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Biosynthesis of silver nanoparticles usingAeromonas sp. THG-FG1.2 and its antibacterialactivity against pathogenic microbes

Hina Singh, Juan Du & Tae-Hoo Yi

To cite this article: Hina Singh, Juan Du & Tae-Hoo Yi (2016): Biosynthesis of silvernanoparticles using Aeromonas sp. THG-FG1.2 and its antibacterial activity against pathogenicmicrobes, Artificial Cells, Nanomedicine, and Biotechnology

To link to this article: http://dx.doi.org/10.3109/21691401.2016.1163715

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Biosynthesis of silver nanoparticles using Aeromonas sp. THG-FG1.2and its antibacterial activity against pathogenic microbes

Hina Singh, Juan Du and Tae-Hoo Yi

Department of Oriental Medicine Biotechnology, College of Life Science, Kyung Hee University Global Campus, Yongin-si, Republic of Korea

ABSTRACTSilver nanoparticles were prepared through green route with the aid of Aeromonas sp. THG-FG1.2 asreductant. Visual observation, ultraviolet–visible spectroscopy, transmission electron microscopy, elemen-tal mapping, energy dispersive X-ray spectroscopy, selected area diffraction pattern (SAED), and X-raydiffraction (XRD) were used to characterize the synthesized silver nanoparticles. UV visible studies indi-cated the surface plasmon resonance at 400 nm which depicts the formation of silver nanoparticles. TheTEM images show spherical silver nanoparticles of 8–16 nm. XRD and SAED fringes revealed the structureof silver nanoparticles as face centered cubic (fcc). These silver nanoparticles also tested for their anti-microbial potential and showed effective antimicrobial activity against tested pathogens and thus applic-able as potent antimicrobial agent. Furthermore, the nanoparticles potential has been reconnoitered fortheir enhanced synergistic effect with antibiotics against multidrug resistant bacteria. Thus, the silvernanoparticles synthesized by Aeromonas sp. THG-FG1.2, were effective in inhibition of pathogenicmicrobes and also show enhanced antibacterial activity with antibiotics.

ARTICLE HISTORYReceived 27 January 2016Revised 4 March 2016Accepted 6 March 2016Published online 29 March2016

KEYWORDSAeromonas sp. THG-FG1.2;antimicrobial effect;biosynthesis; silvernanoparticles

Introduction

Nanotechnology is a method for the synthesis of various kindsof nanoparticles. It is one of the important fields with manyapplications in the revolutionary medicine (Song and Kim2009). On the other hand, nanobiotechnology deals with theuse of biological system for the synthesis of nanoparticles andit is an emerging and promising area in the modern medicaland agricultural science. Nanoparticles are gaining importanceand are being sufficiently applied in areas such as mechanics,biomedical sciences, magnetics, catalysis, optics, and energyscience. An important and exciting matter is the developmentof a consistent approach for the synthesis of nanoparticlesover a range of shapes, sizes, and chemical compositions, andwith high monodispersity (Singh et al. 2015a). Chemical andphysical methods for the synthesis of metal nanoparticles arewell known, but the methodologies are expensive and notenvironmental friendly, thus limiting the applications of metalnanoparticles in biological and medical platforms. To overcomethe limitation of physiochemical methodologies, the simple,low-cost, and eco-friendly technologies are needed wherebythese nanoparticles can be synthesized while avoiding the useof toxic and expensive chemicals and solvents (Singh et al.2015b). As a result, the green synthesis has received consider-able attention due to the growing need to develop eco-friendlytechniques for nanoparticles synthesis (Singh et al. 2015a). Agreat deal of effort has been put into the biosynthesis ofinorganic materials, especially metal nanoparticles using micro-organisms, plants, yeast, fungi, etc. (Singh et al. 2015a).Biosynthesis of nanoparticles using microorganisms or plants is

one of the eco-friendly, biocompatible, nontoxic, and cleanapproaches. The microbial enzymes or the plant phytochemicalswith anti-oxidant or reducing properties are usually responsiblefor reduction of metal compounds into their respective nano-particles (Ankamwar et al. 2005). Furthermore, biologically syn-thesized nanoparticles often exhibit water soluble andbiocompatible properties, which are essential for many pharma-ceutical and biomedical applications (Nithya et al. 2011).

