistic pathogen and its expanding

drug resistance is a growing menace to public health. Its ubiquitous nature and
multiple resistance mechanisms make it a difficult target for antimicrobial
chemotherapy and require a fresh approach for developing new antimicrobial
agents against it. The broad-spectrum antibacterial effects of silver nanoparticles
(SNPs) make them an excellent candidate for use in the medical field. However,
attempts made to check their potency against extensively drug-resistant (XDR)
microbes are meager. This study describes the biosynthesis and biostabilization
of SNPs by Helicteres isora aqueous fruit extract and their characterization by
ultraviolet-visible spectroscopy, transmission electron microscopy, dynamic
light scattering, X-ray diffraction, and Fourier transform infrared spectroscopy.
Majority of SNPs synthesized were of 8-20-nm size. SNPs exhibited
dose-dependent antibacterial activities against four P. aeruginosa (XDR-PA)
clinical isolates as revealed by growth curves, with a minimum inhibitory

AUTHOR'S PROOF!
concentration of 300 g/ml. The SNPs exhibited antimicrobial activity against
all strains, with maximum zone of inhibition (16.4 mm) in XRD-PA-2 at
1000 g/ml. Amongst four strains, their susceptibilities to SNPs were in the
following order: XDR-PA-2 > XDR-PA-4 > XDR-PA-3 > XDR-PA-1. The
exposure of bacterial cells to 300 g/ml SNPs resulted into a substantial
leakage of reducing sugars and proteins, inactivation of respiratory chain
dehydrogenases, and eventual cell death. SNPs also induced lipid peroxidation,
a possible underlying factor to membrane porosity. The effects were more
pronounced in XDR-PA-2 which may be correlated with its higher
susceptibility to SNPs. These results are indicative of SNP-induced turbulence
of membranous permeability as an important causal factor in XDR-PA growth
inhibition and death.
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Keywords separated
by ' - '

Extensively drug resistant (XDR) - Pseudomonas aeruginosa - Helicteres isora

- Silver nanoparticles - Antimicrobial agents - Bacterial membrane

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Foot note
information

Nikunj Mapara and Mansi Sharma contributed equally to this work.

The online version of this article (doi:10.1007/s00253-015-6938-x) contains
supplementary material, which is available to authorized users.

Electronic supplementary material

ESM 1
(PDF 131 kb)

AUTHOR'S PROOF!

JrnlID 253_ArtID 6938_Proof# 1 - 31/08/2015

Appl Microbiol Biotechnol

DOI 10.1007/s00253-015-6938-x

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APPLIED MICROBIAL AND CELL PHYSIOLOGY

Antimicrobial potentials of Helicteres isora silver nanoparticles

against extensively drug-resistant (XDR) clinical isolates
of Pseudomonas aeruginosa
Nikunj Mapara 1 & Mansi Sharma 1 & Varsha Shriram 2 & Renu Bharadwaj 3 &
K. C. Mohite 4 & Vinay Kumar 1

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Received: 8 June 2015 / Revised: 6 August 2015 / Accepted: 11 August 2015

# Springer-Verlag Berlin Heidelberg 2015

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Abstract Pseudomonas aeruginosa is a leading opportunistic pathogen and its expanding drug resistance is a growing
menace to public health. Its ubiquitous nature and multiple
resistance mechanisms make it a difficult target for antimicrobial chemotherapy and require a fresh approach for developing new antimicrobial agents against it. The broad-spectrum
antibacterial effects of silver nanoparticles (SNPs) make them
an excellent candidate for use in the medical field. However,
attempts made to check their potency against extensively
drug-resistant (XDR) microbes are meager. This study describes the biosynthesis and biostabilization of SNPs by
Helicteres isora aqueous fruit extract and their characterization by ultraviolet-visible spectroscopy, transmission electron
microscopy, dynamic light scattering, X-ray diffraction, and
Fourier transform infrared spectroscopy. Majority of SNPs
synthesized were of 8-20-nm size. SNPs exhibited dosedependent antibacterial activities against four P. aeruginosa
(XDR-PA) clinical isolates as revealed by growth curves, with

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Keywords Extensively drug resistant (XDR) . Pseudomonas

aeruginosa . Helicteres isora . Silver nanoparticles .
Antimicrobial agents . Bacterial membrane

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Introduction

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Hospital-acquired infections are often associated with a high

rate of morbidity and mortality besides escalating the health
care costs (Ong et al. 2011). Pseudomonas aeruginosa is one
of the leading opportunistic pathogens causing nosocomial
infections and invasive diseases including pneumonia, particularly in critically ill, debilitated, or immunocompromised patients (Porras-Gomez et al. 2012; Atti et al. 2014). Its ubiquitous nature and ability to survive in moist environments make
it prevalent in hospital environments (Porras-Gomez et al.
2012). The innate potential of this gram-negative bacillus in
developing resistance to practically all classes of antibiotics
via multiple intrinsic as well as acquired resistance

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a minimum inhibitory concentration of 300 g/ml. The SNPs

exhibited antimicrobial activity against all strains, with maximum zone of inhibition (16.4 mm) in XRD-PA-2 at
1000 g/ml. Amongst four strains, their susceptibilities to
SNPs were in the following order: XDR-PA-2 > XDR-PA4 > XDR-PA-3 > XDR-PA-1. The exposure of bacterial cells
to 300 g/ml SNPs resulted into a substantial leakage of reducing sugars and proteins, inactivation of respiratory chain
dehydrogenases, and eventual cell death. SNPs also induced
lipid peroxidation, a possible underlying factor to membrane
porosity. The effects were more pronounced in XDR-PA-2
which may be correlated with its higher susceptibility to
SNPs. These results are indicative of SNP-induced turbulence
of membranous permeability as an important causal factor in
XDR-PA growth inhibition and death.

