Green Synthesis and Antimicrobial Activities of Silver Nanoparticles using Cell Free-Extracts of Enterococcus species

Cell-free extracts of six strains of Enterococcus species obtained from fermented foods were used for the green synthesis of silver nanoparticles (AgNPs), which was characterized by UV-Vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The biosynthesized AgNPs were dark brown in colour having surface plasmon resonance in the range of 420-442 nm. The spherical shaped AgNPs had sizes of 4-55 nm, whose formations were facilitated by proteins as indicated by the presence of peaks 1,635-1,637 and 3,275-3,313 cm-1 in the FTIR spectra. The energy dispersive x-ray (EDX) showed prominent presence of silver in the AgNPs colloidal solution, while the selected area electron diffraction was typified by the face-centred crystalline nature of silver. The particles inhibited the growth of multi-drug resistant clinical isolates of Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris, and also potentiated the activities of ampicillin, ciprofloxacin and cefuroxime in the AgNPs-antibiotic synergy studies. In addition, the prospective relevance of the particles as nanopreservative in paints was demonstrated with the inhibition of growth of Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus niger and A. flavus in AgNPs-paint admixture. This report further demonstrates the green synthesis of AgNPs by strains of Enterococcus species.


Introduction
Nanotechnology is a branch of knowledge that is concerned with the synthesis of particles at nano-scale level.The biological routes of synthesis of nanoparticles are important alternatives to the use of chemicals and other methods that often have toxic effects on the ecosystem (Sneha et al., 2011).The importance of nanotechnology is increasing rapidly because of the manipulation of materials at nano-scale level for different applications.Over the years, nanoparticles have been synthesized by physical, chemical and biological methods.Many nanoparticles have been synthesized using bacterial metabolites, and these include gold (Ojo et al., 2016), ZnS (Malarkodi and Annadurai, 2012), TiO2 (Malarkodi et al., 2013), silver (Rajeshkumar et al., 2013;Lateef et al., 2015a, b, c) and silver-gold alloy nanoparticles (Ojo et al., 2016).
Out of the different types of nanoparticles, AgNPs are the most effective, with good antimicrobial efficacy (Gong et al., 2007).They have been widely applied in the production of solar cells and batteries, as catalysts in chemical reactions, bio-labelling and as antimicrobials Preparation of cell-free extracts Brain heart infusion broth was prepared, sterilized and inoculated with fresh culture of the isolates (1 × 10 6 cfu/ml).The cultured flasks were incubated at 37 °C for 72 h.After incubation, centrifugation was carried out at 4,000 rpm at 10 °C for 15 min (Lateef et al., 2015a), and the supernatants were used for the synthesis of AgNPs without further purification.

Synthesis of AgNPs
The bacterial supernatant (1 ml) was added separately to the reaction vessel containing 10 ml of 1 mM silver nitrate (AgNO3) and placed under sunlight for the photoactivation of silver to aid the synthesis of AgNPs.The formation of AgNPs was monitored through visual observation of change in colour, and measurement of the absorbance in the range of 270-800 nm on a UV-Visible spectrophotometer (Genesys 10 UV Thermoelectron Corporation, UK).
The fingerprints of biomolecules responsible for the formation of AgNPs were elucidated using Fourier transform-infrared (FTIR) spectroscopic analysis (BUCK M530 Spectrophotometer, Buck, USA) on the powder sample of AgNPs as previously reported (Lateef et al., 2015a).The morphology and size of the particles were studied using transmission electron microscope JEM-1400 (JEOL, USA) operated at 200kV.Few drops of colloidal AgNPs were applied on the copper grid (3.05 mm) (Agar Scientific, Essex, UK).This was then layered with 0.3% formvar that was prepared in chloroform.After settling of the particles on the copper grid, excess fluid was removed and then air-dried for TEM viewing.

