Synthesis and characterization of silver nanobactericides produced by Aneurinibacillus migulanus 141, a novel endophyte inhabiting Mimosa pudica L.

Accepted Manuscript Synthesis and characterization of silver nanobactericides produced by Aneurinibacillus migulanus 141, a novel endophyte inhabiting Mimosa pudica L Baker Syed, M.N. Nagendra Prasad, S. Satish PII: DOI: Reference:

S1878-5352(16)00006-X http://dx.doi.org/10.1016/j.arabjc.2016.01.005 ARABJC 1828

To appear in:

Arabian Journal of Chemistry

Received Date: Accepted Date:

18 September 2015 9 January 2016

Please cite this article as: B. Syed, M.N. Nagendra Prasad, S. Satish, Synthesis and characterization of silver nanobactericides produced by Aneurinibacillus migulanus 141, a novel endophyte inhabiting Mimosa pudica L, Arabian Journal of Chemistry (2016), doi: http://dx.doi.org/10.1016/j.arabjc.2016.01.005

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Synthesis and characterization of silver nanobactericides produced by Aneurinibacillus migulanus 141, a novel endophyte inhabiting Mimosa pudica L. Baker Syed a Nagendra Prasad M.N b and Satish.Sa,c* a

Bionanotechnological Laboratory, Department of Studies in Microbiology, University of Mysore,

b

Department of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS Institution Camp,

Manasagangothri, Mysore 570006 c

Department of Plant Pathology, University of Georgia, Athens 30602, USA.

e-mail: [email protected] and [email protected]

Corresponding author Dr. S.Satish, [email protected] and [email protected] Bionanotechnological Department of Studies in Microbiology University of Mysore, India

Synthesis and characterization of silver nanobactericides produced by Aneurinibacillus migulanus 141, a novel endophyte inhabiting Mimosa pudica L. Baker Syed a Nagendra Prasad M.N b and Satish.Sa,c* a

Bionanotechnological Laboratory, Department of Studies in Microbiology, University of Mysore,

b

Department of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS Institution Camp,

Manasagangothri, Mysore 570006 c

Department of Plant Pathology, University of Georgia, Athens 30602, USA.

e-mail: [email protected] and [email protected]

Corresponding author Dr. S.Satish, [email protected] and [email protected] Bionanotechnological Department of Studies in Microbiology University of Mysore, India

Abstract Use of nano-size particles against pathogenic bacteria is a rapidly growing area due to their unique physico-chemical properties. The present investigation reports the synthesis of silver nanobactericides by Aneurinibacillus migulanus, a novel endophyte isolated from surface sterilized inner leaf segment of Mimosa pudica L and cultured at large scale to separate cell free extract which was treated with metal salt silver nitrate to synthesize silver nanobactericides. The synthesized nanobactericides were subjected to biophysical characterization using UV- visible spectra with characteristic absorption peaks between 350 to 550 nm. The role of biomolecules mediating the synthesis and stabilizing the nanobactericides was studied with Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance ( 1H-NMR), which suggested the presence of carbonyl, aromatic, amino and secondary aliphatic groups bound to surface of nanobactericides. Bragg’s peaks with different intensities exhibited the standard diffraction pattern of the silver plane, corresponding to the crystalline nature of the nanobactericides. The energy dispersive X ray spectroscopy (EDS) analysis revealed presence of high intense absorption peak at 3 keV is a typical characteristic of nano-crystalline silver which confirmed the presence of elemental silver. Transmission electron microscopy (TEM) showed polydispersity of nanobactericides with size ranging from 20 to 60 nm. The particle size determined by Dynamic light scattering (DLS) method revealed average size to be 24.27 nm. Further, using in vitro assays, silver nanobactericides showed potent activity against five different bacterial species, including human pathogens. The silver nanobactericides caused DNA damage against the test bacteria, suggesting the possible mode of action. To best of our knowledge this is the first report of Aneurinibacillus migulanus as an endophyte and its ability to reduce silver nitrate to synthesize silver nanobactericides. Keywords: Aneurinibacillus migulanus; endophyte; silver nanobactericides; bactericidal activity 1. Introduction Nanomaterials have attracted considerable attention owing to their distinctive properties compared with bulk material (Baker et al., 2013 a&b). In recent years, nano-silver is reported to have traded their applications in all fields of sciences and has been considered as one of the superior metallic nanomaterials (Kundu and Liang, 2011). Nano-silver is more reactive and undergoes relatively fast oxidation as well as aggregation in solution and bears high electric conductivity, enhanced optical properties and possess unique spatial Raman spectroscopic behavior (Kundu et al., 2009). One such size-dependent property has led to the development of drug delivery systems for

