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Journal of Industrial and Engineering Chemistry xxx (2015) xxx–xxx

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Biologically synthesized silver nanoparticles enhances antibioticactivity against Gram-negative bacteria

Sangiliyandi Gurunathan a,b,*a Dept of Animal Biotechnology, Konkuk University, 1 Hwayang-Dong, Gwangin-gu, Seoul 143-701, South Koreab GS Institute of Bio and Nanotechnology, Coimbatore, Tamilnadu, India

A R T I C L E I N F O

Article history:

Received 25 September 2014

Received in revised form 5 March 2015

Accepted 2 April 2015

Available online xxx

Keywords:

Antibacterial activity

Antibiotics

Escherichia coli

Klebsiella pneumoniae

Typha angustifolia

Silver nanoparticles

A B S T R A C T

Here we report a simple, fast, cost-effective, and nonpolluting approach for synthesis of silver

nanoparticles (AgNPs) using leaf extract of Typha angustifolia. We demonstrate the dose-dependent

antibacterial activity of AgNPs and different antibiotics against Escherichia coli and Klebsiella pneumoniae.

Furthermore, we demonstrate the efficacy of AgNPs in combination with various broad-spectrum

antibiotics against E. coli and K. pneumoniae. The results show that combinations of antibiotics and AgNPs

show significant antimicrobial effects at sub-lethal concentrations of the antibiotics. These data suggest

that combinations of antibiotics and AgNPs can be used therapeutically for the treatment of infectious

diseases.

� 2015 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering

Chemistry.

Contents lists available at ScienceDirect

Journal of Industrial and Engineering Chemistry

jou r n al h o mep ag e: w ww .e lsev ier . co m / loc ate / j iec

2728293031323334353637383940414243444546

Introduction

Recently, nanotechnology has emerged as one of the fastestgrowing areas of science and technology. Nanoparticles havegenerated much interest in academia as well as industry becausethey bridge the gap between bulk materials and atomic ormolecular structures [1]. Owing to their unique properties,nanomaterials are increasingly being used in commercial applica-tions in a variety of fields, including optics, electronics, magnetics,mechanics, catalysis, energy science, nanobiotechnology, andnanomedicine, particularly as antimicrobial agents for diagnosticpurposes [1]. In addition, silver nanoparticles (AgNPs) areextensively used for the production of clothing, catheters, electrichome appliances, and biomedical implants [2]. Because of theirwell-known antiseptic activities, silver compounds are used inclinical settings to prevent skin infections, such as in the treatmentof burns (e.g., silver sulfadiazine) and as coatings on varioussurfaces such as catheters [2,3]. Furthermore, nanoparticlespossess dimensions below the critical wavelength of light. Thisrenders them transparent, a property that makes them very useful

474849505152

* Corresponding author at: Department of Animal Biotechnology, Konkuk

University, 1 Hwayang-dong, Gwangin-gu, Seoul 143-701, South Korea.

Tel.: +82 2 450 0457; fax: +82 2 458 5414.

E-mail addresses: [email protected], [email protected]

Please cite this article in press as: S. Gurunathan, J. Ind. Eng. Chem.

http://dx.doi.org/10.1016/j.jiec.2015.04.005

1226-086X/� 2015 Published by Elsevier B.V. on behalf of The Korean Society of Indus

for applications in cosmetics, coatings, packaging, and diagnostics[4]. Because of the high demand, one trillion dollars’ worth ofnanotechnology-based products is expected on the market by theyear 2015 [5].

The most widely used methods for synthesis of metallicnanoparticles are traditional physical and chemical methods.Conventional physical methods tend to yield low amounts ofnanoparticles, while chemical methods are often toxic, consume alot of energy, and require the use of stabilizing agents such assodium dodecyl benzyl sulfate or polyvinyl pyrrolidone (PVP) toprevent agglomeration of the nanoparticles [4,6]. Therefore, a cost-effective, simple, rapid, high-yield, and environmentally friendlyapproach for the synthesis of metallic nanoparticles is needed.Among several possible approaches, biological synthesis ofnanoparticles is particularly promising owing to the readyavailability of resources including viruses, bacteria, fungi, algae,plants, and plant products [4].

Recently, several microorganisms have been exploited forsynthesis of silver and gold nanoparticles. For example, a silver-resistant bacterial strain isolated from silver mines, Pseudomonas

stutzeri AG259, accumulates AgNPs within the periplasmic space[7,8]. Parikh et al. [9] found that Morganella sp. RP-42 producedextracellular crystalline AgNPs of 20 � 5 nm when exposed to silvernitrate. Lactobacillus spp. produced microscopic gold, silver, and gold–silver alloy crystals of well-defined morphology when exposed tohigh concentrations of metal ions [10]. Bacillus licheniformis produces

(2015), http://dx.doi.org/10.1016/j.jiec.2015.04.005

trial and Engineering Chemistry.


