INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH …ijprbs.com/issuedocs/2015/8/IJPRBS 1110.pdf · In the current study aqueous leaf extract of Psidium guajava was used to synthesize

Research Article CODEN: IJPRNK ISSN: 2277-8713 Marathe R. J., IJPRBS, 2015; Volume 4(4): 186-199 IJPRBS

Available Online at www.ijprbs.com 186

BIOGENICALLY SYNTHESIZED SILVER NANOPARTICLES EXHIBITS ANTIBACTERIAL

AND ANTIBIOFILM ACTIVITY AGAINST PSEUDOMONAS AERUGINOSA 6A (BC4)

MARATHE R. J1, DHUMAL C. D1, SONAWANE A. M2

1. Department of Microbiology, S.P. College Shardanagar, Baramati, Maharashtra, 413115 2. School of Biotechnology, Campus-11, KIIT University, Bhubaneshwar, Orissa751024, India

Accepted Date: 02/08/2015; Published Date: 27/08/2015

Abstract: Today the development of reliable and ecofriendly processes for the synthesis of nanomaterial is an important aspect of nanotechnology. One of the most important approaches used for the biosynthesis of nanoparticles is is by using plant bioactive molecules. Most of the plant extracts contain phenolic compounds, or tannins which act as both reducing and capping agents forming stable NPs. The present study deals with the synthesis of silver nanoparticles (AgNPs) using the leaf extract of Psidiumguajava. Addition of Psidiumguajava leaf extract to aqueous silver nitrate solution resulted in rapid reduction of the silver ions indicated the formation of AgNPs in solution. The characterization of synthesized nanoparticles was done by UV-visible spectroscopy (Uv–vis) and transmission electron microscopy (TEM). The TEM analysis showed that the size of the AgNPs ranges from 10-50 nm, whereas UV-Visible analysis showed the absorption spectra of AgNPs peak at 430nm.These biogenically synthesized AgNPs exhibited antioxidant and antibacterial activity against Pseudomonas aeruginosa 6A(bc4). Confocal Laser Scanning microscopy analysis (CLSM) showed that these AgNPs exhibit potent antibiofilm activity of Pseudomonas aeruginosa 6A (bc4). Exposure to these AgNPs also retarded swimming; swarming and twitching motility of P aeruginosa 6A.In conclusion our data suggest AgNPs as a promising template for the design of novel antibacterial agents.

Keywords: Pseudomonas aeruginosa, nanoparticles, antibacterial activity, antibiofilm activity

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Marathe R. J., IJPRBS, 2015; Volume 4(4): 186-199



INTRODUCTION

Pseudomonas aeruginosa exhibit wide spread occurrence in nature due to its nutritional

versatility. It is one of the bacteria found to be resistant to multiple antibiotics. Though it does

not cause infections in healthy individuals, it is emerging as an opportunistic pathogen in

immunocompromised humans that contributes to the high rate of mortality and morbidity. The

spectrum of disease caused by this bacterium continues to expand from urinary tract infections

to septicemia, osteomyelitis and endocarditis posing new challenges (Goossens et al,; Bahareh

Salehi et al,2013). Various factors such as resistance to multiple antibiotics, production of

extracellular toxins, enzymes, exopolysaccharides and biofilm formation are involved in the

establishment of infections (Ostertonet al,1987; Costerton et al. 2002)

Silver (Ag) has been used as an antimicrobial agent for years before the discovery of antibiotics

(Chopra et al , 2007). Silver materials have some advantages with respect to antibiotics. Various

antibiotics have been discovered, however, due to their reduced effectiveness against drug

resistance strains, development of new therapeutic strategies is required. (Campocciaet.al,

2006; Altet al, 2004). Ag is one of the potent bactericides and recently it has received increasing

attention because of its broad antibacterial spectrum including antibiotic resistant bacteria,

non-cytotoxicity at suitable doses, improved stability, and less possibility to develop drug

resistant strains(Zhao et al, 2009;Hardes et al, 2007;.Ramstedt et al,2009). It has been reported

that a certain range of Ag concentrations can kill bacteria without impairing mammalian cell

functions (Agarwalet al, 2010;Chen et al, 2006;Alt et al,2004)