treating tumor cells and for developing dressing materials incorporated with nano-sized silver as it bears broad spectrum antibacterial activity against an array of pathogens which enhance the wound healing property of the dressing materials (Zamani et al., 2013). In recent years, large numbers of pharmaceutical products have been introduced into market with profound and enhanced activity using nano-sized silver which exhibit low toxicity (Gupta and Kumar, 2014). One such prime focus of the pharmaceutical sector is to develop potent antimicrobial drugs to combat drug-resistant pathogens which are growing at alarming pace with limited choice of available drug treatment (Sosa et al., 2010). Hence, scientific communities are engaged in designing rational strategies to develop potent antimicrobial agents. Interestingly, applications of nanoparticles have generated tremendous with their size dependent properties and have emerged as “nanobactericides”. Nanobactericides are antibacterial agents synthesized at nanoscale. These nano-sized bactericides are generally more active compared to macroparticles (Wang et al., 2013). Nanobactericides can have multiple modes of action on pathogens, for instance they can easily penetrate pathogen cell wall and cause plaques resulting in loss of cellular content, prevention of DNA replication and inactivation of vital enzymes/proteins. Unlike other antimicrobial agents, pathogenic microorganisms cannot easily develop resistance to nanobactericides due to its multiple mode of action (Zhou et al., 2012). Nanobactericides can be produced by physical, chemical and biological methods. Majority of these methods are bound with various limitations for instance chemical synthesis involves use of toxicants and physical synthesis requires high-end and costly instruments. On other hand, biological synthesis of nanobactericides provide potential advantageous like eco-friendly, cost effective and usually one step process to synthesize nanobactericides with desired size and shape (Basavegowda et al., 2014). Biological synthesis can be achieved by employing simple prokaryotic bacteria to multicellular eukaryotic organisms including plants. Among the biological entities, use of microorganisms has become one of the most popular choices in current scenario (Nazeruddin et al., 2014). The concept of using microorganisms to synthesize particles at nanoscale can be traced back to the work of Beveridge and Murray in 1980 who reported the synthesis of nano-sized gold by using Bacillus subtilis (Klaus et al., 1999). Since then, microorganisms have become one of the ideal choices for reducing metals into nano-sized particles by secreting unique bioactive molecules (Li et al., 2011). Even though there has been extensive research on microbial mediating synthesis of nanobactericides,

scanty reports are available on synthesis of nanobactericides from endophytes (Baker et al., 2015; Azmath et al., 2015). Endophytes are microorganisms which reside inside healthy tissue of almost all plant species and are reported to perform innumerable biological application and influences plant growth and development (Strobel 2003). Endophytes secrete unique bioactive metabolites which are reported to have high significance and majority of the endophytes are yet to be explored (Alvin et al., 2014). In particular, endophytes may play major roles in reducing metal salts due to their unique metabolic diversity (Zin et al., 2010). Interference of endophytes with nanoparticles is one of the interest areas which can open new avenue in reporting novel applications (Baker and Satish, 2012). Based on these considerations, the present study reports the isolation of the novel endophyte Aneurinibacillus migulanus 141 from Mimosa pudica L. and its evaluation for synthesizing silver nanobactericides with potent activity against important pathogenic bacteria. 2. Experimental procedures 2.1. Isolation of endophyte Healthy plant materials of Mimosa pudica L. were collected, washed under running tap water and subjected to sequential surface sterilization by immersing plant materials in 3.15% sodium hypochlorite for 120 seconds followed by 70% ethanol for 60 seconds. Tissues were subsequently washed with double distilled sterile water and dried using sterile blotter sheets. The outer tissue of surface sterilized plant segments were excised using a sterilized scalpel, cut into 0.5-1.0 cm blocks and placed on the surface of nutrient agar supplemented with 250 µg/ml of cycloheximide and incubated for 48 hrs to observe colonies of endophytic bacteria (Webster et al., 2001). Sterility checks were performed by transferring the aliquots of washed distilled water onto nutrient agar which served as control plate. 2.2. Screening of endophytic bacteria for synthesis of silver nanobactericides Endophytic bacteria were cultured in nutrient media amended with 1mM silver nitrate (AgNO 3) and incubated at 37 oC until visible growth was observed. Colonies growing abundantly on this media were subjected to large-scale fermentation for 72 hrs under optimized conditions as per the protocol described by Baker et al. (2015). The fermentation broth was centrifuged at 8000 rpm at 4 oC for 20 minutes and supernatant was assessed for synthesis of silver nanobactericides by applying 1 mM silver nitrate and incubating until change in color was observed. Samples were drawn periodically and monitored using UV-visible spectrophotometry to confirm the synthesis of silver