Among various kinds of nanoparticles, silver nanoparticlesare one of the most important commercialized nanoparticlesbecause of its potential applications in different fields such asoptics, optoelectronics, drug delivery, gene therapy, biomedi-cine, biosensor, and oxidative catalysis (Darroudi et al. 2010,Nam et al. 2003, Parak et al. 2003, Schultz 2003, Wang et al.2015a). On the other hand, in recent years, most of thesepathogenic microorganisms have acquired resistance to anti-microbial agents (Gajbhiye et al. 2009, Shahverdi et al. 2007).It is well known, that silver is an effective antimicrobial agentand possesses a strong antibacterial activity against bacteria,viruses, and fungi, although the mechanism and the mannerof action are still not well known (Sharma et al. 2009, Singhet al. 2015a). Several main mechanisms underlie the biocidalproperties of silver nanoparticles against microorganisms. Forinstance, disruption of cell membrane or cell wall (Marambio-Jones and Hoek 2010, Nel et al. 2009) or by damaging DNA,proteins, and other phosphorus- and sulfur-containing cellconstituents (AshaRani et al. 2009, Marambio-Jones and Hoek2010, Nel et al. 2009).

The study focused on silver nanoparticles, due to theirutmost applicability. In the present study, biological route for

CONTACT Tae-Hoo Yi drhoo@khu.ac.kr Department of Oriental Medicine Biotechnology, College of Life science, Kyung Hee University Global Campus, 1732Deokyoungdaero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea� 2016 Informa UK Limited, trading as Taylor & Francis Group.

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the synthesis of silver nanoparticles was adopted since it isecofriendly, facile, and cost effective. Furthermore, the synthe-sized silver nanoparticles were evaluated for their applicabilityin antimicrobial activities against pathogens. Recently, silvernanoparticles were demonstrated enhanced antimicrobialactivity with antibiotics (Naqvi et al. 2013, Singh et al. 2015d).For this reason, we also explored the enhanced activity of sil-ver nanoparticles in combination with antibiotics against drugresistant bacteria.

Materials and methods

Materials

All the media were purchased from Difco, MB Cell, Seoul,Korea. Analytical grade silver nitrate (AgNO3) was obtainedfrom Sigma-Aldrich Chemicals, St. Louis, MO. The standardantibiotics discs: erythromycin (E15) 15 lg/disc, novobiocin(NV30) 30 lg/disc, lincomycin (MY15) 15 lg/disc, penicillin G(P10) 10 lg/disc, vancomycin (VA30) 30 lg/disc, and oleando-mycin (OL15) 15 lg/disc were purchased from Oxoid Ltd.,Basingstoke, England. The pathogenic microorganismsSalmonella enterica [ATCC 13076], Pseudomonas aeruginosa[ATCC 6538], Escherichia coli [ATCC 10798], Vibrio parahaemoly-ticus [ATCC 33844], Bacillus cereus [ATCC 14579], Bacillus subti-lis [KACC 14741], Staphylococcus aureus [ATCC 6538], Candidaalbicans [KACC 30062], and Candida tropicalis [KCTC 7909]were obtained from Korean Agricultural Culture Collection(KACC) and Korean Collection for Type Cultures (KCTC). Thebacterial strains were cultured on nutrient agar (NA) media at28 �C and preserved as a suspension in nutrient broth (NB)with glycerol (25%, w/v) and stored at �80 �C for furtherstudy. C. albicans and C. tropicalis were cultured onSabouraud dextrose agar (SDA) at 28 �C and preserved at�80 �C in glucose yeast peptone broth glycerol stock vials.

Isolation and identification of bacterial strain

Soil sample was obtained in sterile bag from SuwonFortress, South Korea. To obtain isolated strain, 1 g of soilsample was suspended in 10 mL of 0.85% (w/v) saline solu-tion, serially diluted, and spread on NA plates. For furtherscreening, the individual colonies were streaked on NAplate supplemented with 1 mM filter-sterilized silver nitrate(AgNO3) solution. The plates were incubated at 28 �C for48 h and observed for bacterial growth. The colonies resist-ant to silver nitrate were subculture and obtained in thepure form. Molecular identification of the isolated strainwas carried out using 16S rRNA sequencing based method.Genomic DNA was extracted and purified using a commer-cial Genomic DNA extraction kit (Solgent, Daejeon, Korea).The 16S rRNA gene was amplified using the universal bac-terial primer sets including 27F/1492R (Lane 1991) and518F/800R (Weisburg et al. 1991). The purified PCR prod-ucts were sequenced by Solgent Co. Ltd. (Daejeon, Korea).The 16S rRNA gene sequences of related taxa wereobtained from the GenBank database and EzTaxon-e server(Kim et al. 2012).

