Synthesis of silver nanoparticles by endosymbiont Pseudomonas fluorescens CA 417 and their bactericidal activity

Accepted Manuscript Title: Synthesis of silver nanoparticles by endosymbiont Pseudomonas fluorescens CA 417 and their bactericidal activity Author: Baker Syed Nagendra Prasad M.N. Dhananjaya B.L. Mohan Kumar K. Yallappa S. Satish S. PII: DOI: Reference:

S0141-0229(16)30201-0 http://dx.doi.org/doi:10.1016/j.enzmictec.2016.10.004 EMT 8991

To appear in:

Enzyme and Microbial Technology

Received date: Revised date: Accepted date:

3-3-2016 3-10-2016 5-10-2016

Please cite this article as: Syed Baker, M.N.Nagendra Prasad, B.L.Dhananjaya, K.Mohan Kumar, S.Yallappa, S.Satish.Synthesis of silver nanoparticles by endosymbiont Pseudomonas fluorescens CA 417 and their bactericidal activity.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2016.10.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of silver nanoparticles by endosymbiont Pseudomonas fluorescens CA 417 and their bactericidal activity Baker Syed a,b, Nagendra Prasad M.Nc Dhananjaya B.Ld, Mohan Kumar Ke,f, Yallappa Sg, Satish. Sa* a

Bionanotechnological Laboratory, Department of Studies in Microbiology, University of

Mysore, Manasagangotri, Mysore b

Laboratory of Biotechnology of New Materials, Siberian Federal University, Siberia.

c

Department of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS Technical

Institutional Campus, Mysore 570006 d

Toxicology and Drug Discovery Unit, Centre for Emerging Technologies (CET), Jain

University, Ramanagara 562 112, India e

Universidad Nacional Autonoma de Mexico, Instituto de Ciencias Fisicas, Avenida Universidad

s/n, 62210 Cuernavaca, MOR, Mexico f

Department of Chemistry, Madanapalle Institute of Technology & Science, Post Box No: 14,

Kadiri Road, Angallu (V), Madanapalle-517325. Chittoor District, Andhra Pradesh, India g

BMS R and D Centre, BMS College of Engineering, Bangalore 560 019, India

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

1

Abstract The present study emphasizes on biogenic synthesis of silver nanoparticles and their bactericidal activity against human and phytopathogens. Nanoparticle synthesis was performed using endosymbiont Pseudomonas fluorescens CA 417 inhabiting Coffea arabica L. Synthesized nanoparticles were characterized using hyphenated spectroscopic techniques such as UV-Visible spectroscopy which revealed maximum absorption 425nm. Fourier transform infrared spectroscopy (FTIR) analysis revealed the possible functional groups mediating and stabilizing silver nanoparticles with predominant peaks occurring at 3346 corresponding to hydroxyl group, 1635 corresponding carbonyl group and 680 to aromatic group. X-ray diffraction (XRD) analysis revealed the Bragg’s diffraction pattern with distinct peaks at 38o 44o, 64o and 78o revealing the face-centered cubic (fcc) metallic crystal corresponding to the (111), (200), (220) and (311) facets of the crystal planes at 2θ angle. 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. TEM analysis revealed the size of the nanoparticles to be in the range 5 to 50 nm with polydisperse nature of synthesized nanoparticles bearing myriad shapes. The particle size determined by Dynamic light scattering (DLS) method revealed average size to be 20.66 nm. The synthesized silver nanoparticles exhibited significant antibacterial activity against panel of test pathogens. The results showed Klebsiella pneumoniae (MTCC 7407) and Xanthomonas campestris to be more sensitive among the test human pathogen and phyto-pathogen respectively. The study also reports synergistic effect of silver nanoparticles in combination with kanamycin which displayed increased fold activity up to 58.3% against Klebsiella pneumoniae (MTCC 7407). The results of the present investigation are promising enough and attribute towards growing scientific knowledge on development of new antimicrobial agents to combat drug resistant microorganisms. The study provides insight on emerging role of endophytes towards reduction of metal salts to synthesize nanoparticles.