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S. Gurunathan / Journal of Industrial and Engineering Chemistry xxx (2015) xxx–xxx2

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NPs with an average size of 50 nm both intracellularly [11] andtracellularly [12]. Sweeney et al. [13] demonstrated that Escherichia

li spontaneously formed cadmium sulfide semiconductor nano-ystals when incubated with cadmium chloride and sodium sulfide.coli also produces AgNPs with an average size of 50 nm [14].wshik et al. [15] demonstrated that MKY3, a silver-tolerant yeastecies, produced AgNPs ranging in size from 2 to 5 nm, andukherjee et al. [16] described the synthesis of intracellular AgNPsing the fungus Verticillium sp. In addition, plants have been used ine synthesis of nanoparticles. Shankar et al. [17] reported thetracellular synthesis of AgNPs by reduction of aqueous Ag+ ionsing extract of geranium leaves, and extract from lemongrassymbopogon flexuosus) was used to synthesize triangular goldnoprisms [18]. Interestingly, the synthesis of AgNPs using planttracts is fairly rapid compared with synthesis using bacteria orngi [17]. However, few plants have been exploited for the synthesis

silver or gold nanoparticles. Therefore, we attempted to use aeviously unexplored species, Typha angustifolia, for AgNP synthesis.angustifolia is a monocot found in tropical and temperate regions ofe world in marshes and wetlands of various depths. It is a commonant of wetlands and is an unexploited taxon that can be used as aurce of food, medicines, and fibers as well as for the synthesis ofnomaterials. Londonkar et al. [19] reported that crude aqueoustracts of the aerial part of T. angustifolia plants contained alkaloids,nnins, steroids, phenols, saponins, and flavonoids. Based on theesence of these compounds, we expected that the proteins,lysaccharides, or secondary metabolites of T. angustifolia leaftracts would reduce Ag+ ions to the Ag0 state, resulting in thermation of silver nanoparticles. Recently, Singhal et al. [20]nthesized AgNPs using Ocimum sanctum leaf extract and foundat these nanoparticles showed significant antibacterial activityainst E. coli and Staphylococcus aureus. Although several studiesve demonstrated the antibacterial activity of AgNPs, studies of thembination of AgNPs and antibiotics are warranted.Several studies have shown not only an increasing number of

fections caused by gram-negative bacteria worldwide but alsoounting rates of resistance. In a study of 1265 intensive care units

75 countries, Vincent et al. [21] found that gram-negativecteria were present in 62% of patients with an infection, whileam-positive bacteria were present in 47% of these patients.am-negative bacteria are highly adaptive pathogens that canvelop resistance to antibiotics through several mechanisms; thissistance is a serious concern in terms of public health and healthre costs [22–26]. Gram-negative bacteria are common causes oftra-abdominal infections, urinary tract infections, nosocomialeumonia, and bacteremia [27]. Tamma et al. [28] reported thatmbination antibiotic therapy may have benefits other than theevention of resistance during definitive treatment. We selected

coli and Klebsiella pneumoniae as model gram-negative bacteriacause both bacteria cause infections in the abdominal andinary tracts. In light of the observations described above, we firstvestigated the extracellular synthesis of AgNPs using leaf extract T. angustifolia. Second, we investigated the antibacterial effect ofe prepared AgNPs against E. coli and K. pneumoniae. Finally, wevestigated the effect of combinations of selected antibiotics withNPs against E. coli and K. pneumoniae.

aterials and methods

agents, bacterial strains, and culture conditions

Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), silvertrate, gentamicin, cefotaxime, and meropenem were purchasedm Sigma-Aldrich (St. Louis, MO, USA). All other chemicals wererchased from Sigma-Aldrich unless otherwise stated. The E. coli

Please cite this article in press as: S. Gurunathan, J. Ind. Eng. Chem

and K. pneumoniae strains used in the present study were from ourculture collections.

Bacterial culture and media preparation were carried outaccording to previously described methods [14]. Briefly, E. coli andK. pneumoniae were grown aerobically at 37 8C in MHB. Thecultures were maintained by streaking the organisms on LB agarplates and subculturing every fortnight. Pure colonies wereisolated and stored at �80 8C. Cells were harvested by centrifuga-tion at 6000 rpm for 10 min and resuspended in sterile LB mediumto obtain an optical density at 600 nm of 1.0.

Synthesis of AgNPs

T. angustifolia leaves were collected from a marshy area aroundCoimbatore, Tamilnadu, India, and stored at 4 8C until needed.Twenty grams of leaves were washed thoroughly with double-distilled water and then sliced into fine pieces, approximately1–5 cm2, using a sharp stainless steel knife. The finely cut leaveswere suspended in 100 mL of sterile distilled water and boiled for5 min. The resulting mixture was filtered through Whatman filterpaper (grade No. 1). The filtered extract was used for the synthesisof AgNPs by adding 10 mL of extract to 100 mL of 1 mM aqueousAgNO3 solution and stirring the mixture at 37 8C for 15 min. Thebioreduction of AgNO3 was monitored spectrophotometrically at420 nm.