Biosynthesis of silver nanoparticles

Silver nanoparticles was synthesized following the methodalready described by Jo et al. (2015) and Singh et al. (2015b).Briefly, NB medium was prepared, sterilized, and inoculatedwith a fresh growth of bacterial isolate. The cultured flaskswere incubated in a rotating shaker set at 120 rpm for 24 hand 28 �C. After the incubation time, the culture was centri-fuged at 10,000 rpm for 10 min to remove the bacterial pellet.The supernatant was used for the synthesis of silver nanopar-ticles. The culture supernatant was separately added with1 mM filter-sterilized AgNO3 solution. Further, the culturesupernatant with 1 mM AgNO3 was incubated in an orbitalshaker for 48 h, at 120 rpm and 28 �C. The bioreduction of theAgþ ions in the solution was monitored by color change.After completion of the incubation period, the mixture wasfirst centrifuged at 2000 rpm for 5 min to remove any compo-nents of the medium and then the silver nanoparticles werecollected by high speed centrifugation at 14,000 rpm for20 min. The resulting silver precipitate was washed four tofive times with deionized water. Finally, the silver nanopar-ticles were collected in the form of a pellet and were air driedand kept for future experiments.

Characterization

Absorption spectra was recorded on an ultraviolet–visiblespectrophotometer (UV–Vis) (Ultrospec 2100 Pro, Amersham,Biosciences, UK). Transmission electron microscopy (TEM),elemental mapping, energy dispersive X-ray spectroscopy(EDX), and selected area diffraction pattern (SAED) measure-ments were made on a high resolution TEM JEM-2100F(JEOL) operated at an accelerating voltage of 200 kV. Thesamples for FE-TEM characterization were prepared by plac-ing a drop of silver nanoparticles on carbon coated coppergrid and dried at room temperature. The X-ray diffraction(XRD) analyses were performed on X-ray diffractometer, D8Advance, Bruker, Germany, operated at 40 kV, 40 mA, withCuKa radiation, at a scanning rate of 6�/min, step size 0.02,over the 2h range of 20–80�. The XRD sample was preparedby drying the silver nanoparticles collected after frequentwashing with water.

Analysis of antimicrobial activity

The antimicrobial activity of the silver nanoparticles was meas-ured against pathogenic microorganisms such as B. cereus, B.subtulis, S. aureus, S. enterica, E. coli, P. aeruginosa, V. parahae-molyticus on Mueller-Hinton agar (MHA) plates using the discdiffusion method. SDA plates were used for C. albicans and C.tropicalis. Overnight log culture of each pathogenic strain(100 lL, optical density of 0.5 at 620 nm) was spread evenlyon MHA and SDA plates and dried properly. Then, 50 lL(500 ppm) of the purified silver nanoparticle solution in waterwas added over each disc and kept for incubation at 28 �C for24 h. After incubation, the zones of inhibition were measuredaround each disc. The study was done in duplicates to checkthe reproducibility.

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Disc diffusion assay to evaluate synergistic effects

A disc diffusion method was used to assay the various anti-biotics for bactericidal activity against test strains on MHAplates. The standard antibiotics discs of erythromycin, novo-biocin, lincomycin, penicillin G, vancomycin, and oleandomy-cin were used. To determine the combined effects, eachstandard paper disk was further impregnated with 30 lL ofthe freshly prepared silver nanoparticles solution in water ata final content of 15 lg/disc. A single colony of strains P.aeruginosa, E. coli, S. enterica, and V. parahaemolyticus weregrown overnight in Muller-Hinton liquid medium on a rotaryshaker (120 rpm) at 28 �C. The inocula were prepared bydiluting the overnight cultures so that the final optical dens-ity is 0.5 at 620 nm. Hundred microliters of overnight log cul-ture were applied to the MHA plates along with thestandard (only antibiotics, control) and prepared disks con-taining silver nanoparticles. After incubation at 28 �C for 24 h,the zones of inhibition were measured. The assays were per-formed in triplicate.

Results and discussion

Screening and identification of strain

The screening results after incubation period showed thegrowth of bacterial strain THG-FG1.2 on NA plate supple-mented with 1 mM of AgNO3, which suggest that the strainTHG-FG1.2 was capable of tolerating silver salt. The 16SrRNA gene sequence of the strain determined in this studywas a continuous stretch of 1425 bp. According to theEzTaxon-e server, strain THG-FG1.2 shared 97.5% similaritywith Aeromonas schubertii. The 16S rRNA sequence of THG-FG1.2 has been submitted to NCBI with accession numberKU523692.