Nikunj Mapara and Mansi Sharma contributed equally to this work.

Electronic supplementary material The online version of this article
(doi:10.1007/s00253-015-6938-x) contains supplementary material,
which is available to authorized users.
* Vinay Kumar
vinaymalik123@gmail.com
1

Department of Biotechnology, Modern College of Arts, Science and

Commerce, S. P. Pune University, Ganeshkhind, Pune 411 016, India

Department of Botany, Prof. Ramkrishna More Arts, Commerce and

Science College, S. P. Pune University, Akurdi, Pune 411044, India

Department of Microbiology, B. J. Government Medical College,

Pune 411001, India

School of Energy Studies, Department of Physics, Savitribai Phule

Pune University, Ganeshkhind, Pune 411007, India

AUTHOR'S PROOF!
JrnlID 253_ArtID 6938_Proof# 1 - 31/08/2015

Appl Microbiol Biotechnol

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bioimaging, biosensors, biolabels, and biomedicines (Raju

et al. 2013). Compared with other metals, silver exhibits
higher toxicity to microorganisms and is less toxic to mammalian cells (Li et al. 2010). Silver nanoparticles (SNPs) have
emerged as the most effective owing to their potent antimicrobial efficacy against multiple bacterial and fungal species,
regardless to their susceptibility or resistance to common
drugs (Allaker and Memarzadeh 2014). The strong antimicrobial properties of silver nanostructures have been attributed to
(1) their high surface area-to-volume ratio, (2) their direct
effect on bacterial cell owing to their highly reactive nature,
and (3) the activity of silver ions constantly released from their
surface (Sondi and Salopek-Sondi 2004; Krychowiak et al.
2014). Recent investigations showed that SNPs could serve
as an excellent alternative to antibiotics in containing the
MDR microbial infections (Rai et al. 2012; Singh et al.
2013; Singh et al. 2014b).
Though there are various chemical and physical methods
for the synthesis of metal nanoparticles, they are eco-unfriendly, posing possible health hazards besides yielding bigger particles. Therefore, the development of biological approaches
for the synthesis of nanoparticles is evolving into an important
branch of nanotechnology, as the synthesis of SNPs through
biological methods offer various advantages including being
rapid, eco-friendly, non-toxic, and cost-effective (Raju et al.
2013; Bhati-Kushwaha and Malik 2014). Moreover, biologically prepared nanoparticles can easily be coated with lipid/
protein layer that confers physiological solubility and stability,
which are considered crucial for biomedical applications and
are bottleneck of other synthesis methods (Gurunathan 2014).
Amongst various biological means, plants have shown tremendous potential in novel SNP biosynthesis and are considered as biocompatible as they secrete functional biomolecules
which actively reduce the metal ions (Kumar and Yadav 2013;
Rai and Yadav 2013). Moreover, unlike some other biological
counterparts, the use of plant extracts drop the elaborate process of maintaining cell cultures and can also be suitably
scaled up for large-scale nanoparticle synthesis (Jeeva et al.
2014). The most crucial advantage though might be that the
plant extracts, unlike antibiotics, do not contribute to the emergence of resistant bacterial strains when used as antibacterial
agents (Krolicka et al. 2008). Helicteres isora, commonly
known as East Indian screw tree is an important subdeciduous medicinal plant possessing hypolipidemic, hypoglycemic, anti-nociceptive, antioxidant, and DNA damage
protection activities (Kumar et al. 2013).
Though there are numerous reports on assessing antibacterial activities of SNPs, few disseminate their efficacy against
MDR pathogens and very few against XDR/PDR isolates.
Therefore, the current study is the first such attempt to demonstrate an efficient biological synthesis of SNPs by H. isora
and an attempt to evaluate the efficacy of biosynthesized and
stabilized SNPs as a potent source of nanomedicine against

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mechanisms makes it a difficult target for antimicrobial chemotherapy (Lim et al. 2011; Singh et al. 2014a). Its inherent
resistance mechanisms including low permeability of outer
membrane, activation of drug efflux pumps, and production
of antibiotic inactivating enzymes (Moore and Flaws 2011)
are major contributors for the emergence of multidrugresistant (MDR), extensively drug-resistant (XDR), or even
pandrug-resistant phenotypes.
Various definitions have been used so far in medical literature for antimicrobial resistance (Paterson and Doi 2007;
Falagas and Karageorgopoulos 2008), causing a considerable
confusion amongst researchers as well as clinicians. Therefore, a recent consensus was developed by a group of international experts through a joint initiative by the European Centre
for Disease Prevention and Control and the Centers for Disease Control and Prevention to create a standardized international terminology for describing the acquired resistance profiles in major pathogenic bacteria (including P. aeruginosa),
often responsible for healthcare-associated infections as well
as being prone to multidrug resistance (Magiorakos et al.
2012). According to these harmonized definitions, multidrug
resistance is the acquired non-susceptibility to at least one
agent in three or more antimicrobial categories recommended
by this panel using documents and breakpoints from the Clinical Laboratory Standards Institute (CLSI), the European
Committee on Antimicrobial Susceptibility Testing
(EUCAST), and the US Food and Drug Administration
(FDA). Accordingly, extensive drug resistance was described
as non-susceptibility to at least one agent in all but two or
fewer antimicrobial categories, whereas pandrug resistance
(PDR) has been defined as non-susceptibility to all agents in
all antimicrobial categories.
In recent times, several P. aeruginosa nosocomial lifethreatening outbreaks have been reported in children and
adults with the progressive increase in the number of highly
drug-resistant P. aeruginosa strains (Kerr and Snelling, 2009;
Lanini et al. 2011; Atti et al. 2014). The wide spectrum of
diseases caused by this bacterium continues to grow from
urinary tract infection to septicemia, osteomyelitis, and endocarditis and thus posing new challenges as resistance to current empirical therapy limits the available treatment options
(Kerr and Snelling; 2009). Ironically, very few effective antibiotics are available for treating these infections, and, in some
cases, none at all (Pena et al. 2012). All this necessitates the
urgent need for fresh approaches to develop new bactericidals
to replenish the otherwise drying arsenal of anti-infective
agents against drug-resistant P. aeruginosa strains
(Amirulhusni et al. 2012; Rai et al. 2012; Singh et al. 2014a).
Nanotechnology provides a sound platform to alter the
physicochemical properties of metals to generate effective antimicrobials (Seil and Webster 2012). The synthesis of inorganic nanoparticles has gained unprecedented attention in recent years, due to their vast array of applications including