Several methods such as chemical reduction in aqueous and non aqueous solvents,

photochemical reduction and sol–gel methods can be used to prepare stable AgNPs ( Sharmaet

al, 2009). It has been demonstrated that AgNPs retain their bactericidal properties

(Vaidyanathanet al.;Kviteket al,2008.)when included in coatings on orthopedic and other

implantable devices(Stevens, 2009;Vasilevet al. 2010; Jones et al2004; Nairet al. 2008;

andMonteiroet al. 2009), bandages for burn healing, and dressing materials for wound repair

(Jones et al.2004). It is widely believed that AgNPs are incorporated in the cell membrane,

which causes leakage of intracellular substances and eventually cause the cell death. Some of

the AgNPs also penetrate in to the cells. It is reported that the bactericidal effect of Ag-Nps

decreases as the size increases and is also affected by the shape of the particles.

Chemical synthesis ofAgNPs has many disadvantages such as toxicity, pollutes environment,

and deposition. On the other hand green synthesis of AgNPs has many advantages over it like it

is ecofriendly and nontoxic at specific dose (Anstaset al, 1998.). Green synthesis of Ag-NPs has

been reported using extracts of various plants such as Ocimum sanctum (Singhalet al,



2011),Bryophylum pinnatum (DebabratBaishyaet al.2012). In the current study aqueous leaf

extract of Psidium guajava was used to synthesize Ag-Nps.

Here, we evaluated the antibacterial activity of the biogenic AgNPs using various susceptibility

assays against Pseudomonas spp. Pseudomonas aeruginosa 6A (bc4) isolated from wastewater

has many potential abilities. Recently, we have shown that P aeruginosa 6A (bc4) is able to

synthesize siderophores and also promotes growth of plants (Marathe et al.2014). Here, we

have shown that AgNPs exhibit antibacterial and antibiofilm activities and also modulates the

synthesis of exopolysaccharides by Pseudomonas aeruginosa6A (bc4). We have also shown that

treatment with AgNPs reduce bacterial motility, increase hydrophobicity and also affect the

integrity of bacterial cell membrane, which lead to leakage of cytoplasmic proteins.

MATERIALS AND METHODS:

Bacterial strain, plant material and reagents

Pseudomonas aeruginosa 6A(bc4) was isolated from the waste water as described previously

(Marathe et al). Pseudomonas aeruginosa 6A(bc4) was grown on Muller Hinton agar medium

at 370c for 24 h. All chemicals were reagent grade and purchased from Fluka, Sigma Chemical or

Fisher Scientific. Plant material was collected from the nearby regions of Baramati,

Maharashtra, India. Plant was identified by expert Taxonomist from Department of Botany

Savitribai Phule Pune University, Pune.

Synthesis of Ag-Nps

Fresh 4 gm Guava (Psidium guajava

C for 1 h. After incubation it was filtered through

Whatmann filter paper. The filtered Guava plant extract was added to 1 mM solution of silver

nitrate at a 4:10 ratio. Then the solution was kept overnight at room temperature, which

resulted in the colour change from dark yellow to brownish red indicating the formation of Ag-

Nps.

Characterization of Ag NPs:

Synthesized nanoparticles were characterized by Ultraviolet-visible spectroscopy (Epoch,

Biotek) at a resolution of 10 nm from 350 nm to 530 nm. For TEM, a drop of aqueous solution

of Ag-NPs was placed on the carbon coated copper grids. The samples were dried and kept

overnight under a desiccators before loading them onto a specimen holder. The TEM

measurement was performed on JEM-2100, HRTEM, JEOL, JAPAN operating at 200 kV.



