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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
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Springer-Verlag Berlin Heidelberg 2015 (This will be the copyright line in the final PDF)
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Applied Microbiology and Biotechnology
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Corresponding Author
Kumar Vinay
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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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Ganeshkhind, Pune 411 016, India
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[email protected]
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Mapara
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Nikunj
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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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Ganeshkhind, Pune 411 016, India
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Sharma Mansi
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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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Ganeshkhind, Pune 411 016, India
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Shriram Varsha
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S. P. Pune University
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Department of Botany, Prof. Ramkrishna More Arts, Commerce and Science College
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Akurdi, Pune 411044, India
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Bharadwaj Renu
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B. J. Government Medical College
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Department of Microbiology
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Pune 411001, India
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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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Received
8 June 2015
Revised
6 August 2015
Accepted
11 August 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 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 μg/ml. The SNPs exhibited antimicrobial activity against all strains, with maximum zone of inhibition (16.4 mm) in XRD-PA-2 at 1000 μg/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 μg/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. 57
Keywords separated by ' - '
Extensively drug resistant (XDR) - Pseudomonas aeruginosa - Helicteres isora - Silver nanoparticles - Antimicrobial agents - Bacterial membrane
58
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
1 3 2
4 5 Q1 6
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
10 11
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 μg/ml. The SNPs exhibited antimicrobial activity against all strains, with maximum zone of inhibition (16.4 mm) in XRD-PA-2 at 1000 μg/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 μg/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
[email protected] 1
Department of Biotechnology, Modern College of Arts, Science and Commerce, S. P. Pune University, Ganeshkhind, Pune 411 016, India
2
Department of Botany, Prof. Ramkrishna More Arts, Commerce and Science College, S. P. Pune University, Akurdi, Pune 411044, India
3
Department of Microbiology, B. J. Government Medical College, Pune 411001, India
4
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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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 Kα (1.54 Å) 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 16–20 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 (μg/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 cm−1 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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AUTHOR'S PROOF! JrnlID 253_ArtID 6938_Proof# 1 - 31/08/2015
Appl Microbiol Biotechnol 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 (μg/ml)
S
I
R
PA-1
Amikacin Gentamicin Antipseudomonal cephalosporins Ceftazidime Antipseudomonal fluoroquinolones Levofloxacin Phosphonic acids Fosfomycin Polymyxins Colistin Antipseudomonal penicillins + β-lactamase inhibitors Piperacillin-tazobactam Antipseudomonal carbapenems Meropenem
≤16 ≤4 ≤8 ≤2
32 8 16 4
≥64 ≥16 ≥32 ≥8
a
Aminoglycosides
>250 >250 100 >100 – ≤2 4 ≥8 >50 ≤16/4 32/4–64/4 ≥128/4 >200/4 ≤2 4 ≥8 >10
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t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11
MIC interpretive criteria (μg/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 μg/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 μl 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 16–20 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 μg/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 μg/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 4–5 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 μl 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 μg/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 μg/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 μM).
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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)
AUTHOR'S PROOF! JrnlID 253_ArtID 6938_Proof# 1 - 31/08/2015
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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 cm−1 indicating O–H group in polyphenols or protein/enzymes or polysaccharide. A small peak at 2927 cm−1 is due to CH– stretching of alkanes. The bands seen at 1625 cm−1 is a characteristic of –C=O carbonyl group and –C=C stretching. The bands obtained at 1381, 1224, and 1078 cm−1 correspond to –C–N 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 10–20 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 2θ 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-Scherrer’s 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 μg/ml) of H. isora SNPs for different time periods (0–24 h)
antipseudomonal cephalosporins, antipseudomonal fluoroquinolones, phosphonic acids, polymyxins, antipseudomonal penicillins + β-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
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Table 2 Antibacterial activity of varying concentrations of SNPs biosynthesized via H. isora fruit extract
Concentration of SNPs (μg/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 μg/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 μg/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
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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 μg/ 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 μg/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 μg/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 μg/ml of H. isora SNPs. The amount of proteins is expressed as μg/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 μg/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 μg/ml SNPs, respectively (detailed
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Fig. 7 Effect of 300 μg/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 μM 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 Scherrer’s 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 O–H 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 μg/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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656 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 μg/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 μg/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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AUTHOR'S PROOF! AUTHOR QUERIES AUTHOR PLEASE ANSWER ALL QUERIES.
U N C O R R EC TE D
PR O O F
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.