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Appl Microbiol Biotechnol

Materials and methods

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Plant material and extract preparation

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Mature pods of H. isora, growing in the forest areas of

Khopoli regions in Maharashtra, India, were collected and
samples were authenticated at Anantrao Pawar College, Pune,
and specimen voucher (No. APCP/21/2012-13) was submitted. The fruit extract was prepared by putting 5 g of thoroughly washed and finely grounded fruit (pod) powder into 100 ml
of sterile, triple deionized water in a 250-ml Erlenmeyer flask,
and then boiling the mixture for 5 min, the solution was
decanted and then filtered through a Whatman no. 1 filter
paper. The filtrate was collected and stored at 4 C for further
use.

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Biosynthesis and purification of SNPs

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Silver ions were reduced by addition of 5 ml of H. isora

fruit extract to 95 ml of 1 mM aqueous AgNO3 (SigmaAldrich, Germany) solution. The reaction mixture was
incubated at 40 C for 5 h and observations were made
every hour to encompass the change in color from yellowish brown to dark brown. The solution containing
SNPs was centrifuged at 12,000 rpm for 15 min to obtain
the pellet, which was then re-dispersed in sterile, double
distilled water to remove the un-interacted biological molecules. This process of centrifugation and re-dispersion
was repeated five times to ensure better separation of
the SNPs.

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Characterization of biosynthesized SNPs

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The biosynthesis of SNPs was monitored by observing the

absorbance spectra of each sample after every hour on a
UV-VIS spectrophotometer (UV-1800, Shimadzu Corp.,
Japan). The surface morphology and size of the
bioreduced SNPs were determined using a transmission
electron microscope (TEM, Tecnai 12 Cryo, FEI, Eindhoven, Netherlands). The size of the particles in 3 ml of the
reaction mixture was analyzed using dynamic light scattering (DLS) equipment (Zetasizer Nano-2590, Malvern
Instruments Ltd., Worcestershire, UK) in a polystyrene
cuvette. Phase formation was studied using X-ray diffraction. The diffraction data for thin and thoroughly dried
nanoparticle films on glass slides were recorded on an
X-ray diffractometer (D8 Advanced, Bruker, Germany)
with a Cu K (1.54 ) source. After the complete
bioreduction process, before nanoparticle extraction, the

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Clinical isolates of P. aeruginosa

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Four clinical isolates were obtained from the Department of

Microbiology, B. J. Government Medical College, Pune, India. The sources of the clinical isolates were pus (strain numbers PA-1 and PA-4), wound (strain number PA-2), and urine
(strain number PA-3) of four different patients. The purity as
well as identity of each isolate was confirmed in the laboratory
conditions by standard microbiological methods. The isolates
were further identified as P. aeruginosa on the basis of 16S
ribosomal RNA (rRNA) gene sequence homology (detailed
data not shown). The 16S rRNA gene sequences of all the four
isolates were submitted to GenBank with the accession numbers KR677082, KR677083, KR677084, and KR677085 for
PA-1 to PA-4, respectively. The cultures were submitted to the
Microbial Culture Collection (MCC), National Centre for Cell
Science, Pune, India (ref. nos.: D-15-028, D-15-029, D-15030, and D-15-031 for PA-1 to PA-4, respectively).

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Antibiotic resistance pattern of P. aeruginosa isolates

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The resistance pattern of four clinical isolates of

P. aeruginosa was performed by agar dilution method.
The antibiotic concentrations, growth conditions, medium, and inoculum size were selected according to CLSI
guidelines (2014). Briefly, Mueller-Hinton Agar (MHA)
was used for agar dilution with 0.5 McFarland standard
as inoculums and 1620 h incubation at 35 2 C. The
antibiotics used were one or more from each antimicrobial
category prescribed for P. aeruginosa as per the recommendations of the international consensus document
(Magiorakos et al. 2012) to characterize and confirm their
XDR nature. Along with this, the other antibiotics were
also used to check whether these strains have developed
resistance against antibiotics which are not prescribed by
CLSI to be used against them. The minimum inhibitory
concentration (MIC) interpretive criteria (g/ml) (susceptible, intermediate, and resistant) were used according to
the CLSI guidelines (2014) and are those required to
achieve plasma drug exposures on which breakpoints
were derived. Also, multiple fold concentrations as that
of resistance concentration were used to check the extent
of resistance. The antibiotics used to check the susceptibility of P. aeruginosa strains to confirm their XDR

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small volume of the supernatant from the reaction flask

was collected. The supernatant was dried using speed vacuum and subjected to Fourier transform infrared (FTIR,
IRAffinity-1, Shimadzu Corp., Tokyo, Japan) spectroscopic analysis performed in the range of 500
4000 cm1 using the potassium bromide pellet technique
to ascertain the plant peptides possibly coated on SNPs
during their biosynthesis.