In vitro killing assay

To determine the antibacterial activity of AgNPs, overnight grown bacterial culture was

centrifuged at 5000 r.p.m. for 5 minutes and washed with 1 X PBS. Finally the pellet was

resuspended in LB medium. Then optical density of the sample was adjusted to 0.1 at 600 nm.

Bacteria were exposed to various concentrations of AgNPs in LB medium.Bacteria were

harvested at the indicated time points and the bacteria were enumerated by colony-forming

units (CFUs) assay. Medium with silver nitrate was used as positive controls. All samples were

plated in triplicate and values were averaged from three independent experiments.

Biofilm Assay

Overnight grown bacterial culture was diluted 1:100 in fresh LB medium. Diluted culture (200

μL o 96-well microtiter plate and incubated at 37°C for 24 hours without

shaking.The wells were then washed with 1X PBS to remove unbound cells. Different

concentrations of AgNPs were added to the wells and incubated at 37°C for 48 hours.

Thereafter, the medium was removed and the wells were thoroughly washed with 1X PBS and

. % ( / y o ( μL fo 2 . T y o

was removed and washed thoroughly with 1X PBS. For quantification of attached cells, the

crystal violet was solubilised in absolute ethanol and the absorbance was measured at 575 nm.

Reduction of the biofilm was compared with the untreated cells.

Effect of antioxidants on bactericidal activity of silver nanoparticles

To determine the effect of free radicals in the bactericidal activity of Ag-Nps, LB agar plates

were supplemented with 50 and l00 µg/ml of AgNPs. Antioxidants such as N-acetylcysteine

(NAC) and ascorbic acid (AA) were added to these plates at 10 mM final concentration. Then

plates were inoculated with 1x105 CFU per ml of Pseudomonas aeruginosa by spread plating

and the number of surviving bacteria were counted after 18 h of incubation at 370C. Plates

without AgNPs and antioxidants were used as controls.

Effect of Ag-Nps on protein leakage from bacterial cell membraneProtein leakage from

g B fo ’ p o y. 5 / of

g/ o o of g C for 6h. After the incubation period,

1ml of culture samples were centrifuged at 300g for 30 min and the protein leakage was

f o p g B fo ’ g O.D 9

nm.



Quantification of exopolysaccharides

The total carbohydrate assay was performed for the determination of EPS. Sterile glass slides

were immersed in P. aeruginosa culture with different concentrations of AgNPs in 24-well

polystyrene plates followed by 24 h incubation. After the incubation period, the glass slides

were removed and washed with 0.9% NaCl. The cell suspensions in 0.9% NaCl were transferred

to tubes containing equal volume of 5% phenol to which 5 volumes of concentrated

H2SO4 containing 0.2% of hydrazine sulphate was added. The mixture was incubated in dark for

1 h, centrifuged at 10,000 g for 10 min and the absorbance was measured at 490 nm.

Effect of nanoparticles on motility of bacteria

To determine the effect of AgNPs on the motility of the bacteria, overnight grown bacterial

culture was centrifuged at 5000 r. p. m. for 5 minutes, washed with 1X PBS and the optical

density of the sample was adjusted to 0.1 at 600 nm. T

o o of g fo 6 c. One loopful of bacterial culture was taken

from each Ag NP treated bacterial culture and spot was created at the centre of the 0.3%, 0.5%,

and 1 % LB agar plates. Then swim, swarm and twitching type of motility was checked after 24

h of incubation at 37oc. respectively.

Hydrophobicity assay

To study bacterial aggregation, bacterial adhesion to hydrocarbons (BATH) test was performed.

Mid-exponential culture of Pseudomonas aeruginosa 6A (bc4) was pelleted and the O.D. was

j o . 6 S o ’ . 7 cfu/ml bacteria were inoculated into

of S o ’ oC. After 5 days of incubation, 1 ml culture was

removed and added to equal volume of toluene and incubated overnight to form partitioning at

the aqueous-hydrocarbon interface. The hydrocarbon interaction affinity of the bacteria was

spectrophotometrically quantified by taking absorbance of the upper aqueous phase at 595nm.