Keywords: Endosymbiont; Silver nanoparticles; Pseudomonas fluorescens CA 417; Coffea arabica L.; Antibacterial activity; Kanamycin

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1. Introduction Nanoparticles are defined as ultrafine particles which are considered as particles of the century owing to their unique size dependent and physicochemical properties [1]. In a world marked by technological advances, these nanoparticles are playing significant role with myriad applications in multidisciplinary field of sciences [2]. Synthesis of nanoparticles can be grouped into top down and bottom up process but majority of these processes are bound with one or more limitations such as generation of high energy, use of toxic materials thus causing environmental pollutions and restricted use in biomedical applications [3-5]. In recent years, advance scientific domains have envisioned new era which unfolds the bio-nano revolution wherein biological entities are employed for facile and safe synthesis of desired nanoparticles [6,7]. These biological entities may vary from prokaryotes to multicellular eukaryotes among which microbial resources are largely explored to synthesize different nanoparticles [7-9]. The use of microorganisms can be traced down since ancient times with its role in bioremediation of metals [10,11]. But in recent years this unique property of microorganisms has been envisaged to recognise them as nanofactories [12]. Hence, scientific communities are exploring different biological niches to isolate novel species bearing unique redox potential to reduce metal salts and synthesize nanoparticles [13,14]. One such area gaining tremendous importance includes plant associated microorganisms which are reported to play a vital role in protecting them from invading pathogens and promote plant growth by secreting bioactive metabolites [15,16]. In early nineties, burgeoning scientific interest on plant science revealed plants harbour untold number of microorganisms in the form of epiphytes and endophytes [17].

Among these

microbial plethora, endosymbionts residing inside plants tissues were termed as endophytes and interest on endophytes rapidly expanded with large scientific literatures revealing the untold roles of endophytes thus forming one of the novel sources of bioactive compounds [18]. Even though there has been significant progress on endophytes, their role in synthesis of nanoparticles is still in infancy [19]. Some reports suggest that the interference of endophytes and nanomaterials can open new avenue towards facile, eco friendly synthesis of nanoparticles with desired size and shape bearing biological activities [20]. One such successful area is evaluation of nanoparticles as potent antimicrobial agents as these nanoparticles by virtue of their unique properties can easily penetrate across the cell wall of microorganisms and can interfere with the vital components of the pathogens preventing it from cell proliferation [14, 22]. During the post 3

antibiotic resistant era, nanoparticles can offer new hope to combat drug resistant pathogenic microorganisms [23]. In most cases, conventionally synthesized nanoparticles has restricted their evaluation in biomedical applications and hence nanoparticles synthesized using biogenic principles offers alternative approach towards producing

compatible nanoparticles bearing

activity [7,24]. Hence the crux of present study reports synthesis of silver nanoparticles by endophytic bacterium inhabiting Coffea arabica L. Synthesized nanoparticles displayed significant bactericidal activity against test pathogens via disc diffusion, well diffusion, broth dilution and colony forming unit (CFU) assays. The study also reports synergistic effect of synthesized nanoparticles with standard antibiotic kanamycin which resulted in increase fold activity against the test pathogen thus highlighting the combined effect which can address the drug resistant issue across the globe. 2. Materials and Methods 2.1. Isolation of endophyte and surface sterilization Endophyte was isolated from intensive surface sterilization with 3.15% sodium hypochlorite and 70% ethanol as disinfectant as described by Baker et al (2015b) [13]. In each step of surface sterilization, plant materials were washed in sterile double distilled water. To confirm the efficacy of sterilization, sterility checks were carried out for each sample to monitor the effectiveness by imprint method and plating the final rinse aliquot on nutrient agar which served as control plate and all surface sterilized plant segments were placed onto the media enriched with cycloheximide and bavistin [25,26]. 2.2. Molecular characterization of potent endophytic bacteria Potent endophytic bacterial strains were inoculated to sterile 3 mL of LB medium and incubated overnight at 37º C in a rotary incubator for 24 hours. 2 mL of the resulting bacterial suspension (OD at 0.6) was pelleted at 13,000 rpm for 3 minutes and the total cellular DNA was extracted using the CTAB method outlined in Wilson, (1990) [27].