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XDR clinical isolates of P. aeruginosa (XDR-PA), understanding the underlying antibacterial mechanism of SNPs.

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Table 1 Antibiotic resistance pattern of Pseudomonas aeruginosa clinical isolates against antibiotics to categorize their level of resistance as MDR/
XDR or PDR

t1:2

Antimicrobial categorya

Antimicrobial agent

MIC of P. Aeruginosa (PA) strains

(g/ml)

PA-1

Amikacin
Gentamicin
Antipseudomonal cephalosporins
Ceftazidime
Antipseudomonal fluoroquinolones
Levofloxacin
Phosphonic acids
Fosfomycin
Polymyxins
Colistin
Antipseudomonal penicillins + -lactamase inhibitors Piperacillin-tazobactam
Antipseudomonal carbapenems
Meropenem

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4
8
2

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16
4

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8

Aminoglycosides

>250
>250
100
>100

2
4
8
>50
16/4 32/464/4 128/4 >200/4
2
4
8
>10

t1:4
t1:5
t1:6
t1:7
t1:8
t1:9
t1:10
t1:11

MIC interpretive criteria

(g/ml)b

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t1:1

PA-2

PA-3

PA-4

>1000
>1000
>1000
>100
>350
>50
>200/4
>10

>1000
>1000
>100
>100
300
>50
>200/4
>10

>1000
>1000
>1000
>100
>350
>50
>200/4
>10

t1:3

The antibiotics were selected as recommended by the International Consensus (Magiorakos et al. 2011) for characterizing the MDR/XDR or PDR
nature of P. aeruginosa

b
The MIC interpretive criteria for characterizing microbial strains as susceptible (S), intermediate (I), and resistant (R) were followed (CLSI,
2014). The strains were characterized as extensively drug resistant owing to their non-susceptibility against at least one agent from all but two or
fewer antimicrobial categories

nature are given in Table 1. Additionally, the resistance

pattern of the isolates was also checked against various
antibiotics (other than the recommended for P. aeruginosa)
and are given in Supplementary Table S1.

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Antibacterial assays of SNPs

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Ten different concentrations (100, 200, 300, 400, 500, 600,

700, 800, 900, and 1000 g/ml) of SNPs were prepared in
sterile Milli-Q water. The antibacterial activities were determined by agar well diffusion assay (Perez et al. 1990). Twenty
milliliters of MH medium was dispensed in each presterilized
Petri plates in a biosafety chamber under aseptic conditions.
Bacterial suspension was then spread with the help of a cotton
swab onto the solidified medium. The media was then
punched with 6-mm-diameter hole and filled with 20 l of
different concentrations of H. isora SNPs. Respective AgNO3
and H. isora extracts were used as controls. The plates were
then incubated at 35 2 C for 1620 h. The diameter of the
zone of inhibition was measured as clear area around the well.
To examine the MIC of H. isora SNPs and the growth
curves of XDR-PA strains exposed to varying concentrations
of SNPs, the bacterial cells and SNP solutions were added
separately to 20 ml cultures in conical flasks of 50-ml capacity, resulting in final concentrations of SNPs as 0, 30, 50, 100,
150, 200, 250, and 300 g/ml, respectively, as described earlier by Li et al. (2010). The cultures were incubated at
35 2 C and shaken at 150 rpm. The growth rates were
determined by measuring optical density at 600 nm (OD600,
where 0.6 OD600 corresponds to 108 cells per ml) through time
series on a microtiter plate reader (iMark, Bio-Rad, USA).

Measurement of the effect of SNPs on membrane leakage

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In order to detect the leakage of reducing sugars and proteins

through bacterial membrane, the strains were subjected to the
treatments of 300 g/ml SNPs; P. aeruginosa cells were added
into 10 ml cultures with a final concentration of 106 cells/ml.
Control experiments were conducted without SNPs. The cultures were incubated at 35 2 C with shaking at 100 rpm.
One-milliliter aliquots were sampled from the cultures when
SNPs were added and kept for 3 h. The cultures were maintained
in ice bath immediately after treatment and harvested within the
next 45 min for measurement of membrane leakages, and this
is mentioned as 0 h treatment. The samples were centrifuged at
12,000 rpm, and supernatant was stored at 20 C for further use
(Li et al. 2010). The concentration of reducing sugars and
proteins were determined by the Miller (1959) and Bradford
(1976) methods, respectively. The respective concentration
blanks were used while recording the absorbance.