Hydrophobicity index of each strain was determined by comparing the absorbance before and

after addition of toluene

RESULTS

Synthesis of Ag-NPs:

After incubation at room temperature a prominent brown color change was observed in the

solution containing a reaction mixture of 1mM aqueous silver nitrate and the leaf extract of

guava plant .In contrast there was no such color change observed in aqueous silver nitrate

incubated without leaf extract under the same conditions. It is well known that Ag-Nps exhibit

dark brown color in aqueous solution due to excitation of surface plasma vibration in Ag-Nps.



The color change from yellow to brown is the indication of formation of Ag-Nps and it is due to

reduction of silver ions. Almost all the herbal mediated silver nano solutions after incubation

time showed the color change from light to dark color.

Characterization of Ag-Nps:

The synthesis of plant derived Ag-Nps was confirmed by the UV-visible spectrophotometry. The

UV-visible spectrum of biosynthesized Ag-Nps showed a well observable sharp peak at 440nm,

the characteristic wavelength range of Ag-Nps (Figure 1a). The observable peak might be a

result of the excitation of longitudinal plasmon vibrations in Ag-Nps in the solutions. The

stability of the synthesized Ag-Nps was checked by measuring its absorption spectrum at

specific time interval for one month. Within this period no significant change in absorbance

were observed.

Size and shape of the synthesized Ag-NPs were determined by TEM study. The typical TEM

micrograph of the Ag-Nps is depicted in Figure 1b.The zeta potential of Ag-Np was measured

and found to be positive. By TEM analysis the average size of the synthesized Ag-Nps was found

to be 40 nm.

a b

Figure1: Characterization of Ag-NPs (a)UV-visible spectroscopic analysis to determine max

of Ag-NPs. (b)Transmission electron micrograph of Ag-Nps

In vitro killing assay

The antbacterial effect of Ag-NP was checked by exposing bacteria to varying concentrations of

Ag-NPs and number of CFU was determined. As shown in Figure 2, AgNPs exhibited dose

dependent killing effect on P. aeruginoase 6A such as at .....dose.......% of bacteria were killed.



Figure 2- CFU assay after 1 and 3 hour.

Biofilm Assay

P. aeruginosa have been studied in great detail with respect to their ability to form biofilms.

CLSM analysis showed development of dense biofilm formation on glass slides by Pseudomonas

aeruginosa 6A(bc4)strain(Fig.3a), while treatment with Ag-NPs showed disintergrated

recalcitrant biofilm structure. The crystal violet staining also showed dose dependent inhibition

of biofilm formation Treatment for 24 hours resulted in a decrease of more than 50% and 90 %

of of fo o μg/ μg/ o o , p y( g. .

The inhibition of biofilm formation by Ag-NPs was also studied with microtiter plate assay.

As shown in Figure 3b, AgNPs exhibited dose dependent biofilm formation.

% hydrophobicity

The percentage of hydrophobicity index was also decreased after treatment with Ag-NPs such

that 40 %hydrophobicity inhibition of P. aeruginosa was observed as compared to untreated

(Figure3c)

P aeruginosa (AuSno2)

1hrs

3hrs

0

20

40

60

80Control

1

2

5

10

20

50

Time (h)

CF

U

10

8



Controll 50ug/ml 100ug/ml 100ug/ml Controll

a c

C 1 2 5 10 20 50 100 C 1 2 5 10 20 50 100

Concentration (ug/ml) Concentration (ug/ml)

b1 b2

Figure3- Biofilm inhibition and Hydrophobicity assay (a)Confocal microscopy images of

effect of Ag-Nps on biofilm formation (b)Biofilm inhibition bymicrotiter plateassay (b1)after

24 hour (b2) after 48 hour( c) Determination of hydrophobicity index of P. aeruginosa after

treatment with 100μg/ml Ag-NPs for 24 h. Experiments were performed in triplicates