Synthesis and characterization of nanoparticles

Nanoparticles synthesized by Aeromonas THG-FG1.2 were con-firmed by visual observation with the appearance of colorchange in the reaction mixture. The color reaction wasobserved in which pale yellow supernatant and AgNO3 solu-tion changed into dark brown color which indicates the syn-thesis of silver nanoparticles, as the silver nanoparticles causesurface plasmon resonance due to which brown color appears(Naqvi et al. 2013, Singh et al. 2015c). The absorption spec-trum of this sample displayed in Figure 1 shows a well-defined plasmon band at 400 nm, characteristic of nanosizedsilver (Martınez-Castanon et al. 2008, Singh et al. 2015d, Wanget al. 2015a, 2015b). Figure 2(A and B) shows a representativeTEM image of the silver nanoparticles that were synthesizedby treating the AgNO3 solution with bacterial supernatant.The silver nanoparticles were spherical in shape with the sizerange of 8–16 nm.

The results of elemental mapping of the biosynthesized sil-ver nanoparticles showed the distribution of elements in theelectron micrograph of purified silver nanoparticles.Figure 2(C) corresponds to the electron micrograph of silvernanoparticles and Figure 2(D) shows the distribution of

elemental silver in the electron micrograph. The distributionof elemental silver was clearly visible in the elemental mapsand was found to be the predominant element in the respect-ive nanoproduct (Singh et al. 2015b, Wang et al. 2015a,2015b). The silver nanoparticles display an optical absorptionband peak at approximately 3 keV (Figure 3A), which is typicalof the absorption of silver nanoparticles due to surface plas-mon resonance (Singh et al. 2015e). The other metal iongroups also appeared in the EDX spectrum which correspondto the TEM grid utilized for study. The silver nanoparticles arecrystalline, as can be seen from the SAED recorded from oneof the nanoparticles in the aggregates (Figure 3B). Figure 3(C)shows the XRD pattern of nanoparticles, exhibited extremepeaks in the whole spectrum of 2h value ranging from 20 to80 and this pattern was analogous to the Braggs’s reflectionof silver nanocrystals (Gurunathan et al. 2014, Singh et al.2015d). The obtained XRD results were similar with previousstudy which showed synthesis of silver nanoparticles by bac-terial strains (Singh et al. 2015e, Vivek et al. 2011).

Antimicrobial and enhanced synergistic activity

To analyze the results, the zone of inhibition was measuredaround each disc, after the incubation period (Singh et al.2015b). The results indicated that the extracellularly synthe-sized silver nanoparticles have antimicrobial activity. Asshown in Figure 4(A–C); clear inhibition zones surroundedthe discs impregnated with silver nanoparticles (50 lL,500 ppm). The silver nanoparticles showed highest antimicro-bial activity against C. albicans followed by P. aeruginosa, V.parahaemolyticus, S. aureus, C. tropicalis, B. cereus, B. subtulis,E. coli., and S. enterica. The results of zone of inhibition areinterpreted in Table 1. The results were comparable and fol-lowed the previous studies based on antimicrobial activityof silver nanoparticles (Naqvi et al. 2013, Singh et al.2015b,2015d).

Additionally, the combination effect of silver nanoparticleswith different antibiotics was also investigated against P. aeru-ginosa, E. coli, S. enterica, and V. parahaemolyticus using thedisc diffusion method. The diameter of inhibition zones (in

Figure 1. UV–vis spectra of reaction mixture containing silver nanoparticles.

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Figure 2. TEM image of spherical shaped silver nanoparticles at 10 nm (A) and 20 nm (B). Elemental mapping results indicate distribution of silver elements, TEMmicrograph of silver nanoparticles solution (C), and silver nanoparticles (D).

Figure 3. EDX spectra of the whole scan area showing major peak of silver nanoparticles at 3 keV (A), SAED pattern of silver nanoparticles (B), and X-ray diffraction pat-terns of silver nanoparticles (C).

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millimeters) around the different antibiotic discs with or with-out silver nanoparticles against test strains are shown in Table2. The strains P. aeruginosa, E. coli, and S. enterica are com-pletely resistant to the antibiotics, but showed a zone ofinhibition when treated with silver nanoparticles. The otherpathogenic strain, V. parahaemolyticus, showed sensitivity tostandard antibiotics and enhanced zone of inhibition whenthe antibiotics discs were impregnated with silver nanopar-ticles. Silver nanoparticles displayed enhanced antibacterialactivity with antibiotics (Figure 5A–D). The highest foldincreases in area were observed for E15 and NV30 against S.enterica, for P 10 against P. aeruginosa and for MY15 againstV. parahaemolyticus. Thus the biologically synthesized silvernanoparticles also showed a similar potent bactericidal activityagainst pathogens as reported previously (Chen et al. 2011,Elbeshehy et al. 2015, Saravanan et al. 2014, Singh et al.2015b,2015d, Wang et al. 2015b).