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Measurement of the effect of SNPs on malondialdehyde

content

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Thiobarbituric acid-reactive substances (TBARS) assay was performed to determine the concentration of malondialdehyde
(MDA, an indicator of lipid peroxidation) generated in the culture media (Dutta et al. 2012) with some modifications. Briefly,
an aliquot of 1 ml of culture medium was collected from the cells
subjected to SNP treatments for 0 and 3 h, as in case of determining the membrane leakages, and 200 l of 10 % SDS was
added and swirled vigorously. Two milliliters of freshly prepared
TBA was added to the reaction mix and incubated at 95 C for

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Measurement of the effect of SNPs on respiratory chain

dehydrogenase activity

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The activities of respiratory chain dehydrogenases of

P. aeruginosa strains were determined by iodonitrotetrazolium
chloride method as described by Li et al. (2010). In brief, the
enzyme activity was determined by the change of the spectrophotometric value of dark red iodonitrotetrazolium formazan.
The final reaction volume of 10 ml contained 300 g/ml SNP
concentration and 106 cells per ml of XDR-PAs. The SNPs
were absent in the controls. The reaction mixture containing
bacterial cells boiled for 20 min (to inactivate the enzyme
completely) served as negative control, whereas in case of positive controls, the cells were not boiled to maintain the native
activities of their enzymes.

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Cytotoxicity of SNPs

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The biosynthesized SNPs were tested for their cytotoxicity

against human (HeLa) cells using standard methylthiazole tetrazolium (MTT) test as described earlier by Shriram et al.
(2010). Briefly, the cells were seeded at the density of 15,
000 per well into 96-well plates and allowed to adhere for
24 h at 37 C. The next day, cells were treated with various
concentrations (0, 100, 200, and 300 g/ml) of SNPs of
H. isora in 1 % dimethyl sulfoxide (DMSO) for 24 h in triplicates. Absorbance was taken at 570 nm using 630 nm as
reference filter. Absorbance given by untreated cells (without
DMSO and SNPs) was taken as 100 % cell survival and the

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Results

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Biosynthesis and characterization of silver nanoparticles

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Bioreduction of Ag+ to Ag0 mediated by H. isora fruit extract

was monitored by recording the absorption spectra as a function of time. A change in color was observed with increasing
time from yellowish to dark brown (Fig. 1 inset). The UV-VIS
absorbance spectra of reaction mixture revealed that there was
no significant synthesis for the initial 1 h of incubation; however, the rate of synthesis increased sharply after 1 h and
maximum absorbance was recorded at 440 nm after 5 h of
incubation (Fig. 1) at 40 C with constant shaking at
150 rpm indicating the successful synthesis of SNPs.
The shape and size of the resultant particles were elucidated
with the help of TEM. The TEM images (Fig. 2a) confirmed

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results were compared with standard compound cisplatin

(35 M).

60 min. The reaction was allowed to cool to 25 C and centrifuged at 5000 rpm for 10 min, and the absorbance of the supernatant was measured at a 530-nm wavelength and respective
concentration blanks were used while recording the absorbance.

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Fig. 1 The UV-VIS absorption spectra of SNPs biosynthesized by

H. isora fruit extract with varying incubation times

Fig. 2 Transmission electron micrographs of SNPs of H. isora at

different scales (a) and histogram for SNP particle size distribution (b)

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Extensive drug resistance profile of P. aeruginosa isolates

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A bacterial isolate of P. aeruginosa is considered as XDR if it

shows non-susceptibility to at least one agent in all but two or
fewer antimicrobial categories including aminoglycosides,

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Fig. 3 X-ray diffraction (XRD)

pattern (a) and FTIR spectrum of
SNPs of H. isora (b)

as capping agents. FTIR analysis of SNPs from fruit extracts

of H. isora is shown in Fig. 3b. FTIR showed the strongest
peak at 3435 cm1 indicating OH group in polyphenols or
protein/enzymes or polysaccharide. A small peak at
2927 cm1 is due to CH stretching of alkanes. The bands
seen at 1625 cm1 is a characteristic of C=O carbonyl group
and C=C stretching. The bands obtained at 1381, 1224, and
1078 cm1 correspond to CN stretching vibrations. The
overall FTIR spectral analysis confirms the presence of plant
proteins in the sample of SNPs.

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the biosynthesis of SNPs; the notable finding of this investigation was the particle size and a large proportion of synthesized SNPs were nanospheres in the size range of 1020 nm,
which was further confirmed by the size distribution histogram (Fig. 2b), determined by DLS. Further evidence of
SNP biosynthesis was provided by the XRD pattern as illustrated in Fig. 3a. The peak positions with 2 values confirmed
the typical planes (111, 200, 220, and 311; indicated by the red
lines in Fig. 3a) of silver and the obtained data matched with
the Joint Committee for Powder Diffraction Set and confirmed
face-centered cubic structure for the SNPs. These findings
confirmed the crystalline nature of SNPs, whereas their sizes,
as calculated using the Debye-Scherrers formula, as well as
observations under TEM reaffirmed the particle size.
The FTIR spectrum confirmed the presence of functional
groups in the sample, which coat-cover the SNPs and known

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Fig. 4 Growth curves of XDRPA clinical isolates: XDR-PA-1

(a), XDR-PA-2 (b), XDR-PA-3
(c), and XDR-PA-4 (d) exposed
to different concentrations (0
300 g/ml) of H. isora SNPs for
different time periods (024 h)

antipseudomonal cephalosporins, antipseudomonal

fluoroquinolones, phosphonic acids, polymyxins,
antipseudomonal penicillins + -lactamase inhibitors, and
antipseudomonal carbapenems (Magiorakos et al. 2012). All
four clinical isolates were examined for their resistance profile against antibiotics belonging to these categories and the
results are presented in Table 1. The results confirmed the
XDR nature of all the isolates and the MICs were significantly higher than the prescribed MIC interpretive criteria by
CLSI.