Characterization of AgNPs

The synthesized nanoparticles were characterized according topreviously described methods [14]. Briefly, the prepared AgNPswere characterized primarily by UV–vis spectroscopy, which hasproved to be a very useful technique for the analysis of AgNPs.UV–vis spectra were obtained using a Biochrom (Cambridge, UK)WPA Biowave II UV–vis spectrophotometer. The synthesized AgNPswere freeze-dried, powdered, and analyzed by X-ray diffraction(XRD) spectroscopy. The spectra were produced using an X’Pert-MPD X-ray diffractometer (Philips, the Netherlands) and Cu Karadiation (l = 1.5405 A) over an angular range of 10 to 808 at 40 kVand 30 mA. The dried powder was diluted with KBr at a ratio of 1:100and analyzed by Fourier transform infrared spectroscopy (FTIR)using a Spectrum GX spectrometer (Perkin Elmer Inc., USA) withinthe range of 500 to 4000 cm�1. The size distribution of the dispersedparticles was measured using a Zetasizer Nano ZS90 (MalvernInstruments Ltd., UK). Transmission electron microscopy (TEM) wasused to determine the size and morphology of the AgNPs. A smallamount of aqueous dispersion was dropped on copper grids, whichwere dried and examined in the transmission electron microscope(JEM-1200EX).

Determination of minimum inhibitory concentrations of AgNPs and

antibiotics

To determine the minimum inhibitory concentrations (MICs) ofAgNPs and antibiotics, bacterial strains were cultured in MHB. Cellsuspensions were adjusted to obtain standardized populationsby measuring the turbidity with a spectrophotometer (DU530,Beckman, Fullerton, CA, USA). Susceptibility tests were performedby twofold microdilution of the antibiotics and AgNPs in standardbroth according to the Clinical and Laboratory Standards Institute(CLSI) guidelines (CLSI, 2003). The bacterial strains were grown inMHB to mid-log phase (1 � 106 cells/mL) and diluted in fresh MHB,and 0.1 mL of the diluted cell suspension was dispensed into eachwell of a 96-well microtiter plate. E. coli and K. pneumoniae werethen exposed to different concentrations of AgNPs or antibiotics.Growth was assayed by monitoring absorbance at 600 nm using amicroplate reader (EMax, Molecular Devices, Sunnyvale, CA, USA).

. (2015), http://dx.doi.org/10.1016/j.jiec.2015.04.005


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Fig. 1. Synthesis and characterization of AgNPs produced using leaf extract of Typha

angustifolia. The inset image shows containers with samples of AgNO3 (1), leaf

extract (2), and a mixture of AgNO3 and leaf extract at 15 min (3) and 30 min (4)

after mixing. The color of the mixture changed from green to dark brown 15 min

after mixing, indicating the formation of AgNPs. The absorption spectrum of the

AgNPs exhibited a strong, broad peak at 420 nm. This band was attributed to the

surface plasmon resonance of the AgNPs. (For interpretation of the references to

color in this figure legend, the reader is referred to the web version of this article.)

S. Gurunathan / Journal of Industrial and Engineering Chemistry xxx (2015) xxx–xxx 3

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The MICs of the AgNPs and antibiotics were determined as thelowest concentrations that inhibited visible growth of the bacteria.Antibiotic or AgNP concentrations that reduced the number ofsusceptible cells by less than 20% after 24 h of incubation weredesignated as ‘‘sublethal.’’ Viability assays were carried out withdifferent concentrations of antibiotics or AgNPs alone or withcombinations of sublethal concentrations of antibiotics and AgNPs.

Disc diffusion assay

An agar diffusion assay was performed as described previously,using MHA [29,30]. Conventional and broad-spectrum antibioticswere selected to assess the effect of combined treatment withantibiotics and AgNPs. Based on the CLSI standard, the followingconcentrations of antibiotics were used: gentamicin, 10 mg/mL;cefotaxime, 30 mg/mL; and meropenem, 10 mg/mL. For the combi-nation treatments, each standard antibiotic disc was impregnatedwith 1.4 mg of AgNPs. A single colony of each test strain was grownovernight in MHB on a rotary shaker (200 rpm) at 37 8C. The inoculawere prepared by diluting the overnight cultures with 0.9% NaCl to a0.5 McFarland standard. Inocula were applied to the plates alongwith control discs and discs containing different antibiotics. Similarexperiments were carried out with AgNPs alone. After incubation at37 8C for 24 h, zones of inhibition (ZOIs) were determined bysubtracting the disc diameter from the diameter of the totalinhibition zone. The assays were performed in triplicate. Antibacte-rial activity was quantified by the equation (A–B)/A � 100, where A

and B are the ZOIs for antibiotic and antibiotic with AgNPs,respectively.

In vitro killing assay

An in vitro killing assay was performed as described previously[31] with suitable modifications. Cells were grown overnight inMHB at 37 8C and then regrown in fresh medium for 4 h beforebeing collected by centrifugation and suspended in saline. A cellsuspension consisting of 106 cells/mL was incubated with variousconcentrations of antibiotics, AgNPs, or combinations of AgNPswith an antibiotic and incubated at 37 8C without shaking. Aliquots(100 mL) were withdrawn at specific time intervals and spread onMHA plates undiluted or after 10-fold serial dilution to determinethe number of colony forming units (CFUs). Experiments wereperformed with various controls including a positive control(AgNPs and MHB, without inoculum) and a negative control (MHBand inoculum, without AgNPs). All samples were plated intriplicate and values were averaged from three independentexperiments. The experiments with sublethal concentrations ofantibiotics or AgNPs, or combinations of AgNPs and antibiotics,were performed for 4 h at 37 8C.