Figure 4. Zones of inhibition of 50 lL of silver nanoparticles against Candida albicans [KACC 30062] (A1), Candida tropicalis [KCTC 7909] (A2), Bacillus cereus [ATCC14579] (B1), Bacillus subtulis [KACC 14741] (B2), Staphylococcus aureus [ATCC 6538] (B3), Salmonella enterica [ATCC 13076] (C1), Escherichia coli [ATCC 10798] (C2),Pseudomonas aeruginosa [ATCC 6538] (C3), and Vibrio parahaemolyticus [ATCC 33844] (C4). Note: AgNPs are silver nanoparticles solution (in water).

Table 1. Antimicrobial activity of silver nanoparticles against pathogenicmicroorganism.

Pathogenic strains Zone of inhibition (mm)a

Bacillus cereus [ATCC 14579] 13.5 6 0.5Bacillus subtulis [KACC 14741] 13 6 1.0Staphylococcus aureus [ATCC 6538] 15.5 6 0.5Escherichia coli [ATCC 10798] 13 6 0.2Pseudomonas aeruginosa [ATCC 6538] 16 6 0.1Vibrio parahaemolyticus [ATCC 33844] 16 6 0.1Salmonella enterica [ATCC 13076] 11 6 0.2Candida albicans [KACC 30062] 20 6 0.1Candida tropicalis [KCTC 7909] 15 6 0.5aMean diameter of zone of inhibition around each disc.

Table 2. Zone of inhibition (mm) of different antibiotics against test strains (inabsence and in presence of AgNPs at concentration of 15 lg/disc).

Salmonella enterica

Antibiotics (lg/disc) Antibiotics (a) AntibioticsþAgNPs (b)b FAc

Erythromycin (E15) 0a 12.5 0.77Lincomycin (MY15) 0a 10.5 0.61Novobiocin (NV30) 0a 12.5 0.77Penicillin G (P10) 0a 10.5 0.61Vancomycin (VA30) 0a 10.5 0.61Oleandomycin (OL15) 0a 10.5 0.61

Pseudomonas aeruginosaErythromycin (E15) 0a 12.5 0.77Lincomycin (MY15) 0a 12.5 0.77Novobiocin (NV30) 0a 13.5 0.85Penicillin G (P10) 0a 14.5 0.93Vancomycin (VA30) 0a 12.5 0.77Oleandomycin (OL15) 0a 12.5 0.77

Escherichia coliErythromycin (E15) 0a 12 0.73Lincomycin (MY15) 0a 14 0.89Novobiocin (NV30) 0a 11.5 0.69Penicillin G (P10) 0a 10.5 0.61Vancomycin (VA30) 0a 11 0.65Oleandomycin (OL15) 0a 11.5 0.69

Vibrio parahaemolyticusErythromycin (E15) 18 21.5 1.49Lincomycin (MY15) 21.5 25.5 1.81Novobiocin (NV30) 29.5 12.5 0.77Penicillin G (P10) 45.5 10.5 0.61Vancomycin (VA30) 11 10.5 0.61Oleandomycin (OL15) 20 10.5 0.61aIn the absence of bacterial growth inhibition zones, the discs’ diameters

(6 mm) were used to calculate the fold increase.bThe mean of inhibition zone diameter around the antibiotics disc containing

AgNPs (15 lg/disc). All experiments were done in triplicate and standard devi-ations were negligible.

cIncrease in fold area (FA) was calculated as (b2�a2)/a2.

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Conclusion

The study highlights the cost effective and environmentalfriendly biological synthesis of silver nanoparticles by thestrain Aeromonas THG-FG1.2, isolated from soil. The formedsilver nanoparticles were well characterized by UV–Vis spectra,transmission electron micrographs, elemental mapping, EDX,and XRD. The biosynthesized silver nanoparticles were crystal-line, spherical, and stable with particle size of 8–15 nm. Thesilver nanoparticles revealed good antimicrobial activityagainst the selected pathogenic microorganisms. Additionally,they also displayed the enhanced bactericidal activity with dif-ferent antibiotics. This biosynthesis approach appears to be afacile, cost-effective, non-toxic, ecofriendly alternative to theconventional physical and chemical methods. These silvernanoparticles may also be used in effluent treatment processfor reducing the microbial load, wound healing, and othermedical applications.

Disclosure statement

The authors report no conflicts of interest. The authors alone are respon-sible for the content and writing of this article.

Funding information

This work was conducted under the industrial infrastructure program (No.N0000888) for fundamental technologies which is funded by the Ministryof Trade, Industry and Energy (MOTIE, Korea).

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