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Antibacterial activities of SNPs

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The antibacterial investigations were performed in the solution and on Petri dishes. The biosynthesized SNPs
showed dose-dependent antibacterial activities against all
the selected XDR-PA isolates as revealed by the growth

t2:1
t2:2

t2:4
t2:5
t2:6
t2:7
t2:8
t2:9
t2:10

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Table 2 Antibacterial activity of

varying concentrations of SNPs
biosynthesized via H. isora fruit
extract

Concentration of SNPs (g/ml)

Control (plant extract)

Control (AgNO3)
600
700
800
900
1000

curves (Fig. 4). No bacterial growth was observed when

XDR-PA strains were exposed to SNPs at a 300 g/ml
concentration, indicating it as the MIC of SNPs to all four
XDR-PA strains. These findings hold significance since
SNPs showed remarkably lower MIC than the standard
antibiotics (Table 1).
Five different concentrations (600, 700, 800, 900, and
1000 g/ml) of H. isora SNPs were used to determine
their antibacterial effect against all the tested XDR pathogens by agar well diffusion method. The SNPs exhibited
antimicrobial activity against all the tested pathogens and
the zones of inhibition measured in a range of 2 to
16.4 mm (Table 2). Amongst four XDR-PA strains,
XDR-PA-2 was found to be most susceptible to SNPs
followed by XDR-PA-4, XDR-PA-3, and XDR-PA-1, respectively, as revealed by their corresponding zone of inhibition (Table 2, Supplementary Figure. S1). Once again,

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Zone of inhibition in mm against P. aeruginosa XDR strains

XDR-PA-1

XDR-PA-2

XDR-PA-3

XDR-PA-4

0.0
0.0
2.0
4.4
10.0
12.0
13.0

0.0
0.0
2.0
8.1
12.0
14.1
16.4

0.0
0.0
3.0
4.1
6.6
12.1
13.6

0.0
0.0
2.8
6.0
10.0
14.0
15.0

t2:3

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Fig. 5 Leakage of reducing

sugars from XDR-PA clinical
isolates: XDR-PA-1 (a), XDRPA-2 (b), XDR-PA-3 (c), and
XDR-PA-4 (d) treated with
H. isora SNPs. The amount of
reducing sugar is expressed as g/
mg dry weight of cells. Bar
represents standard error (n = 2)

the antibacterial activities were dose-dependent and increased with the SNP concentrations.

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Effect of SNPs on membrane leakage of reducing sugars

and proteins

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In order to determine the impact of SNPs on the membrane

integrity and leakage of membrane reducing sugars as well as
proteins, the cells were treated with SNPs at MIC. It can be
postulated from the results given in Fig. 5 that the SNPs significantly elevated the membrane leakage of reducing sugars.
Initially (at 0 h), there was no considerable reducing sugar
leakage from both the control and treated cells of all the
strains. The SNP treatment for 3 h resulted into multifold
increase in amount of reducing sugars leaked from the membranes. Maximum leakage was noticed in XDR-PA-2
(143 3.6 g/mg dry cell mass) followed by XDR-PA-1
(109 2.4), XDR-PA-4 (99 2.7), and XDR-PA-3
(75 2.1), respectively.
Similarly, SNP treatment for 3 h induced protein leakage
by increasing the membrane permeabilities of XDR-PA
strains. On the other hand, there was no significant change
in the amount of protein leakage from the non-treated control
groups (Fig. 6). The magnitude of protein leakage was highest
in XDR-PA-2 with 3.94 times higher than their control cells,
whereas the lowest leakage level was observed in XDR-PA-3
(2.3-fold higher than control). These findings suggest that
XDR-PA-2 cells failed to maintain their membrane integrity
and their comparatively higher membrane permeability than
other strains seems to be responsible for their higher susceptibility to SNPs.

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Effect of SNPs on MDA content

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The effects of H. isora SNPs were evaluated on the rate of

lipid peroxidation in bacterial cell membranes in terms of
TBARS level, an indicator of MDA which in turn is a
degradation product of lipid peroxidation. As shown in
Fig. 7, the levels of MDA were significantly higher in
SNP-treated cells over their respective controls in all four
pathogenic strains and increased further after 3 h of incubation. However, the magnitude of increase varied
amongst the strains, where XRD-PA-2 and XDR-PA-1
showed the highest and lowest MDA contents, respectively. These results hold importance since MDA content is
usually correlated with the amount of free radicals generated in the cells.

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Effect of SNPs on respiratory chain dehydrogenases

of P. aeruginosa

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Figure 8 reveals a substantial increase in respiratory chain

dehydrogenase activities with time in (+) control cells; on
the contrary, no significant change was observed in enzyme
activities of () control cells of P. aeruginosa boiled for
20 min. The exposure of bacterial cells of all pathogenic
strains to SNPs brought a sharp decrease in dehydrogenase
activities in a time-dependent manner. After an incubation of
cultures for 40 min treated with 300 g/ml SNPs, XDR-PA-2
showed 70 % lesser enzyme activity, against 56 % in XDRPA-3, 38 % in XDR-PA-4 followed by 27 % in XDR-PA-1
than their respective control counterparts.