Extracted DNA was

amplified using universal primers, Forward primer UPF 5ˈ-AGTTTGATCCTGGCTCA-3ˈ and Reverse primer UPR 5ˈ-ACGGCTACCTTGTTACGACTT-3ˈ. Polymerase chain reaction (PCR) amplification was carried out by preparing 50 µl PCR reaction mixture containing 4µl extracted DNA with 100 ng/ml concentration, 5.0 µl of 10× PCR buffer (Merck Genei, India), 5 µl of 2.0 mM dNTP mix, 2.0 µl of 0.2 µM of each primer and 2.0 µl Taq DNA polymerase (1 U) 4

(Fermentas) and 30 µl sterile deionized water. PCR amplification was performed with an Eppendorf Master cycler Gradient (Eppendorf, Germany) using the following program: Initial denaturation at 95° C for 4 minutes, followed by 30 cycles of denaturation at 95° C for 1 minute, annealing temperature at 55° C for 1 minute and extension at 72° C for 1 minute with a final extension at 72° C for 10 minutes [28]. The PCR products were electrophoresed in 0.8% (w/v) agarose gel containing ethidium bromide (0.5μg/ml) and the amplicons were visualized under a gel documentation system (Bio Rad). The gel section with the desired band was carefully excised under UV light and subjected to extraction using a gel band purification kit (Qiagen) and sequenced bidirectional way in an ABI 3730 sequencer (Applied Biosystems, United States) using the UPF and UPR primers. Sequences were processed using BioEdit [29] and subjected to BLAST search at NCBI (www.ncbi.nlm.nih.gov) to assign putative identity, designation of operational taxonomic units based on sequence similarity measures and phylogenetic inference. Partial nucleotide sequences were deposited in NCBI Gen Bank and authentic accession number were obtained. Phenogram was constructed with Clustalw [30] software by grouping the isolates deposited at GenBank to reveal the relationships of identified strain with taxonomically similar bacteria by constructing the phylogenetic tree. Alignments were manually edited wherever necessary and a phylogenetic analysis was performed to assess phylogenetic affiliation.

2.3. Screening of endophytes for synthesis of silver nanoparticles The endophytes were screened for synthesis of nanoparticles by culturing on to media supplemented with metal salt silver nitrate. The abundantly growing endophyte was further subjected to secondary screening and cell free extract was assessed for synthesis of nanoparticles [21]. Actively growing colonies of the selected endophytic bacterial isolates were cultured in nutrient broth and incubated for 72 hours. Later the culture broth was centrifuged at 8000 rpm at 4o C for 20 minutes to obtain the cell free supernatant and was challenged with 1mM of silver nitrate and incubated on rotary incubator with 180 rpm under optimized conditions and samples were drawn every 2 minutes of interval and monitored for synthesis of silver nanoparticles. The synthesis protocol was optimized by varying different variables for instance pH was varied from acidic to alkaline, effect of temperature was studied by varying between 20 °C to 90 °C. Similarly, concentration of silver nitrate was varied from 0.5 mM to 2 mM . 5