nanobactericides by recording the spectra between 200 to 800 nm using a Shimadzu double beam spectrophotometer (Shimadzu Corp., Kyoto, Japan). 2.3. Biophysical characterization of silver nanobactericides (FTIR, NMR, XRD and XDS) Biophysical characterization of silver nanobactericides was carried out by precipitating silver nanobactericides followed by washing with sterile deionized water and drying under vacuum. The processed silver nanobactericides were subjected to Fourier transform infrared spectroscopy (FTIR) analysis, which was carried out with a JASCO FT-IR 4100 (Jasco, Easton, MD, USA) at a resolution of 4 cm-1 to predict the functional group of biomolecules in supernatant responsible for reducing metal salts and stabilizing of silver nanobactericides. Diffraction pattern of the silver nanobactericides were studied for X-ray diffraction analysis by coating the dried silver nanobactericides on a grid and recording the spectra with a Rigaku Miniflex-II Desktop X-ray diffractometer operating at a voltage of 30 kV (RigakuCorp., Tokyo, Japan). Nuclear Magnetic Resonance (NMR) analysis (both 1H and

13

C NMR spectra) were recorded on a Bruker DRX-500

spectrometer using deuterated DMSO-d6 as solvent and trimethylsilane (TMS) as internal standard. 1

H NMR was measured at 300 MHz. Size and morphology of silver nanobactericides was analyzed

using transmission electron microscopy (TEM); an aliquot of silver nanobactericides was transferred onto a carbon-coated copper TEM grid and scanned using a TECNAI-T12 JEOL JEM-2100 (JEOL Ltd., Akishima-Shi, Japan). The TEM was operated at a voltage of 120 kV with a Bioten objective lens. Subsequently, the particle size was ascertained using a Gatan CCD camera (Gatan, Pleasonton, CA, USA) (Baker et al., 2015). The average size and stability of the nanoparticles were determined using Dynamic Light Scattering (DLS) experiments were performed at 25 °C on a Malvern using Zetasizer Nano ZS (Malvern Instruments; UK). Finally, the synthesized Ag-NPs were dried, drop coated on to carbon film, and tested using energy dispersive X-ray (EDS) analyzer (XL 30; Philips).

2.4. Evaluation of silver nanobactericides against pathogenic bacteria Bactericidal activity was evaluated via well diffusion, disc diffusion and micro broth dilution assay. In brief pre-warmed Mueller-Hinton agar plates were seeded with 106 CFU (colony forming unit) suspensions of selected test bacteria which includes Pseudomonas aeroginosa (MTCC 7903) followed by Escherichia coli (MTCC 7410), Staphylococcus aureus (MTCC 7443), Bacillus subtilis (MTCC 121) and Klebsiella pneumoniae (MTCC 7407). The selected test pathogens were swabbed uniformly and using a sterile cork borer 10 mm diameter of agar was removed and 50 µl of 10