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Fig. 6 Leakage of proteins from

XDR-PA clinical isolates: XDRPA-1 (a), XDR-PA-2 (b), XDRPA-3 (c), and XDR-PA-4 (d)
treated with 300 g/ml of H. isora
SNPs. The amount of proteins is
expressed as g/mg dry weight of
cells. Bar represents standard
error (n = 2)

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Cytotoxicity of SNPs

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Notably, the biosynthesized SNPs did not show any cytotoxic

activities against human HeLa cells at the concentrations ranging from 0 to 300 g/ml indicating their suitability for clinical
purposes. The SNPs did not affect cell survival or induced any
considerable cytotoxicity to the cells used. The cells showed
100, 98 2, 97 2, and 97 2 % cell survival when treated
with 0, 100, 200, and 300 g/ml SNPs, respectively (detailed

Fig. 7 Effect of 300 g/ml of

H. isora SNPs on MDA levels in
clinical isolates XDR-PA-1 (a),
XDR-PA-2 (b), XDR-PA-3 (c),
and XDR-PA-4 (d). The quantity
of MDA is expressed as nmol/mg
dry weight of cells. Bar represents
standard error (n = 2)

data not given). On the contrary, only 5 % cells survived when

treated with 35 M concentration of the standard compound
cisplatin.

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Discussion

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Owing to growing bacterial resistance to classical antibiotics,

there is an unprecedented upsurge in the investigations on

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indicating the complete synthesis of SNPs, as reported earlier

by Dar et al. (2013). TEM and DLS revealed the size of SNPs
and notable achievement was that the average size was below
20 nm (Fig. 2). The preparation of nanosized drug particles
with uniform size and shape has been described a critical
factor in the formulation of new and effective pharmaceutical
products (Sondi and Salopek-Sondi 2004; Martinez-Castanon
et al. 2008). Thus, the striking antibacterial activities of
biosynthesized SNPs in the present study may be attributed
to their small and uniform size and shape, as pointed out
earlier by Sondi and Salopek-Sondi (2004) and Singh et al.
(2014a).
The XRD pattern confirmed the crystalline nature of SNPs,
whereas their sizes, as calculated using Scherrers formula, as
well as observations under TEM reaffirmed the particle size,
as described previously (Abboud et al. 2014; Thombre et al.
2014). Therefore, the XRD pattern (Fig. 3a) proved strong
evidence in support of UV-VIS spectra as well as electron
microscopic images for the presence and desired size of SNPs.
Further, FTIR spectroscopic analysis was carried out to identify the possible biomolecules of H. isora responsible for
bioreduction, capping, as well as stabilization of
biosynthesized SNPs. Since different functional groups absorb characteristic frequencies of IR radiation, IR spectroscopy is one of the most popular tools for structural elucidation as
well as compound identification. IR spectrum confirmed the
presence of such compounds in the sample, which coat-cover
the SNPs and known as capping agents. Figure 3b indicates
the presence of OH group in polyphenols or protein/enzymes
or polysaccharide, besides plant proteins as reported

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identifying novel antimicrobial agents, and nanomaterials (1

100 nm), particularly the SNPs, have emerged as a strong
candidate for their powerful antimicrobial efficacies (Dar
et al. 2013). Moreover, the green synthesis of SNPs using
biological sources like fungi, bacteria, algae, and plants has
become the method of choice due to its environment-friendly
and cost-effective aspects (Chowdhury et al. 2014). Therefore,
present study was a step in this direction with an objective to
biosynthesize SNPs of H. isora and evaluates their antibacterial activities against XDR human pathogens of P. aeruginosa.
The SNPs were successfully biosynthesized and stabilized
by aqueous fruit extracts of an important medicinal plant
H. isora. This plant is distributed widely in forests throughout
India and is known for its wide-spectrum therapeutic properties including strong antimicrobial efficacies (Shriram et al.
2010). The MDR-reversal activities via curing the Rplasmids have been reported from fruit extracts previously
by our group (Shriram et al. 2010). However, to our knowledge, this is the first attempt for use of this plant to synthesize
SNPs and testing their efficacy against XDR pathogens. A
simple, robust, and cost-effective method which can be easily
scaled up and eco-friendly in nature was developed for synthesis of SNPs via reducing the aqueous AgNO3 solution by
H. isora fruit extract. The color of extract containing 1 mM
AgNO3 changed from yellowish brown to dark brown due to
the surface plasmon resonance phenomenon and provided a
convenient signature to indicate the formation of SNPs (Fig. 1,
inset) as reported previously (Gurunathan 2014; Singh et al.
2014a). The synthesis was further confirmed by UV-VIS absorption maxima at 440 nm after 5 h of incubation (Fig. 1)
Fig. 8 Effect of 300 g/ml of
H. isora SNPs on respiratory
chain dehydrogenase activities in
XDR-PA-1 (a), XDR-PA-2 (b),
XDR-PA-3 (c), and XDR-PA-4
(d). The positive (C(+)) and
negative (C()) controls represent
the unboiled and boiled XDR-PA
cells, respectively

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Acknowledgments The authors acknowledge the use of facilities created under the DST-FIST program implemented at Modern College,
Ganeshkhind, Pune. The authors sincerely thank the Dean, B.J. Govt.
Medical College, and the Principal, Modern College, for permitting to
work and utilize the necessary facilities.
Conflict of interest The authors declare that they have no competing
interests.