2.4. Characterization of nanoparticles Samples were periodically drawn and monitored for synthesis by using various hyphenated techniques such as double beam UV-visible spectroscopy of Shimadzu double beam spectrophotometer with spectra recording at 200 to 800 nm. Similarly, FTIR analysis was performed with JASCO FT-IR 4100 instrument at room temperature with a resolution of 4 cm-1 and XRD pattern of the synthesized nanoparticles was recorded by Rigaku Miniflex-II Desktop X-ray diffractometer instrument operating at a voltage of 30 kV. Morphological characteristics of nanoparticles was studied using Transmission Electron Microscopy whose measurement was scanned using a TECHNAI-T12 JEOL JEM-2100 Transmission electron microscope operated at a voltage of 120 kV with Bioten objective lens. Subsequently, the particle size was ascertained using a Gatan ccd Camera [13]. The average size and stability of the nanoparticles were determined using Dynamic Light Scattering (DLS) performed at 25 °C on a Malvern using Zetasizer Nano ZS (Malvern Instruments; UK). Finally, the synthesized Ag-NPs were dried, drop coated onto carbon film, and tested using energy dispersive X-ray (EDS) analyzer (XL 30; Philips). 2.5. Test pathogens All the test human pathogens viz., Bacillus subtilis (MTCC 121), Staphylococcus aureus (MTCC 7443), Pseudomonas aeruginosa (MTCC 7903), Klebsiella pneumoniae (MTCC 7407), Escherichia coli (MTCC 7410) were procured from MTCC- Chandigarh, India which were preserved and maintained at stock of Department of Studies in Microbiology, University of Mysore. The phyto-pathogens Xanthomonas campestris, Xanthomonas oryzae, Xanthomonas vesicatora were procured from DANIDA laboratory, Department of Studies in Biotechnology, University of Mysore. 2.6. Antibacterial activity of nanoparticles Silver nanoparticles were separated and concentrated by repeated centrifugation at 10,000 x g replacing the supernatant with sterile distilled water. Antimicrobial activity of synthesized nanoparticles was evaluated against important human pathogens via disc diffusion assay, well diffusion assay, micro dilution assay, CFU plate method and minimal inhibitory concentration. In brief pre-warmed MHA (Mueller-Hinton agar) plates were seeded with 106 CFU (colony forming unit) suspensions of each test organism and swabbed uniformly by using 6

sterile cork borer 10 mm diameter of agar was punched and 50 µl of 10 mg/ml nanoparticles were added to each well and incubated at 37° C for 24 hours. After incubation, the zone of inhibition was measured and interpreted with kanamycin at 1µg/mL concentration. Disc diffusion assay was performed similarly to that of well diffusion assay, instead of punching well the disc impregnated with 50 µl of 10 mg/mL nanoparticles were placed and incubated as mentioned above [2,13]. Statistical analyses of results were obtained using IBM SPSS version 20 (2011). One way ANOVA (analysis of variance) at value p<0.001 followed by Tukey’s Post Hoc test with p ≤ 0.05 was used to determine the significant differences between the results obtained in each experiment. Minimal inhibitory concentration was carried out based on the protocol described by Sarkar et al., (2007) with slight modification. The resazurin solution was prepared by dissolving a 270 mg tablet in 40 mL of sterile distilled water. Plates were prepared under aseptic conditions. A sterile 96 well plate was labelled. A volume of 100 μL of test material (nanoparticles) was dissolved in sterile saline and pipette into the first row of the plate. To all other wells, 50 μL of nutrient broth was added. Serial dilutions were performed using a multichannel pipette. To each well 10 μL of resazurin indicator solution was added. Later 30 μL of isosensitised broth was added to each well to ensure the final volume was a single strength of the nutrient broth. Finally, 10 μL of bacterial suspension (5 × 106 CFU/mL) was added to each well to achieve a concentration of 5 × 105 CFU/mL. The plate was wrapped with cling film and each plate was prepared with a set of controls. Kanamycin served as positive control and placed in an incubator at 37° C for 18 to 24 hours. The color change was then assessed visually. Any colour changes from purple to pink or colourless were recorded as positive. The lowest concentration at which colour change occurred was recorded as the MIC value [31]. 2.7. Synergistic activity Synergistic activity was carried out as per the protocol of Fayaz et al., (2010) [32] with slight modification and standard kanamycin disc was impregnated with freshly prepared silver nanoparticles with a final concentration of 10 µg of silver nanoparticles per disk and increase fold activity was calculated using the formula: b-a/a x 100, wherein ‘a’ is antibiotic and ‘b’ referred to nanoparticles in combination with antibiotic kanamycin. 3. Results and discussion