mg/ml silver nanobactericides were added into the well and simultaneously the sterile agar disc was impregnated with silver nanobactericides and placed onto the agar incubated at 37 °C for 24 hrs. After incubation, the zone of inhibition was measured and interpreted with gentamicin (1mg/ml) as standard. Micro broth dilution assay was performed whereby different concentrations of silver nanobactericides varying from 25 μg/ml to 100 μg/ml were suspended in sterile saline and each aliquot was suspended in test tubes with 10 ml of Muller Hinton broth seeded with 150 µl of test bacterial cells (5 x 106 CFU/ml) and incubated at 37 oC on a shaker (150 rpm) for 20 to 24 hrs. Absorbance at 600 nm was subsequently measured to quantify bacterial growth. One positive and negative control was maintained to distinguish the activity of the silver nanobactericides based on the optical density of the control (Baker et al., 2015). 2.4. Mode of action of silver nanobactericides on DNA The mode of action of silver nanobactericides was studied using a DNA damage assay according to the protocol of Vahdati and Sadeghi (2013) with slight modification. In brief, silver nanobactericides (10mg/ml) was treated with DNA (10 ng) isolated from Pseudomonas aeroginosa (MTCC 7903) and incubated for 30 min. Further, DNA without treatment of silver nanobactericides served as a control. Both treated and untreated DNA was subjected to electrophoresis using 1% agarose gel at 75 V for 30 min (Vahdati and Sadeghi, 2013). 2.5.

Genotypic characterization of endophyte The endophyte isolate producing the most active silver nanobactericide was subsequently

characterized. Total genomic DNA was extracted using CTAB method and DNA pellets were resuspended in sterile TE buffer and stored at -200C until further use. Amplification of the 16S rRNA

gene

and

sequencing

was

performed

by

using

universal

primers,

fP1

(5ˈ-

AGTTTGATCCTGGCTCA-3ˈ) and rP2 (5ˈ-ACGGCTACCTTGTTACGACTT- 3ˈ). The amplicons were purified and the sequence was processed at NCBI to reveal its homology according to the protocol of Liu et al., (2006). Based on sequence similarity measures and phylogenetic inference, partial nucleotide sequences were deposited in NCBI GenBank to avail the accession number. Sequences were then aligned with other similar sequences retrieved from GenBank using Clustal W, and alignments were manually edited and phylogenetic analyses were performed to assess phylogenetic affiliation with other deposited sequences. 3. Results and discussion

Selection of plant Mimosa pudica L. was carried out based on the earlier reports on endophytes and their biological applications. It is reported that Mimosa pudica L. is geographical restricted short prickly plant which is not found ubiquitous. Ancient records highlights the traditional values of this plant in curing various ailments and use of Mimosa pudica L still persist by tribal as it hails medical properties (Kaur et al., 2011 and Varnika et al., 2012). To best of our knowledge the present investigation forms the first reports on isolation of bacterial endophytes from this plant and its evaluation for synthesis of nanoparticles. Perusal of scientific literatures perceives endophytes as one of the rich novel source of secondary metabolites bearing activity (Strobel and Daisy, 2003). Majority of these scientific reports are pertaining to fungal endophytes compared to its counter symbionts bacterial endophytes. Recent studies on bacterial endophytes highlight its abundant colonization in healthy plant tissues when compared to fungal endophytes (Emiliani et al., 2014). Isolation and evaluation of bacterial endophytes are more advantageous for instance bacterial endophytes can easily reproduce with less generation time and are capable of secreting structurally diverse metabolites (Brader et al., 2014). Though large scientific literatures report synthesis of diverse nanomaterials using microbial entities but one of the major constrain associated with majority of microbial species is prolong reaction time for synthesis of nanomaterials (Kavitha et al., 2013). This might be due to the fact that lack of secretion of unique secondary metabolites required for reducing metal salts. In present investigation, targeting endophytic plethora as subject of interest resulted in overcoming constrains of other microbial species and silver nanobactericides were synthesized within 25 minutes. This can be attributed with the fact that endophytes are capable of secreting unique metabolites bearing redox potentials (Sunkar and Nachiyar, 2013; Azmath et al., 2015). Based on these facts, the present study was executed to evaluate novel endophytic bacterium for synthesis of nanoparticles. Screening of bacterial endophytes from Mimosa pudica L. was successful with the use of cycloheximide which suppressed the growth of fungal endophytes resulting in isolation of only bacterial endophytes from surface sterilized stems and roots. The endophyte capable of synthesizing silver nanobactericides was screened based on the abundant growth onto the enriched nutrient media with silver nitrate and the isolate was cultured at large scale and evaluated for synthesis. 3.4.