Measurement of reactive oxygen species generation

Reactive oxygen species (ROS) generation was measured asdescribed earlier [32]. A quantitative assay for superoxide anionswas carried out using an in vitro toxicology assay kit based on XTTsodium salt (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetra-zolium-5-carboxanilide inner salt; catalog number Tox-2) pur-chased from Sigma-Aldrich, according to the manufacturer’sinstructions. E. coli and K. pneumoniae were grown in MHB in thepresence of meropenem, AgNPs, or a combination of meropenemand AgNPs. Cells were then washed with phosphate-buffered saline(PBS) and resuspended in PBS at a concentration of 2�106 viable cells(determined as CFUs) per milliliter. XTT was added to the cellsuspension at a concentration of 125 mM from a stock solution(7.5 mM) prepared in PBS. Cell suspensions were incubated at 30 8Con a rotary shaker for 24 h, aliquots were spun in a microfuge, and


the absorbance of the supernatant was measured at 450 nm. XTTreduction in the absence of cells was always determined as a controland subtracted from the values observed in the presence of cells.

Statistical analysis

All experiments were carried out in triplicate and repeated atleast three times. The results are presented as means � SD. Allexperimental data were compared using Student’s t test. A p-valueless than 0.05 was considered statistically significant.

Results and discussion

Synthesis and characterization of AgNPs

To synthesize AgNPs, the leaf extract from T. angustifolia wasused as both reducing and stabilizing agent. In a typical reaction,10 mL of T. angustifolia leaf extract was added to final volume100 mL contains of 1 mM aqueous AgNO3 solution with 10 mL ofleaf extract, and the mixture was stirred with a magnetic stirrer atroom temperature for 15 min. The green mixture of silver nitrateand leaf extract changed rapidly after 15 min at 37 8C to a brownsuspension, whereas silver nitrate without leaf extract showed nocolor change (Fig. 1 inset). This indicated that AgNPs can besynthesized using T. angustifolia leaf extract. Aqueous extracts ofleaves of T. angustifolia are known to contain alkaloids, tannins,steroids, phenols, saponins, and flavonoids, and these secondarymetabolites could induce the formation of nanoparticles byserving as reducing agents. Chandran et al. [33] synthesizedsilver nanoparticles by incubating Aloe vera leaf extract withsilver nitrate for 24 h. Interestingly, in our experiment the colorchange was observed within 15 min of incubation; however, thereaction was carried out for 30 min. The reduction of silver ionsusing leaf extract occurs more rapidly than with bacterial culture[11,14].

We confirmed the synthesis of AgNPs using UV–vis spectros-copy. The strong surface plasmon resonance (SPR) of the AgNPsproduced a peak centered near 420 nm. The SPR band at 420 nmindicated that formation of AgNPs had occurred (Fig. 1). Mulvaney(1996) Qreported that AgNPs exhibit a yellowish-brown color inwater owing to excitation of surface plasmon vibrations.



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D analysis

To confirm the crystalline nature of the AgNPs, XRD analysisas performed. The XRD spectrum of the AgNPs showed fourtense peaks with 2u values of 23.618, 29.908, 33.878, and 46.738ig. 2). The five diffraction peaks in the standard pattern of silvere indexed as reflections of the (1 1 1), (2 0 0), (2 2 0), (3 1 1), and 2 2) planes of face-centered cubic (fcc) silver (JCPDS file No. 04-83). The (2 0 0), (2 2 0), (3 1 1), and (2 2 2) Bragg reflections are

eak and broadened relative to the intense (1 1 1) reflection. Thisature indicates that the nanocrystals are (1 1 1) oriented [17,34].

The Full Width at Half Maximum (FWHM) values measured for1 1, 2 0 0, 2 2 0, and 3 1 1 planes of reflection were used with thebye–Scherrer equation to calculate the size of the nanoparticles.e particle sizes obtained from XRD line broadening agreed well

ith that obtained from TEM. From these the average particle sizeas found to be around 8 nm. A comparison of our XRD spectrumith the standard spectrum confirmed that the silver particlesrmed in our experiment were in the form of nanocrystals, as theaks at 2u values of 23.618, 29.908, 33.878, and 46.738 corresponded

reflections of the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of fccver, respectively. A few additional intense and unassigned peaksere also observed in the area of the characteristic peaks of silver.ese additional and unidentified sharp Bragg peaks may havesulted from bioorganic compounds or proteins present in the leaftract or may be related to amorphous and crystalline organicases on the surface of the nanoparticles. Similar results weretained for silver nanoparticles synthesized using geranium leaftract [17], Krishna tulsi (O. sanctum) leaf extract [35], Ipomoea

dica flowers [36], carob leaf extract [37], and mushroom extract8].