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The emerging roles of endophytes in mediating the synthesis of silver nanoparticles accentuate the unique metabolic diversity of endophytes compared to normal microbial flora which makes them one of the superior sources [33-35]. Till date, most of the reports on endophytes were related to fungal endophytes compared to bacterial endophytes which are known to be more dominant colonizing symbionts. The use of surfactants resulted in isolation of potent endophytes residing inside the plant tissues. The isolation studies suggested that use of cycloheximide suppressed the growth of fungal endophytes to yield only bacterial endophytes. Further no colonies were observed with control plates which indicated successful surface sterilization. One of the typical characteristics of endophyte is their emergence from surface sterilized plant segments and slow growth rate initially. All the obtained bacterial endophytes were subjected to primary screening for synthesis of silver nanoparticles by growing onto the enrich media supplemented with 2mM silver nitrate. The bacterial colony growing luxuriantly was further grown and supernatant was treated with silver nitrate. Among the isolated strains, endophytic bacterium which is capable of synthesizing nanoparticles within shorter time interval was selected and treated with metal salts which resulted in rapid synthesis of nanoparticles and these results were in accordance with the earlier findings. One such study reports extracellular synthesis of gold nanoparticles using endophytes isolated from the medicinal plant Bauhinia variegata L. among the isolates screened, Pencillium citrinum was capable of synthesizing nanoparticles with the reduction time in minutes compared to the other isolates [36]. In the present investigation selected strain Pseudomonas fluorescens strain CA 417 was characterized and sequence was submitted to Genbank with accession number KC 480603. Phylogram was constructed using ClustalW software to reveal the affiliation of Pseudomonas fluorescens strain CA 417 with taxonomically similar bacteria based on the 16S rRNA gene sequences (Fig 1). Bacterial endophytes colonize inside the host tissues in large number and scientific reports envision isolation of culturable endophytic bacteria. Among the diverse classes of endophytic bacteria one of the dominant species comprises of Pseudomonas. During the course of time Pseudomonas are known to invade plant roots and reach host tissues and inhabit as endophytes [37]. Perusal of scientific literatures report Pseudomonas species are capable of secreting extracellular secondary metabolites bearing biological activities [38]. In present investigation, emphasis was attributed to synthesize silver nanoparticles by treating 1 mM silver nitrate with cell free supernatant secreted by Pseudomonas fluorescens 8

strain CA 417 and under optimized conditions. The optimization of variables resulted in maximum synthesis and it was observed that elevated temperature 80o C, alkaline pH 8 and 1 mM silver nitrate influenced rapid synthesis (Fig.2a,b,c). The obtained results are in accordance with previous findings suggesting the vital role of pH and temperature influencing the synthesis of nanoparticles [13,21]. The compatibility of rapid synthesis was compared by synthesizing silver nanoparticles by popular chemical method of sodium citrate reduction. The results suggested that chemically synthesis of nanoparticles was achieved at 25 minutes and endosymbiotic synthesis was completed within 15 minutes of incubation (Fig.3a,b). In the present investigation, change in colour from the reaction mixture to dark brown indicated the synthesis of silver nanoparticles. The further confirmation of silver nanoparticles was achieved using UV-Visible spectra recorded at different time intervals which resulted in maximum absorbance at 425 nm (Fig 3a) due to surface plasmon resonance [40]. The fate of nanoparticles and their applications remain infancy without accurate and wellresolved characterization of nanoparticles [39]. Characterization of nanoparticles ensures and determines the application based on their properties. For metallic nanoparticles such as silver and gold initial preliminary confirmation can be achieved by visual observation with a change in colour [4]. Further synthesized nanoparticles were subjected to characterization using hyphenated techniques. FTIR analysis of supernatant prior to exposure to silver ion was recorded to reveal predominant peaks occurring at 3346 corresponding to the primary amines [41], 1738 and 1365 corresponds to the carbonyl group and 1217 corresponds to C-H bonding, 1635 corresponding hydroxyl group [42] and 680 to aromatic group [43] as depicted in Figure 4. Interestingly, FTIR analysis of synthesized silver nanoparticles displayed absence of vibrational stretching at 1738, 1365 1217 which indicated the involvement of functional groups in mediating and stabilizing silver nanoparticles. In general, the stability of silver nanoparticles is more significant for exploring their applications in biomedicine. Consequently, the silver nanoparticles are normally stabilized by using stabilizing agents under various conditions. However, in present investigation, silver nanoparticles were more stable owing to in situ bio-capping by the organic moieties present in the supernatant which is responsible for the synthesis and stabilization of silver nanoparticles thereby preventing agglomeration or so called in situ stabilization [44]. Crystalline nature of the silver nanoparticles synthesized was confirmed by peaks at 38o 44o, 64o and 78o which corresponds to the (111), (200), (220) and (311) facets of 9