Genotypic characterization of endophyte

Genotypic characterization based on 16S rRNA sequencing revealed 99% homology to Aneurinibacillus migulanus when matched at NCBI GenBank using BLAST tool. A phenogram was

constructed with Clustal W software by grouping the isolates deposited at GenBank to reveal its relationships with taxonomically similar bacteria (Fig.1) and the sequence was deposited at Genbank (KF 606762). To the best of our knowledge, this is the first report of Aneurinibacillus migulanus as an endophyte and its role in reducing silver nitrate. An earlier study demonstrated the ability of Aneurinibacillus migulanus to secrete the antibiotic gramicidin (Berditsch et al., 2007). But no previous studies reported synthesis of nanobactericides from this bacterium this might be due to the fact that Aneurinibacillus migulanus being novel species, it is largely unexplored and has been less reported and research studies on Aneurinibacillus migulanus is in its infancy stage and untraced roles of this novel bacterium can open new avenue in coming decades. 3.5.

Biosynthesis of silver nanobactericides

When cell-free supernatant was treated with 1mM silver nitrate, there was gradual change in color of the reaction mixture which turned to dark brown color due to surface plasmon resonance (Li et al., 2012; Gopinath et al., 2013). Change in color served as preliminary confirmation for synthesis of silver nanobactericides and further formation process of silver nanobactericides was confirmed by monitoring UV‒Visible spectra (Fig.2) at different intervals for surface plasmon resonance peak between 350 to 550 nm. The intensity of reaction mixture increased steadily with incubation time up to 25 minutes and no further color change was observed which indicated attainment of saturation in the bio-reduction process of silver nanobactericides. However, during the process of synthesis, bioorganics or bioactive metabolites present in cell free supernatant spatially controlled the nucleation and growth of particles and hindered the reduction when the desired size and shape were obtained (Baker et al., 2015). It is noteworthy that elevated temperature above 50 oC influenced the reduction of silver nitrate and alkaline pH 8 showed maximum synthesis which clearly indicated influence of different variables in synthesis of silver nanobactericides. Previous studies also describe influence of different variables on synthesis of nano silver (Qian et al., 2013; Khan et al., 2013; Khodashenas and Ghorbani, 2015). 3.6.

Biophysical characterization (FTIR, NMR, XRD and XDS)

The possible role of biomolecules present in the supernatant responsible for reduction of silver nitrate to silver nanobactericides was assessed with FTIR analysis which displayed predominant peaks (Fig. 3) occurring at 3339 cm-1 corresponding to NH stretching (Devi and Gayathri, 2010). 1634 cm-1 corresponding to C-N stretching (Gunasekaran et al., 2009) and 669 cm-1 may correspond to C-H stretching (Gunasekaran et al., 2009). The 1H-NMR