IR analysis of AgNPs

FTIR measurements were used to identify the biomolecules ine leaf extract that were responsible for the reduction of the Ag+

ns and the capping of the bioreduced silver nanoparticles [17].ter complete reduction of the Ag+ ions and formation of theNPs, the silver nitrate solution was centrifuged at 10,000 rpm for

min to isolate the AgNPs from free proteins or other compoundsesent in the solution, and the centrifugate was collected for FTIRalysis [17]. Fig. 3A shows the FTIR spectra of the T. angustifolia

af extract. The FTIR spectra reveal the presence of differentnctional groups. The IR bands observed at 3314 and 1635 cm�1 in

Fig. 2. XRD pattern of silver nanoparticles sy


dried leaf are characteristic of the O–H and C55O stretching modesfor the OH and C55O groups possibly of secondary metabolitespresent in the leaf extract.

The FTIR spectrum of the AgNPs exhibited peaks at 1728 and1635 cm�1, which could be assigned to ester C55O groups ofchlorophyll [17,40] and the amide I bond of proteins [20,41],respectively. The water-soluble fraction of T. angustifolia leavescontains large amounts of alkaloids, tannins, steroids, phenols,saponins, and flavonoids [19], and these secondary metabolitescould induce the formation of nanoparticles by serving as reducingagents. It is possible that terpenoids also contribute to thereduction of silver ions and, in the process, are oxidized to carbonylgroups, resulting in the band at 1728 cm�1. Upon formation of theAgNPs, the peak corresponding to the amide I band at 1635 cm�1

broadened, indicating capping of the AgNPs by proteins (Fig. 3B).The absorption peak at 1635 cm�1 arises from the carbonyl stretchin proteins, while the peak at 3409 cm�1 is due to OH stretching inalcohols and phenolic compounds [20,41]. The absorption peak at1635 cm�1 is close to that reported for native a protein [42], whichsuggests that proteins are interacting with the biosynthesizednanoparticles and that their secondary structure is not affectedduring reaction with the Ag+ ions. It is known that proteins canbind to gold nanoparticles through either free amine groups orcysteine residues [43]. A similar mechanism is possible for silvernanoparticles, since the leaf extract from T. angustifolia capped thesilver nanoparticles, thereby stabilizing them. Similar FTIRpatterns were observed for silver nanoparticles synthesized usinggeranium leaf extract [17] or O. sanctum leaf extract [20,35].

XPS analysis of AgNPs

X-ray photoelectron spectroscopy (XPS) was utilized toinvestigate the chemical state of the leaf extract of T. angustifolia

leaves mediated synthesis of AgNPs. The quantitative Ag/C atomicratios of the samples were determined using the peak area ratio ofthe corresponding XPS core levels and the sensitivity factor (SF) ofeach element in XPS [39]. Fig. 4 shows high resolution XPS spectraof the C (1s) core level for the AgNPs. As shown in Fig. 4, two peakslocated at the binding energies of 368.4 and 373.6 eV wereobserved, which confirming the successful formation of Ag(0) byleaf extracts. The binding energies of Ag(3d5/2) and Ag(3d3/2)peaks were found at binding energies of 368.4 and 373.6 eV,respectively. To further understand the chemical state of the AgNPson the surface, a detailed deconvolution of the Ag(3d) peak was

nthesized using T. angustifolia leaf extract.



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Fig. 3. FTIR pattern of T. angustifolia leaf extract (A) and silver nanoparticles synthesized using T. angustifolia leaf extract (B).


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also performed. The binding energy of the Ag(3d5/2) core level forAg, Ag2O, and AgO is 368.4, 368.3, and 367.5 eV, respectively. Basedon the Ag (3d5/2) peak analysis, we have found that about 94% ofthe silver atoms on the surface were in the Ag0 (metallic) state,while only about 1% and 5% of the silver atoms were in the Ag+ andAg2+ chemical states, respectively. These results are in goodagreement with earlier report suggested that synthesis of silverusing Allophylus cobbe leaf extracts [39].

Dynamic light scattering analysis

Dynamic light scattering (DLS) as a useful technique to evaluateparticle size, size distribution, and the zeta potential of nanoma-terials in solution [14,38,39]. The characterization of nanoparticlesin solution before assessment of in vitro toxicity is a high priority


[44] because particle size, size distribution, particle morphology,particle composition, surface area, surface chemistry, and particlereactivity in solution can affect nanoparticle toxicity [44]. In thepresent study, we used DLS in conjunction with TEM to evaluatethe size distribution of the synthesized silver nanoparticles. TheDLS pattern revealed that the AgNPs synthesized using T.

angustifolia leaf extract had an average size of 8 � 4 nm (Fig. 5).Singhal et al. [20] reported that silver nanoparticles synthesized usingO. sanctum leaf extract had an average diameter of 22.38 nm.

TEM analysis of AgNPs

TEM is one of the most valuable tools for direct and accurateanalysis of the size and structure of nanoparticles. TEM has beenused previously to obtain essential information on primary



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Fig. 4. XPS spectra of silver nanoparticles synthesized using T. angustifolia leaf

extract.

Fig. 5. Size distribution analysis by DLS. The sizes of the AgNPs ranged from 2 to

15 nm. The average particle size was 8 nm.