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References

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657Q4=Q5

Abboud Y, Saffaj T, Chagraoui A, El Bouari A, Tanane O, Ihssane B

(2014) Biosynthesis, characterization and antimicrobial activity of

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negative bacteria under the influence of SNPs. These results

are indicative of SNP-induced turbulence of membranous permeability as an important contributing factor for bacterial
growth inhibition. Similarly, our experimental results
(Fig. 7) indicated that SNP-induced bacterial growth inhibition may also be attributed to their abilities in extracting hydrogen from the allylic positions of unsaturated fatty acids,
since these allylic free radicals react with oxygen molecules
to form lipid peroxide radicals, which forms MDA via undergoing the rearrangements (Dutta et al. 2012). The peroxidation
of lipids might also have contributed in making the membranes porous. This elevated production of free radicals could
have been caused by an impeded electronic transport along the
respiratory chain in the damaged plasma membrane, as suggested by Su et al. (2009). Our results further reaffirm these
hypotheses, and the respiratory chain dehydrogenase activity
was inhibited in the bacterial cells treated with H. isora SNPs
(Fig. 8). These findings revealed the abilities of
biosynthesized SNPs in depressing the respiratory chain dehydrogenases and thus inhibiting the respiration ultimately
leading to cell death, with relatively higher rate in XDR-PA2 than other pathogenic strains, which may be correlated with
the greater antibacterial activities against XDR-PA-2. Similarly, Li et al. (2010) observed the inhibition of enzyme activity
in E. coli cells exposed to SNPs. Further, the cytotoxic profile
of SNPs revealed that the SNPs did not show notable cytotoxicity against human cells and therefore have the potential to be
used for drug applications.
Overall, through a systemic approach, the SNPs were
biosynthesized and stabilized by H. isora fruit extracts and
the synthesized SNPs successfully inhibited the growth of four
different XDR-PA clinical isolates apparently by targeting
permeable membranes, the key intrinsic resistance mechanism. The SNPs might have penetrated the bacterial cells via
enhanced membrane permeability and caused subsequent
leakage of proteins and reducing sugars besides generation
of lipid peroxidation. The bactericidal effects of SNPs may
also be attributed to their ability to inhibit the cellular respiration via destroying the respiratory chain dehydrogenases.

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previously by Shanmugam et al. (2014). It has been suggested

earlier that proteins bind to nanoparticles either through their
free amine groups or via cysteine residues (Thombre et al.
2014).
Though there are countless reports on antimicrobial potential of SNPs biosynthesized using various plants and/or microbes, only few of them described their antibacterial effects
against MDR/XDR strains. The present work holds significance as the biosynthesized SNPs proved a good source of
potential, eco-friendly, and cost-effective growth inhibitory
and bactericidal agents against XDR clinical isolates. Results
presented in Fig. 4 postulates that H. isora SNPs possess
strong bactericidal activity against XDR-PA strains. The cultures were maintained for 7 days for confirming the bactericidal effects of SNPs; however, no bacterial growth was observed after 24 h of incubation with 300 g/ml SNPs (Fig. 4;
detailed data not given). Interestingly, the MICs of SNPs were
significantly lesser than the standard antibiotics, indicating
that these SNPs have the potential of being developed into
effective nanomedicine against XDR-PA pathogens.
It has been suggested that the SNPs release the silver ions
and have the ability to anchor and penetrate the bacterial cell
wall and induce structural changes in the cell membrane
through increasing the cell permeability (Sondi and SalopekSondi 2004; Prabhu and Poulose 2012). However, the acting
mechanism of SNPs causing antibacterial effect is not very
well understood (Singh et al. 2014a). Therefore, in addition
to assessing the antibacterial activities of SNPs against XDRPA isolates, the underlying resistance mechanisms targeted by
the SNPs were also investigated. P. aeruginosa is a Gramnegative opportunistic nosocomial pathogen responsible for
broad range of infections and shows its non-susceptibility to
virtually all available commercial antibiotics. The outer membrane of these Gram-negative bacteria outside their peptidoglycan layer serves as a selective permeability barrier,
protecting bacteria from harmful agents such as drugs, detergents, toxins, and degradative enzymes and penetrating nutrients to sustain the bacterial growth (Li et al. 2010). Besides,
outer membrane porins are major constituents of multidrug
efflux systems. Therefore, the bacterial membrane plays a
pivotal role in making these organisms intrinsically resistant
to a wide variety of antibiotics (Meletis and Bagkeri 2013).
The antibacterial activities against four different isolates of
XDR-PA had a clear relevance with the abilities of SNPs in
disturbing the membrane permeability and its physiological
nature.
The integrity or stability of intracellular membranes was
notably affected in all the four XDR-PA isolates when they
were exposed to 300 g/g concentrations of SNPs. Results
showed that the SNPs apparently enhanced the permeability
of membranes for reducing sugars and proteins (Figs. 5 and 6).
In the same vein, Li et al. (2010) and Gurunathan (2014)
reported enhanced rate of membrane leakages from Gram-

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Moore NM, Flaws ML (2011) Antimicrobial resistance mechanisms in
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Ong DS, Jongerden IP, Buiting AG (2011) Antibiotic exposure and resistance development in Pseudomonas aeruginosa and Enterobacter
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Pena C, Gomez-Zorrilla S, Suarez C, Dorninguez MA, Tabau F, Arch Q,
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AUTHOR'S PROOF!
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Q1. Please check captured article title, if appropriate.

Q2. Please check if the edit to the sentence According to these harmonized definitions retained the
intended meaning of the text.
Q3. Please check if the edit to the sentence Therefore, the current study is the first such retained
the intended meaning of the text.
Q4. Please check whether in the references all species names are typeset in italics.
Q5. References "Jeeva et al, 2013, Kumar & Yadav, 2009, Shriram et al, 2010a, Shriram et al, 2010b"
were not cited anywhere in the text. Please provide a citation. Alternatively, delete the items from
the list.