spectrum of silver

nanobactericides is depicted in Fig. 4. The signals appearing at δ 7.21 ppm and δ 5.87 ppm could be due to aromatic groups, between δ 2.23 to δ 2.7 ppm due to carbonyl groups, and δ 1.82 and δ 1.355 ppm due to amino and secondary aliphatic groups. These results are consistent with the FTIR prediction. Interestingly these results also coincide with the majority of earlier findings which state presence of amides, aliphatic, carbonyl and aromatic groups mediating the synthesis and stabilization of nanobactericides (Yilmaz et al., 2011; Chandran et al., 2014; Islam et al., 2015). Generally, the stability of nano-silver is more significant for exploring their applications in biomedicine. Consequently the nano-silver is normally stabilized by using stabilizing agents under various conditions. However, in present investigation, nano-silver was more stable owing to in situ biocapping by the organic moieties present in supernatant which are responsible for the synthesis and stabilization of silver nanobactericides thereby preventing agglomeration, so called in situ stabilization. X-ray diffraction displayed Bragg’s peaks at 38.22, 44.52, 64.58 and 77.59 conferring the 111, 200, 220, and 311 facets of the face centered cubic symmetry of nanobactericides suggesting that these nanobactericides were crystalline in nature (Fig. 5a). The diffraction pattern was in agreement with the standard pattern of the silver plane of JCPDS file no. 04-0783 (Fig. 5b). The obtained result justifies with previous reports (Jeevan et al., 2012). Table-1 shows experimentally obtained X-ray diffraction and standard diffraction angle. The average crystallite size‘d’ of silver nanobactericides was calculated to be 20-30 nm using Scherer equation: d = Kλ/β cosθ, where K‒shape factor between 0.9 and 1.1, k‒incident X-ray wavelength (CuKα = 1.542 Å), β‒full width half-maximum in radians of the prominent line and θ‒position of that line in the pattern. The obtained diffraction pattern was in agreement with previous scientific reports (Awwad et al., 2013). The energy dispersive X ray spectroscopy analysis revealed presence of high intense absorption peak at 3 keV is a typical characteristic of nano-crystalline silver which confirmed the presence of elemental silver (Fig.5c). The EDS gives both qualitative and quantitative status of elements. The spectrum consisted signals from Cl, C, O and Mo atoms. These signals are likely due to X-ray emission from biomolecules responsible for stabilization of nanobactericides (Devi et al., 2013). The obtained results are in accordance with the characteristic results of elementary nano silver at 3 keV due to their surface plasmon resonance as per the reported scientific literatures (Kundu and Liang, 2011; Ibrahim, 2015; Padalia et al., 2015). 3.7.

TEM and DLS analysis

TEM micrographs of precipitated solid phase revealed the size and shapes of the silver nanobactericides (Fig. 6a) which shows images of biosynthesized silver nanobactericides at different nanometric scale. The TEM analysis also described surface morphological characteristic and polydispersity of the silver nanobactericides with different morphological characteristics varying from spherical, oval, hexagonal, cubic and triangular shapes. The TEM micrographs also highlighted silver nanobactericides were well with a diameter (ɸ) in the range of 20‒25 nm. Interestingly, few silver nanobactericides exhibited scum adsorption on the surface which is due to bio-capping of organic moieties present in supernatant which incongruence with the results of FTIR and NMR analysis. The selected area electron diffraction (SAED) pattern (Fig 6a) of clear bright circular rings corresponds to (111), (200), (220), and (311) planes of the fcc lattice of metallic silver. This was also an evidence for highly crystalline structure of silver nanobactericides. Particle size histogram determined from TEM microgram (Fig. 6b) revealed the distribution of silver nanobactericides from 20 to 60 nm. Similarly, the particle size determined by Dynamic light scattering (DLS) method is in agreement with the TEM result and average size was found to be 24.27 nm as shown in the size distribution graph (Fig.6c). These results are in accordance with previous scientific reports (Kalpana et al., 2014; Jyoti et al., 2015). 3.8.

Bactericidal activity

In general, silver and their by-products are well known for their antimicrobial properties and are used for the treatment of non healing chronic wounds (diabetic, vascular and pressure ulcers), wound dressing in superficial burns (bandages), skin diseases etc. (Lipsky and Hoey, 2009). In recent years, introduction of silver nanobactericides have reported profound activity owing to higher surface area to volume ratio which results significant antimicrobial activity (Basavaraja et. al., 2008). In present investigation, biologically synthesized silver nanobactericides exhibited bactericidal activity via broth dilution assay with drastic decrease in optical density of the broth seeded against different test pathogens. Efficacy was greatest against Pseudomonas aeroginosa (MTCC 7903) followed by Escherichia coli (MTCC 7410), Staphylococcus aureus (MTCC 7443), Bacillus subtilis (MTCC 121) and Klebsiella pneumoniae (MTCC 7407). Further, disc diffusion and well diffusion assays showed significant activity of silver nanobactericides with zone of inhibition across discs and wells (Table 1) against all the test pathogens (Fig. 7a). The minimal inhibitory concentration of silver nanobactericides was found to be 12.5 µg/mL for Pseudomonas aeroginosa (MTCC 7903) followed by Escherichia coli (MTCC 7410), Staphylococcus aureus (MTCC 7443),