Figdis


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noparticle sizes and morphologies [44]. Therefore, we examinede size and morphology of the AgNPs using TEM. The transmissionectron micrographs of the AgNPs revealed that the nanoparticlesere distinct, uniform, spherical, and well separated from eachher. The particle size was estimated from more than 200rticles in TEM images and was found to be between 3 and 18 nm,

ith an average size of 8 nm (Fig. 6A). We also observed that theNPs were evenly distributed in the analyzed sample. The particlee distribution determined from TEM images is shown in

ig. 6B). Shankar et al. [17] reported that the size of nanoparticlesoduced using geranium leaf extract ranged from 16 to 40 nm.terestingly, our data suggest that silver nanoparticles produceding T. angustifolia leaf extract are smaller, which might result intter antimicrobial activity.

termination of MICs and sub-lethal concentrations of AgNPs and

tibiotics

The MICs of the AgNPs and antibiotics were defined as thewest concentrations that completely inhibited visible growth ofcteria after incubation at 37 8C for 24 h. In this study, E. coli and

pneumoniae were used as model gram-negative bacteria toaluate the antibacterial activities of AgNPs and three different

. 6. Determination of AgNP size and shape using TEM. (A) The size and morphology of

tribution determined from TEM images.


antibiotics. Bacterial cells were incubated with different concen-trations of AgNPs for 24 h in MHB. Medium without AgNPs wasused as a control. The E. coli and K. pneumoniae cell counts weresignificantly reduced by treatment with AgNPs in comparison withthe control. As shown in Table 1, the AgNPs showed similar MICs(1.4 mg/mL) towards E. coli and K. pneumoniae. Size, surface area,and surface functionalization are major factors that influence thebiokinetics and toxicity of nanomaterials [45]. The antibiotics alsoshowed similar MICs for E. coli and K. pneumoniae: 1.0 mg/mL forgentamicin, 0.5 mg/mL for cefotaxime, and 0.4 mg/mL for mer-openem (Table 1).

Dose-dependent antibacterial effect of AgNPs

We assessed the dose-dependent effect of AgNPs on E. coli andK. pneumoniae, the relative susceptibility of both species to AgNPs,and the extent of the bactericidal activity of AgNPs. Fig. 7 shows thetoxic effect of the biologically synthesized AgNPs on E. coli and K.

pneumoniae. The bacterial strains were treated with 8 nm AgNPs atconcentrations between 0.2 and 1.4 mg/mL. The introduction ofAgNPs reduced cell viability in comparison with the negativecontrol. Cell viability was reduced further as the concentration ofAgNPs increased. As determined earlier in the MIC experiment, novisible bacterial growth was observed above an AgNP concentra-tion of 1.4 mg/mL for both E. coli and K. pneumoniae (Fig. 7). These

AgNPs were analyzed using TEM. The average particle size was 8 nm. (B) Particle size



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Table 1Determination of MICs and sub-lethal concentrations of antibiotics and AgNPs.

Bacterial species MIC of various antibiotics and AgNPs (mg/ml)

GN CE MPM AgNPs

E. coli 1.0 0.5 0.40 1.4

K. pneumoniae 1.0 0.5 0.40 1.4

Sub lethal concentration of antibiotics and AgNPs

(mg/ml)

GN CE MPM AgNPs

E. coli 0.25 0.15 0.10 0.3

K. pneumoniae 0.25 0.15 0.10 0.3

Fig. 7. Effect of AgNPs on cell survival. E. coli and K. pneumoniae cells were incubated

with various concentrations of AgNPs. Bacterial survival was determined at 4 h by a

CFU count assay. The experiment was performed with various controls including a

positive control (AgNPs and MHB, without inoculum) and a negative control (MHB

and inoculum, without AgNPs). The results are expressed as the means � SD of three

separate experiments, each of which contained three replicates. Treated groups

showed statistically significant differences from the control group by Student’s t test

(p < 0.05).


G Model

JIEC 2472 1–10

results show that the AgNPs synthesized from T. angustifolia leafextract exhibited a strong, dose-dependent antimicrobial activityagainst both test microorganisms (Fig. 7). The antimicrobialactivity of these AgNPs is greater than that of AgNPs from othersources such as bacteria and fungi. Li et al. [46] reported that at aconcentration of 10 mg/mL, commercially prepared silver nano-particles completely inhibited the growth of 107 CFUs/mL of E. coli

Fig. 8. Effect of antibiotics on cell survival. E. coli and K. pneumoniae cells were incubated

survival was determined at 4 h by a CFU count assay. The experiment was performed wit

and a negative control (MHB and inoculum, without AgNPs). The results are expresse

replicates. Treated groups showed statistically significant differences from the control gro


in liquid MHB. Anthony et al. [47] evaluated the toxicity of 40 nmAgNPs prepared from culture supernatant of Bacillus marisflavi andfound that the MIC against Pseudomonas aeruginosa was 10 mg/mL.Several studies have examined the mechanisms underlying theantimicrobial activity of AgNPs from various sources. Shrivastavaet al. [48] studied the interaction of silver nanoparticles with E. coli

and found that the nanoparticles adhered to the bacterial cell walland subsequently penetrated the cell, eventually killing thebacteria by destroying the cell membrane. AgNPs may passthrough the cell wall of bacteria and oxidize surface proteins on theplasma membrane, consequently disturbing cellular homeostasis[45,49]. Several research groups have suggested that AgNPs mayattach to the surface of the cell membrane and disturb membranefunctions such as permeability and respiration [49,50]. Our resultssuggest that AgNPs synthesized using a biological method, such asthe method used in this study, are smaller than nanoparticlessynthesized by other methods. Since smaller nanoparticles aretaken up by cells more easily than larger particles and have a largersurface area available for interaction with bacteria, they may havestronger bactericidal effects.