Effect of antioxidants on bactericidal activity of silver nanoparticles

To check the involvement of reactive oxygen species(ROS)in the antibactericidal activity of Ag-

Nps we used scavengers such as NAC and AA.These antioxidants were used to scavenge the

ROS produced by Ag-Nps in the bacterial cells. The control plate with NAC did not show any

growth inhibition (Figure 4...). But,withAA there was a slight reduction in the number of

surviving colonies.However the AA was able to protect the cells compeletlyfrom the toxicity of

Ag-Nps.This was evident from the 100% survival in plates supplemented with AA and at higher

concentration of Ag-Nps. NAC was able to protect 73% bacteria from the toxicity of Ag-Nps.

These data indicate that both these scavengers i.e NAC andAA protect the bacterial cells from

the toxic effects of silver nanoparticles .From this data we have showed that ROS are involved



in the bactericidal activity of Ag-Nps and these scavengers prevent these activity by interacting

with the ROS

A b c d

Figure 4-Effect of antioxidants on bactericidal activity of silver nanoparticles (a) Control

10mM AA (b) Control 10mM NAC (c)100ugAg-Np+AA(d) 100ugAgNp+AA+NAC

Effect of Ag-Nps on protein leakage from bacterial cell membrane

From this result it was found that Ag-Nps could enhance protein leakage by increasing the

membrane permeability of Pseudomonas aeruginosa cell. Initially protein leakage from the

membrane of the Pseudomonas cells treated with Ag-Nps was almost the same as that from

cells in the control group. After 6 hours incubation, protein leakage from the cells treated with

Ag-Nps was significantly increased compared to that from the cells in the control groups

indicating that Ag-Nps can increases the membrane permeability.

Figure 5- Effect of Ag-Nps on protein leakage from bacterial cell membrane

Quantification of exopolysaccharides

FIRST WRITE ON EXOPOLYSACHARIDE WAS MEASURED AND WHAT WAS THE PURPOSE. As the

concentration of Ag-Nps increases it affect or inhibit the secretion of exopolysaccharides during

bio film formation



Figure 6-Quantification of exopolysaccharides

Effect of nanoparticles on motility of bacteria

Exposure to biogenically synthesized AgNPs also retarded swimming, swarming and twitching

motility of P aeruginosa 6A(bc4) .Figure7 shows how the effect of nanoparticles on swim

,swarm and twitching kind of motility of P aeruginosa in a 0.3%, 0.5% ,and 1% of agar

respectively

Controll 1ug 10ug 100ug a

Controll 1ug 10ug 100ug b



Controll 1ug 10ug 100ug c

Figure 7- Effect of Ag-Nps on motility of bacteria(a)swim type of motility (b) swarm type of motility (c) twitching type of motility

CONCLUSION

The present work has evaluated a common plant not used for synthesis of Ag-Nps yet before.

The biogenically synthesized Ag-Nps were well capped and showed a strong antibacterial

activity, biofilm disruption activity, protein leakage activity which are very important from the

aspects of its bio-medical application. The method described how leaf extract of

Psidiumguajava can become a potential source of reducing and capping agent for the synthesis

of Ag-Nps.

REFRENCES

1. Ahmadi TS, Wang ZL, Green TC, Henglein A, El-Sayed MA: Shape-controlled synthesis of

colloidal platinum nanoparticles. Science 1996; 272: 1924-6.

2. AshaRaniPV.,Mun GLK., Hande MP., Valiyaveettil S. Cytotoxicity and genotoxicity of silver

nanoparticles in human cells. ACS Nano; 3: 279-90, (2009).

3. Athirah Nur A, NavindraKumari P, Zaini M-Z, LiewJian P, Durairaj R: Antibacterial effect of Ag-

Nps on multi drug resistant Pseudomonas aeruginosa. World Academy of Science, Engineering

and Technology 2012; (67):210-213.

4. Bos R, van der Mei CH, Busscher JH: Physico-chemistry of I nitial microbial adhesive

interactions. Its mechanisms and methods for study. FEMS Microbiol Rev 1999; 23: 179-230.

5. Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ: Bacterial

biofilms in nature and disease. Ann Rev Microbiol 1987; 987:435-464.

6. Chau CF, Wu SH, Yen GC: The development of regulations for food nanotechnology. Trend

Food Sci Technol 2007; 18:269-80.

7. Castellano JJ, Shafii SM., Ko F, Donate G, Wright TE, Mannari RJ: Comparative evaluation of

silver containing antimicrobial dressings and drugs. Int Wound 2007 ; 4:114-22.



8. Chen W, Cai W, Zhang L, Wang G, Zhang L: Sonochemical processes and formation of gold

nanoparticles within pores of mesoporous silica.J Colloid Interface Sci 2001; 238:291-5.

9. Ciriono O, Ghiselli R, Tomasinsig L, Orlando F, Silvestri C, Skerlavaj B: Efficacy of LL-37 and

granulocyte colony-stimulating factor in a neutropenic murine sepsis due to Pseudomonas

aeruginosa . Shock 2008; 30: 443-8.

10. Clinical and Laboratory Standards Institute: Performance standards for antimicrobial

susceptibility testing (Wayne, P.A) 1995: 15th informational supplement. Document M100- S15.

; 2000.

11. Chen J, Han CM, Lin XW, Tang ZJ, Su SJ: Effect of nanoparticle dressing on second degree

burn wound. Zhonghua WaiKeZaZhi 2006; 44:50-52.

12. Castrillón Rivera Laura E, Palma Ramos A: Biofilms: a survival and resistance mechanism of

microorganisms, antibiotic resistant bacteria - a continuous challenge in the new millennium,

Dr. Marina Pana (Ed.). 2012. ISBN: 978-953-51-0472-8, (2012).

13. Donlan MR, Costerton W: Biofilms: survival mechanisms of clinically relevant

microorganisms. ClinMicrobRev 2002; 15:167-193.

14. Evanoff DD, Chumanov G: Synthesis and optical properties of Ag-Nps and

arrays.Chemphyschem 2005 ; 6:1221-31.

15. FlemmingHC, Wingender J: The biofilm matrix.Nature Rev Microbiol 2010; 8: 623-633.

16. Friedman L, Kolter R, Two genetic loci produce distinct carbohydrate-rich structural

components of the Pseudomonas aeruginosa biofilm matrix. Journal of Bacteriology

2004;186(14):4457-4465.

17. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO: A mechanistic study of the antibacterial

effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 2000;

52:662-8.

18. Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R: Biogenic synthesis of

silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive

and gram-negative bacteria. Nanomedicine 2010;6: 103-9.

19. Goossens H: Susceptibility of multi-drug-resistant Pseudomonas aeruginosa in intensive

care units: results from the European MYSTIC study group.ClinMicrobiol Infect2003; 9(9):980-

983.

http://www.jnanobiotechnology.com/sfx_links?ui=1477-3155-12-2&bibl=B5






20. Gutierrez FM, Olive PL, Banuelos A, Orrantia E, Nino N, Elpidio Morales Sanchez EM:

Synthesis characterization and evaluation of antimicrobial and cytotoxic effect of silver and

titanium nanoparticles. Nano medicine 2010; 6:681-8.

21. Hall-Stoodley L, CostertonWJ, Stoodley P: Bacterial biofilms: from the natural environment

to infectious diseases. Nat Rev Microbiol 2004; 2:95-108.

22. Hong B, Kai J, Ren Y, Han J, Zou Z, Ahn CH: Highly sensitive rapid, reliable, and automatic

cardiovascular disease diagnosis with nanoparticle fluorescence enhancer and MEMS. Adv Exp

Med Biol 2008; 614:265-73.

23. Hong SH, Jeong J, Shim S, Kang H, Kwon S, Ahn KH, Yoon J: Effect of electric currents on

bacterial detachment and inactivation. Biotechnology and Bioengineering 2008; 100(2):379-

386.