Bacillus subtilis (MTCC 121) and 25 µg/mL for Klebsiella pneumoniae (MTCC 7407). The obtained results justifies with scientific studies highlighting the evaluation of nano- silver and its bactericidal activity (Kundu and Liang, 2011; Azmath et al., 2015). Perusal of literatures suggests potent activity of nano-silver against Gram-positive bacteria compared to Gram-negative bacteria due to difference in the cell wall composition. Interestingly, in the present investigation, highest activity was observed against Gram-negative Pseudomonas aeruginosa which is considered as clinically important as well as environmental pathogen. The results obtained clearly justify its bactericidal activity against both Gram-positive and Gram-negative bacteria 3.9.

Possible mode of action of silver nanobactericides on DNA

The mode of action of silver nanoparticles on the five test pathogens was evaluated by treating isolated DNA with silver nanobactericides followed by electrophoresis using 1% agarose gel. The results showed a cleaved and light band for DNA treated with nanobactericides compared with the control DNA of Pseudomonas aeroginosa (MTCC 7903) as shown in Fig. 7b. These results confirmed the potent bactericidal activity of synthesized nanobactericides targeting the DNA of the pathogen (Vahdati and Sadeghi, 2013; Azmath et al., 2015). 4. Conclusions The present study reports eco-friendly synthesis of silver nanobactericides from novel bacterium Aneurinibacillus migulanus 141 inhabiting Mimosa pudica L. To best of our knowledge this is first report on synthesis of nanobactericides from Aneurinibacillus migulanus 141. The synthesis was rapid compared to other endophytes reported and synthesized nanobactericides displayed significant bactericidal activity against both Gram-positive and Gram-negative pathogens. The study also reports possible mode of action of silver nanobactericides by targeting the DNA of Pseudomonas aeroginosa (MTCC 7903).

The results obtained in present investigation are

promising enough and attributes towards growing scientific knowledge to develop alternative strategies to combat drug resistant pathogens. Acknowledgements Authors are pleased to Dr. Harald Scherm from Department of Plant Pathology for his valuable suggestions and correction of manuscript. Authors are thankful DST-SERB for providing financial assistance. Authors acknowledge ICMR for awarding Research associate fellowship. We also express gratitude to RAMAN FELLOWSHIP UGC-INDIA and CIMO- Finland for providing

opportunity for exchange research program. Authors are thankful to department of UPE, University of Mysore for providing facilities and also than Mr. Adarsh Kumar for his timely technical inputs. Conflict of interest Authors confirm that there is no conflict of interest

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GRAPHICAL ABSTRACT

List of Figures

Figure 1 Phenogram expressing the relationships of Aneurinibacillus migulanus 141 strain to taxonomically similar bacteria based on the 16S rRNA gene sequences

Figure 2 UV-visible spectra of silver nanobactericides

Figure 3 FTIR analyses of silver nanobactericides

Figure 4 NMR analyses of silver nanobactericides

Figure 5a X-ray diffractograph of silver nanobactericides

Figure 5b Comparison of X-ray diffractograph of silver nanobactericides with Standard JCPDS file no. 04-0783

Figure 5c EDS analysis of silver nanobactericides

Figure 6a TEM microgram of silver nanobactericides

Figure 6b Histogram of silver nanobactericides

28

Figure 6c DLS analysis of silver nanobactericides

29

Figure 7a Bactericidal activity of silver nanobactericides against human pathogens with broth dilution assay

30

Figure 7b DNA damage activity of silver nanobactericides

31

Table.1. Bactericidal activity of silver nanobactericides Sl. No

Pathogens

Silver nanobactericides Gentamicin

1.

Bacillus subtilis (MTCC 121)

19mm

18mm

2.

Escherichia coli (MTCC 7410)

18mm

19mm

3.

Klebsiella pneumoniae (MTCC 7407)

17mm

22mm

4.

Staphylococcus aureus (MTCC 7443)

16mm

24mm

5.

Pseudomonas aeroginosa (MTCC 7903)

21mm

17mm

32