Antimicrobial activities of AgNPs
It was carried out by agar diffusion method as previously described (Lateef et al., 2015a;Lateef and Ojo, 2016), using clinical isolates of Proteus vulgaris, Escherichia coli and Klebsiella pneumoniae originally sourced from LAUTECH Teaching Hospital, Ogbomoso.Inoculum obtained as 18-h culture (~10 6 cfu/ml) was used for the seeding of Mueller-Hinton agar (Lab M Ltd., UK) plates.AgNPs of different concentrations were dispensed as 100 µl into wells of 7 mm that were bored on the plates using cork borer.This was followed by the incubation of the plates at 37 °C for 24 h, after which zones of inhibitions were monitored.
In the current work, strains of Enterococcus species previously isolated from traditional fermented vegetable condiments and local cheese 'wara' (Oladipo et al., 2013;2014a, b;2015) that have probiotic potentials were evaluated for the green synthesis of AgNPs, using the cellfree extracts.The study also investigated the biomedical and industrial applications of biosynthesized AgNPs as antimicrobial agents.
The use of Enterococcus species in nanobiotechnology is at infancy as there are few reports of the use of E. faecalis (Chandrakanth et al., 2014) for the synthesis of AgNPs, ZnO nanoparticles (Ashajyothi et al., 2014), AuNPs (Ashajyothi and Chandrakanth, 2014) as well as the synthesis of CdS nanoparticles by marine isolate of Enterococcus sp.(RMAA) (Rajeshkumar et al., 2014).However, the present study further demonstrates the capabilities of different species of Enterococcus for the green synthesis of AgNPs.The study is the first reference to the use of E. gallinarum and E. casseliflavus for the synthesis of AgNPs.

Microorganisms
Six strains of Enterococcus species used in this study were isolated as described by Oladipo et al. (2013).They were characterized using both biochemical and molecular techniques.Molecular identification to species level was performed by 16S rRNA analysis.Genomic DNA of the species were isolated and the DNA fragments were amplified; amplification was carried out in a thermal cycler with each reaction mixture comprising the template DNA along with master mix and primers designated as FD1 (5′-AGAGTT TGATCCTGGCTCAG -3′) forward and RD1 (5′-AAGGAGGTGATCCAGCC-3′) for reverse.The amplified products with the expected sizes were subsequently sequenced and compared with sequences in the database of National Centre for Biotechnological Information (NCBI) (BLAST, 2016).The sequences were subsequently submitted to GenBank for accession numbers.
cultures were observed at 48 h of incubation at 37 °C.The inhibitory zones were examined and interpreted (Chortyk et al., 1993) taken cognizance of the appropriate breakpoints (Andrews, 2005).

Synergistic studies on antibiotic-AgNPs mixtures
Studies were conducted on the antimicrobial synergy between the AgNPs and some commonly dispensed antibiotics such as cefuroxime, ampicillin and ciprofloxacin.The agar-diffusion method as previously described was used (Lateef et al., 2015a;Lateef et al., 2016e).The antibiotics were dissolved in sterile distilled water to obtain concentrations of 500 µg/ml and 1 mg/ml.Then, bacterial isolates were exposed to the antibiotics using 100 µl of each antibiotic dispensed into the wells.The second part of the experiment on antimicrobial synergy was carried out by combining 50 µl of the antibiotic with 50 µl of AgNPs (100 µg/ml), to which the test isolates were exposed.The plates were then incubated and observed for zones of inhibition as previously stated.

Evaluation of antimicrobial properties of synthesized AgNPs as preservative in paint
The potential preservative action of the synthesized AgNPs against bacteria and fungi was carried out through the introduction of AgNPs into emulsion paint as previously described (Lateef et al., 2015a).Commercially available white emulsion paint was procured and prepared according to the instructions of the manufacturer.The paint was dispensed as 19 ml in McCartney bottles and autoclaved at 121 °C for 15 min.Thereafter, the paints were inoculated with 1 ml (~10 6 cfu/ml) of 18 h broth cultures of S. aureus and P. aeruginosa.For Aspergillus flavus and A. niger, 1 ml (~10 6 cfu/ml) of 48 h culture was used.In the control, samples of emulsion paint were inoculated with the test organisms alone, whereas for the test, samples of the paint containing 1 ml of 100 µg/ml of AgNPs were inoculated with the test organisms.The bottles were incubated at 37 and 30 ± 2 °C for 48 h for bacteria and fungi, respectively.After the period of incubation, contents of the bottles were withdrawn and 1 ml was plated on nutrient agar (bacteria) and potato dextrose agar (fungi) using the pour plate method.The incubation of plates was done at 37 °C for bacteria and 30 ± 2 °C for fungi for up to 48 h, and thereafter checked for growth.