24. Jaiswal S, Duffy B, Jaiswal AK, Stobie N, McHale P: Enhancement of the antibacterial

properties of Ag- p g β –cyclodextrin as a capping agent. Int J Antimicrob Agents 2010;

36:280-3.

25. Jefferson KK. Cerca N: Bacterial-bacterial cell interactions in biofilms: detection of

polysaccharide intercellular adhesins by blotting and confocal microscopy. Methods Mol Biol

2006; 341:119-26.

26. Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG: Pseudomonas aeruginosa

displays multiple phenotypes during development as a biofilm. J Bacteriol ; 184: 1140-1154.

27. John LP, Mark ER, Roger GF: Biofilm, Infection, and Antimicrobial Therapy. Boca Raton: CRS

Press , 2006.

28. Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, Kim SH, Park YK, Park YH, Hwang CY, Kim YK,

Lee YS, Jeong DH, Cho MH: Antimicrobial effects of Ag-Nps Nanomedicine: nanotechnology.

Biology and Medicine 2007; 3(1):95-101.

29. Liu P, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR:. Toll-like receptor triggering of a vitamin

D- mediated human antimicrobial response. Science 2006; 311:1770-3.

30. M T, O’Too G: Mechanisms of biofilm resistance to antimicrobial agents.Trends in

Microbiology2001; 9:34.

31. Mudshinge SR, Deore AB, Patil S, Bhalgat CM: Nanoparticles: emerging carriers for drug

delivery. Saudi Pharmaceutical Journal 2011; 19(3):129-141.









32. Overhage J, Campisano A, Bains M, Torfs EC, RehmBH, Hancock RE: Human host defense

peptide LL-37 prevents bacterial biofilm formation. Infect Immun 2008; 76:4176-82.

33. Pan Y, Neuss S, Leifert A, Fischler M, Wen F, Simon U: Size-dependent cytotoxicity of gold

nanoparticles. Small 2007; 3: 1941-9.

34. Salunkhe RB. Patil SV, Salunke BK, Patil CD, Sonawane AM: Studies on silver accumulation

and nanoparticle synthesis by Cochlioboluslunatus .Appl Biochem Biotechnol 2011; 165:221-

34.

35. Shahverdi AR., Fakhimi A, Shahverdi HR, Minaian S: Synthesis and effect of Ag-Nps on the

antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli.

Nanomedicine: Nanotechnology, Biology and Medicine 2007; 3(2):168-171.

36. Travan A, Pelillo C, Donati I, Marsich E, Benincasa M, Scarpa T: Non cytotoxic

silvernanoparticle- polysaccharide nanocomposites with antimicrobial activity.

Biomacromolecules 2009; 10: 1429-35.

37. Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG: Pseudomonas aeruginosa

displays multiple phenotypes during development as a biofilm. J Bacteriol 2002; 184:1140-1154.

38. Vieira SMC, Beecher P, Haneef I, Udrea F, Milne WI, Namboothiry MAG, Carroll DL, Park J,

Maeng S: Use of nanocomposites to increase electric “g ” o . Appl Phys

Lett 2007; 91: 203111-203113.

39. Wei D, Sun W, Qian W, Ye Y, Ma X: The synthesis of chitosan-based Ag-Nps and their

antibacterial activity. Carbohydr Res 2009; 344:2375-82.

40. Yen HJ, Hsu SH, Tsai CL: Cytotoxicity and immunological response of gold and Ag-Nps of

different sizes. Small 2009; 5: 1553-61.

41. Zhang L, Gu FX, Chan JM, Wang AZ, Langer RS, Farokhzad OC: Nanoparticles in medicine:

therapeutic applications and developments. Clin Pharmacol Ther 2008; 83:761-9.



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INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH …ijprbs.com/issuedocs/2015/8/IJPRBS 1110.pdf · In the current study aqueous leaf extract of Psidium guajava was used to synthesize