the crystal planes at 2θ angle exhibiting Braggs reflections that have been indexed on the basis of the face centered cubic structure of silver (Fig.5). Apart from Bragg’s peaks, the recorded XRD pattern showed additional unassigned peaks (marked stars). These peaks might be due to bioorganic or metallo-proteins present in the supernatant which are responsible for stabilization of nanoparticles. Similar observations were reported in majority of previous studies [45]. The average crystallite size‘d’ of silver nanobactericides was calculated to be 20-30 nm using Scherer equation: d = Kλ/β cosθ, where K is the 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 [46]. Further, the purity of silver was depicted with energy dispersive X ray spectroscopy which revealed the presence of high intense absorption peak at 3 keV, a typical characteristic of nano-crystalline silver (Fig.6). Generally, the EDS give 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 nanoparticles [47]. The obtained results are in congruence with the characteristic results of elementary nano silver at 3 keV due to their surface plasmon resonance as per the reported scientific literatures [48-50]. The morphological characteristics of silver nanoparticles were investigated by Transmission electron microscopy at 10,000x and 20,000x magnification which revealed distinct shapes of nanoparticles which that were well separated from each other. The average size of nanoparticles was estimated by measuring 100 nanoparticles from TEM micrograph which displayed particles size between 5 to 50 nm as shown in the figure 7. Synthesized nanoparticles were polydisperse in nature with myriad shapes spherical, near to spherical, hexagonal and triangular. The size distribution was also confirmed with DLS studies which revealed the average size of the nanoparticles as 20.66 nm. The obtained results were in accordance with the previous studies with polydispersity of nanoparticles [51, 52]. Interestingly, synthesized silver nanoparticles displayed bactericidal activity against targeted test pathogens. The activity of silver nanoparticles was validated and interpreted with kanamycin as standard. Kanamycin is referred as broad spectrum antibiotic particularly against Gram –ve pathogenic bacteria. Kanamycin is used in treatment of various bacterial infections including drug resistant pathogens and in management of tuberculosis. Hence Kanamycin was selected as subject of interest in the present investigation. Antimicrobial activity using disc and well 10

diffusion assay was measured as zone of inhibition (Table 1). Furthermore, activity of synthesized nanoparticles was evaluated against phytopathogens such as Xanthomonas campestris, Xanthomonas oryzae, Xanthomonas vesicatora. The obtained results expressed significant activity against all the test phytopathogens which was compared and validated with tetracycline and Xanthomonas campestris was most sensitive among the test phytopathogens (Table-1). Similarly, broth dilution assay resulted in drastic decrease in optical density of the broth seeded with different test pathogens against increase in the concentration of silver nanoparticles. Colony forming assay results showed gradual decrease in the viable colonies as the concentration of nanoparticles increased from 0 to 100 µg/ml. Further minimal inhibitory concentration of silver nanoparticles varied from 31.25-250 µg/ml. The bactericidal activity suggested that synthesized nanoparticles were more effective against Klebsiella pneumoniae (MTCC 7407) compared to other test pathogens followed by Staphylococcus aureus (MTCC 7443), Bacillus subtilis (MTCC 121), Pseudomonas aeruginosa (MTCC 7903) and least activity was observed against Escherichia coli (MTCC 7410). The results were consistent with respect to antibacterial assays and profound activity of silver nanoparticles against Klebsiella pneumoniae (MTCC 7407) compared to other pathogens (Fig. 8). Interestingly, biologically synthesized nanoparticles expressed significant results in comparison with chemically synthesized nanoparticles. This might be owing to the fact that presence of biomolecules on the surface of biologically synthesized nanoparticles which marked the profound activity which confirmed the use of biologically synthesized nanoparticles as one of the alternative against drug resistant pathogens [21]. One of the interesting facets of the present study is that synthesized silver nanoparticles are more sensitive to Gram-ve bacteria. Previous scientific literatures the highlights role of silver and their by-products that 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 to name a few [20,53,54]. The synergistic effect of silver nanoparticles in combination with antibiotic kanamycin resulted in increased fold dilution up to 58% against Klebsiella pneumoniae (MTCC 7407) followed by Pseudomonas aeruginosa (MTCC 7903), Bacillus subtilis (MTCC 121), Staphylococcus aureus (MTCC 7443) and Escherichia coli (MTCC 7410). The synergistic activity of synthesized silver nanoparticles 11