Dose-dependent effects of antibiotics

The dose-dependent effects of three antibiotics on E. coli and K.

pneumoniae were assessed. As shown in Fig. 8, the growth of E. coli

and K. pneumoniae in MHB was continuous in the absence ofantibiotics. As we expected, various concentrations of gentamicin,cefotaxime, and meropenem inhibited growth. Gentamicin wasthe least inhibitory antibiotic for both strains tested. Greaterinhibition was observed with increasing concentrations ofgentamicin (0.01 to 1.0 mg/mL), and complete inhibition wasobserved at 1.0 mg/mL, consistent with the MIC value. In bothstrains, cefotaxime and meropenem completely inhibited growthat concentrations of 0.5 and 0.4 mg/mL, respectively. Among thethree tested antibiotics, meropenem showed complete inhibitionof growth at the lowest concentration (0.4 mg/mL). Therefore,further studies of the synergistic effects of AgNPs and antibioticsfocused on meropenem.

Evaluation of antibacterial effects of antibiotics with AgNPs

The potential additive or synergistic antibacterial effects ofantibiotics and AgNPs were evaluated using the disc diffusionmethod. All three antibiotics tested—gentamicin (10 mg/mL),cefotaxime (30 mg/mL), and meropenem (10 mg/mL)—showed

with various concentrations of gentamicin, cefotaxime, and meropenem. Bacterial

h various controls including a positive control (AgNPs and MHB, without inoculum)

d as the means � SD of three separate experiments, each of which contained three

up by Student’s t test (p < 0.05).



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506

507508509510511512513514515516517518519520521522523524525526527528529530531532533534

Fig. 9. Enhancement of antibacterial activity of antibiotics in the presence of AgNPs.

Antibacterial activities were determined by the agar diffusion method. Growth

inhibition was determined by measuring the zone of inhibition after 24 h.

Experiments were performed in triplicate. The percent enhancement of

antibacterial activity was calculated using the formula (B–A)/A � 100, as

described in the Materials and methods section. The results are expressed as the

means � SD of three separate experiments. Treated groups showed statistically

significant differences from the control group by Student’s t test (p < 0.05).

Figan

MH

co


G Model

JIEC 2472 1–10

nificant (p < 0.05) antibacterial effects against both E. coli and pneumoniae (Fig. 9). The activities of all three antibiotics againstth bacterial strains were increased in combination with AgNPs; significant differences were observed between E. coli and

pneumoniae, perhaps because both species are gram-negative.ong the three antibiotics, meropenem showed the largest

crease in activity (75%) when combined with AgNPs, followed byfotaxime (63%) and gentamicin (48%). Hwang et al. [51] observednergistic activities of silver nanoparticles in the presence ofnventional antibiotics and suggested that bacterial viability wascreased at lower concentrations of antibiotics. Kora and Rastogi0] reported that the combined effects of silver nanoparticles andtibiotics were more prominent with PVP-capped nanoparticlesan with citrate- or SDS-capped nanoparticles in both gram-sitive and gram-negative bacteria.

nergistic antibacterial effect of antibiotics and AgNPs

Next, we explored the possibility of using AgNPs as an antibioticjuvant. Morones-Ramirez et al. [52] reported that Ag+ couldhance the bactericidal effect of antibiotics owing to the

. 10. AgNPs enhance the bactericidal effect of antibiotics. Bacterial strains were treat

d meropenem for 4 h. Bacterial survival was determined at 4 h by CFU assay. The exp

B, without inoculum) and a negative control (MHB and inoculum, without AgNPs). Th

ntained three replicates. Treated groups showed statistically significant differences fro


generation of ROS [52,53]. To analyze the synergistic effect ofantibiotics and AgNPs, we selected a sublethal dose of meropenemand examined the antibacterial activity against both E. coli and K.

pneumoniae. Exponentially growing bacteria were incubated witha sublethal concentration of antibiotic or AgNPs or a combinationof both. Bacteria were harvested at different time points todetermine the number of CFUs. The surviving colonies wereenumerated after 24 h. The CFU assays showed that a sublethalconcentration of antibiotic or AgNPs alone had no significantkilling effect on either tested strain. However, the combination ofsublethal concentrations of meropenem and AgNPs significantlyreduced the viability of E. coli and K. pneumoniae cells by more than75% (Fig. 10). Comparing the antibacterial effects of meropenem,AgNPs, and their combination, the augmented antibacterial effectof the antibiotic in combination with AgNPs is noticeable.