in combination with kanamycin displayed an overall 22.3% increase in fold activity and highest increase fold dilution was observed against Klebsiella pneumoniae with 58% fold increase activity (Table 2,Fig 9a). The synergistic activity was also measured with chemically synthesized nanoparticles which marked increase fold activity to merely 8.8 %. These results were in accordance with other antibacterial assay of present investigation (Table 3). The increase in fold dilution might be due to the conjugation of active sites present in the antibiotic and silver nanoparticles. The possible mode of conjugation is predicted in figure 9b, as kanamycin consists of NH and OH functional groups which can easily form bonding with synthesized nanoparticles via non-conventional H -bond and conventional O-bond. These bonding can lead to multiple modes targeting sites in pathogen. The results obtained are in accordance with findings [32,55] which suggested the use of nanoparticles as alternative strategies to combat drug-resistant pathogens. In recent years, with expansion of drug resistance, there is decrease in the effectiveness of kanamycin hence in order to retain and increase its activity synergistic effect with nanoparticles was conducted in the present investigation which exhibited increase fold dilution in the activity in comparison with individual kanamycin. The obtained results in the present investigation are promising enough to report a benign endosymbiotic synthesis of silver nanoparticles with potent bactericidal activity. Conclusion: The present study envisions eco-friendly and rapid synthesis of silver nanoparticles by endophyte Pseudomonas fluorescens CA 417 inhabiting Coffea arabica L. The characterization of synthesized nanoparticles revealed the polydisperse and crystalline nature. Silver nanoparticles displayed significant antibacterial activity against the panel of test human and phyto pathogens. The study also reports synergistic activity of silver nanoparticles with the standard antibiotic kanamycin which resulted in increase fold activity up to 58%. The study highlights an alternative and emerging strategy to combat drug resistant pathogenic bacteria which has created serious concern across the globe due to limited choice of antibiotic treatment. Hence, the study adds tremendous value to the growing scientific knowledge of developing new antimicrobials.

Acknowledgments

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Authors thank DST-SERB for financial assistance and Department of Studies in Microbiology, University of Mysore for providing facility and infrastructure. Authors express their gratitude to Dr. Harald Scherm for his scientific editing and Mr. Kalyan Chakravarthy for proof reading the manuscript. Authors are thankful for Siberian Federal University for Young scientist award to Dr. Syed Baker.

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Fig. 1. Phylogram expressing the relationships of Pseudomonas fluorescens strain CA 417 to taxonomically similar bacteria based on the 16S rRNA gene sequences

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Fig.2a. Optimization of silver nitrate concentration in synthesis of silver nanoparticles

Fig.2b. Optimization of pH in synthesis of silver nanoparticles

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Fig.2c. Optimization of temperature in synthesis of silver nanoparticles

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Fig.3a. UV-Visible spectrum of silver nanoparticles synthesized by Pseudomonas fluorescens strain CA417

Fig.3b. UV-Visible spectrum of silver nanoparticles synthesized by chemical method

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Fig. 4. FTIR analysis of supernatant and silver nanoparticles synthesized by Pseudomonas fluorescens strain CA 417

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Fig. 5. Comparison of experimental XRD and standard JCPDS file 04-0783

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Fig. 6. EDS analysis of silver nanoparticles