Synergistic effect of antibiotics and AgNPs on ROS generation

Silver and antibiotics are known to enhance the production ofROS by increasing the permeability of bacterial membranes;increased production of ROS induces cell death and may be acommon mechanism of bactericidal antibiotics [53–58]. Recently,several studies have shown that lethal doses of bactericidalantibiotics promote the formation of highly detrimental ROS[53,57]. The mechanisms of cell death, oxidative stress, and ROSformation are some of the key mechanisms of cellular defenseafter particle uptake. Nanoparticles could hasten intracellularoxidative stress by disturbing the equilibrium between oxidantand antioxidant processes [45]. The generation of ROS is a keymechanism of toxicity. Therefore, we investigated the effect ofsublethal concentrations of antibiotics, AgNPs, or combinations ofantibiotics and AgNPs in E. coli and K. pneumoniae. Cells weretreated with an antibiotic, AgNPs, or both, and ROS levels weremeasured. The ROS levels in cells treated with an antibiotic orAgNPs were lower than those in cells treated with a combination ofan antibiotic and AgNPs (Fig. 11). Elevated ROS and free radicallevels are candidate mediators of cell death. The production of ROScould be caused by impeded electron transport along therespiratory chain in the damaged plasma membrane [50]. Theunderlying mechanisms of ROS production could be the reason forcell death. The mechanism underlying the bactericidal effect ofAgNPs against bacteria remains unclear and further studies arerequired. Our results provide some evidence that cell death due toROS generation is one of the mechanisms of AgNPs, antibiotics, or acombination of both antibiotics and AgNPs. On the other hand,AgNPs may attach to the surface of the cell membrane and disturb

ed with a sublethal concentration of meropenem or AgNPs or a combination of AgNPs

eriment was performed with various controls including a positive control (AgNPs and

e results are expressed as the means � SD of three separate experiments, each of which

m the control group by Student’s t test (p < 0.05).



535

536

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541

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576577578579580581582

5835

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5865876

5887

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5905915925935945955965975985996006016026036046056066076086096106116126136146156166176186196206216226236246256266276286296306316326336346356366376388639640641642643644645964664710648

Fig. 11. Synergistic effect of antibiotics and AgNPs on ROS generation. E. coli and K.

pneumoniae were treated with a sublethal concentration of meropenem or AgNPs or

a combination of AgNPs and meropenem for 24 h. ROS generation was measured by

an XTT assay. The results are expressed as the means � SD of three separate

experiments, each of which contained three replicates. Treated groups showed

statistically significant differences from the control group by Student’s t test (p < 0.05).


G Model

JIEC 2472 1–10

membrane functions such as permeability and respiration, and ithas been suggested that the binding of the particles to the bacteriadepends on the surface area available for interaction [59]. Ourstudy supports the idea that smaller nanoparticles may be moreeffective in inducing cell death than larger particles.

Kvitek et al. [60] also suggested that AgNPs may attach to thesurface of the cell membrane, disturbing permeability andrespiration, and that smaller AgNPs having a larger surface areaavailable for interaction would have greater bactericidal effectsthan larger AgNPs. Silver binds to the bacterial cell wall and cellmembrane and inhibits the respiration process [61]. In the case ofE. coli, silver acts by inhibiting the uptake of phosphate and byreleasing phosphate, mannitol, succinate, proline, and glutaminefrom the cells [62]. Recently, Balaji Raja and Singh [63] reportedthat the antibacterial activity of cephalexin against E. coli wasincreased in the presence of AgNPs. Furthermore, the combinationof silver nanoparticles and graphene nanocomposites exhibitedenhanced antibacterial activity against E. coli and S. aureus

compared with individual silver nanoparticles, reduced grapheneoxide nanosheets, or their nanocomposites [63]. Previously,several studies demonstrated that improvement of antibacterialactivity of AgNPs through silver ion release using nanocomposites[64–73]. Altogether, these results consistent with previous studiesand also indicate that the generation of ROS might be a crucialmechanism of cell death mediated by AgNPs or a combination ofantibiotics and AgNPs.

Conclusion

In this work, we describe a green approach for the synthesis ofsilver nanoparticles using leaf extract of T. angustifolia. Theprepared AgNPs were characterized using various analyticaltechniques. The synthesized AgNPs were uniform and had anaverage size of 8 nm. We demonstrated that the antibacterialactivity of AgNPs and antibiotics against E. coli and K. pneumoniae

was dose dependent. Furthermore, the antibacterial activity ofgentamicin, cefotaxime, and meropenem against the tested strainswas increased in the presence of AgNPs. Interestingly, thecombination of sublethal concentrations of an antibiotic andAgNPs significantly decreased cell viability and increased ROSproduction. This study provides evidence of the antibacterialeffects of AgNPs and their synergistic activity with antibioticsagainst gram-negative bacteria. Additionally, these results suggest


that AgNPs synthesized using leaf extract can be used as anantibiotic adjuvant for the treatment of various infectious diseasescaused by gram-negative bacteria. Finally, these results suggest apossible mechanism for the synergistic effects of antibiotics andAgNPs. Thus, our findings support the claim that AgNPs haveconsiderable antibacterial activity that can be used to enhance theaction of existing antibiotics against gram-negative bacteria.

Uncited reference Q

[74].

Acknowledgements

This paper was supported by the SMART-Research ProfessorProgram of QKonkuk University. Dr. Sangiliyandi Gurunathan wassupported by a Konkuk University QKU-Full time Professorship.

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