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Fig.7. TEM microgram of nanoparticles synthesis by Pseudomonas fluorescens strain CA 417

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Fig. 8. Comparison of antibacterial activity of silver nanoparticles synthesized by Pseudomonas fluorescens strain CA 417 using broth dilution and Colony forming unit (Note: OD: Optical density, CFU: Colony forming unit, SA: Staphylococcus aureus, EC: Escherichia coli, BS: Bacillus subtilis, PA: Pseudomonas aeroginosa, KP: Klebsiella pneumoniae )

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Fig. 9a. Zone of inhibition in mm with antibiotic kanamycin, nanoparticles and their synergistic activity

N H

H H H-O

O-H 0

Ag

O

O O-H

Fig. 9b. A schematic illustration of interaction of functional groups of kanamycin interacts with silver nanoparticles. (---- indicates non-conventional H -bond and ─ indicated conventional O-bond)

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Table 1. Mean zone of inhibition (mm) of antibiotic kanamycin, silver nanoparticles (Biological and chemical), silver nitrate, supernatant Human pathogens

Kanamycin

B.subtilis E.coli S.aureus K.pneumonae P. aeruginosa

26.00±1.00 24.66±0.57 24.00±1.00 12.66±0.57 18.66±0.57

Biologically synthesized nanoparticles 12.33±0.57 11.66±0.57 11.00±1.00 08.00±1.00 09.66±0.57

Phyto pathogens

Chemically synthesized nanoparticles 07.33±0.57 09.00±1.00 10.66±0.66 07.00±1.00 07.66±0.57

Silver nitrate

Supernatant

0.00±0.00 0.00±0.00 06.00±1.00 0.00±0.00 0.00±0.00

0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00

Tetracycline Biologically Chemically Silver synthesized synthesized nitrate nanoparticles nanoparticles X. campestris 15.66±0.57 13.00±1.00 09.66±0.57 0.00±0.00 X.oryzae 13.00±1.00 11.66±0.57 10.00±1.00 0.00±0.00 X. vesicaloria 12.66±0.57 11.00±1.00 11.66±0.57 0.00±0.00 Values mentioned in the table are mean of triplicates ±Standard deviation.

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Supernatant

0.00±0.00 0.00±0.00 0.00±0.00

Table 2. Mean zone of inhibition (mm) of antibiotic kanamycin, biologically synthesized silver nanoparticles and combined antibiotic kanamycin with biologically synthesized silver nanoparticles Pathogens Kanamycin B-SNP Kanamycin Fold increase % (a) + [ (b-a)/a]x100 B-SNP(b) KANAMYCIN B.subtilis 26.00±1.00 12.66±0.57 28.66±0.57 7.6 E.coli 24.66±0.57 11.66±0.57 27.00±1.00 12.5 S.aureus 24.00±1.00 10.66±0.57 26.66±0.57 8.3 K.pneumonae 12.66±0.57 08.66±0.57 19.66±0.57 58.3 P. aeruginosa 18.66±0.57 10.00±1.00 21.00±1.00 16.6 Overall synergistic antibacterial effect: 20.66% Values mentioned in the table are mean of triplicates ±Standard deviation.

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Table 3. Mean zone of inhibition (mm) of antibiotic kanamycin, chemically synthesized silver nanoparticles and combined antibiotic kanamycin with chemically synthesized silver nanoparticles Pathogens Kanamycin C-SNP Kanamycin Fold increase (a) + [ (b-a)/a]x100 C-SNP(b) KANAMYCIN B.subtilis 26.00±1.00 07.66±0.57 26.66±0.57 0 E.coli 24.66±0.57 08.00±1.00 24.00±1.00 0 S.aureus 24.00±1.00 10.00±1.00 26.00±1.00 8.3 K.pneumonae 12.66±0.57 07.66±0.57 14.33±0.57 11.1 P. aeruginosa 18.66±0.57 07.00±1.00 20.66±0.57 11.1 Overall synergistic antibacterial effect: 6.1% Values mentioned in the table are mean of triplicates ±Standard deviation.

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%