Bacterial isolates
The strains of E. gallinarum, E. faecium and E. casseliflavus used in the study were isolated from traditionally fermented vegetable condiments (iru, ogiri, okpehe, ugba) and wara.These strains were Gram-positive cocci, catalase negative, oxidase negative, and non-spore formers, with the ability to grow in the presence of 6.5% NaCl at pH 9.6 and at 10 and 45 °C.The strains were able to hydrolyze esculin, pyrrolidonyl-β-naphthylamide and arginine, but were unable to hydrolyze starch.They also showed different sugar fermentation patterns.Further confirmation of identity was carried out by 16S rRNA sequencing, and accession numbers assigned to each strain as shown in Table 1.
Enterococcus species are members of the family of lactic acid bacteria (LAB) that is widely distributed in nature.They have a history of being used as starter cultures in fermented foods largely due to the unique flavours that they produce, and their health-promoting activities (Oladipo et al., 2014b;Oladipo et al., 2015).Some of them may have antimicrobial activities against food deteriorating microorganisms, making them useful in food preservation (Oladipo et al., 2015).Also, different probiotic supplements, for both man and animals, include Lactobacillus and Enterococcus species (Pollmann et al., 2005;Tompkins et al., 2008;Vankerckhoven et al., 2008).To a large extent, there is dearth of information on the use of Enterococcus to produce nanoparticles.Its relevance in nanotechnology include the use of the marine isolate of Enterococcus sp.(RMAA) for the green synthesis of CdS nanoparticles (Rajeshkumar et al., 2014), and E. faecium to synthesize AgNPs (Chandrakanth et al., 2014).However, there is no report on the evaluation of E. gallinarum and E. casseliflavus for the green synthesis of AgNPs.

Biosynthesis and characterization of AgNPs
The formation of AgNPs was characterized with the development of colour, which was produced as a result of the reduction of silver ion by biomolecules present in the cell-free extracts.The intensity of the colour increased as the bio-reduction of silver ions progressed and stabilized when the reaction was completed.The formation of AgNPs was facilitated by the cell-free extracts within a period 10 min, with the dark brown colour which stabilized in 20 min as shown in Fig. 1.Several colours, including light yellow, yellow brown and dark brown, have been reported for colloidal solutions of AgNPs (Das et al., 2014;Emeka et al., 2014;Lateef et al., 2015a, b, c, d;Netala et al., 2016).The variations in colours have been ascribed to the activities of different types of biomolecules that act as bioreductants in the transformation of silver metal to nanoparticles of different sizes and shapes that affect the surface plasmon resonance of the particles.The excitation of surface plasmon vibrations in metal nanoparticles leads to the development of colour (Mulvaney, 1996).The UV-Vis absorption spectra of the biosynthesized AgNPs are as presented in Fig. 2. The biosynthesized AgNPs displayed surface plasmon resonance in the range of 420-442 nm, which is within the range of values previously reported for AgNPs (Shaligram et al., 2009;Kannan et al., 2013;Emeka et al., 2014;Kathiraven et al., 2015;Lateef et al.,2015b, d;Lateef and Adeeyo, 2015;Anandalakshmi et al., 2016).However, using cell-free extract of a strain of E. faecium, Chandrakanth et al. (2014) reported the formation of AgNPs, which absorbed maximally at 309 nm.The particles obtained in the present study were stable without significant changes in absorption spectra when stored for about three months at room temperature.
Microscopic analysis using TEM (Fig. 4a) showed that the biosynthesized AgNPs were nearly spherical, having a size range of 4-33 nm for E. gallinarum T7, 5-38 nm for E. faecium IP11, 12-50 nm for E. gallinarum C103, 5-41 nm for E. gallinarum U8, 10-26 nm for E. faecium T10, and 5-55 nm for E. casseliflavus W14 cell-free extract mediated AgNPs.The polydispersed form of the AgNPs was corroborated by the broadness of the UV-Vis spectra as earlier presented.The EDX patterns (Fig. 4b) showed the predominant presence of silver in the AgNPs solutions with the characteristic ring-like SAED patterns (Fig. 4c) typical of the face-centered cubic crystalline structure of silver (Shameli et al., 2011;Salem et al., 2014;Shankar et al., 2014).From the foregoing, it can be concluded that the cell-free extracts of Enterococcus sp. can be applied for the biogenic and eco-friendly synthesis of AgNPs, which adds to the biotechnological relevance of the bacterial isolates.

Antimicrobial activities of biosynthesized AgNPs
The biosynthesized AgNPs showed inhibitory activities against some clinical isolates of bacteria (Table 2).The AgNPs at concentrations of 60-100 μg/ml inhibited the growth of multi-drug resistant strains of E. coli, P. vulgaris and K. pneumoniae with zones of inhibition of 8-15 mm.The limited inhibitory actions might be due to the multidrug resistant nature of the isolates used in the present study.The antibiotic susceptibility of the isolates showed the drug resistance patterns as follow: K. pneumoniae (Caz, Crx, Gen, Cpr, Ofl, Aug, Amp), E. coli (Caz, Crx, Gen, Cpr, Ofl, Aug, Nit, Amp) and P. vulgaris (Caz, Crx, Cpr, Ofl, Aug, Nit, Amp).The results are in agreement with earlier published works on the antibacterial activities of AgNPs (Salem et al., 2014;Shankar et al., 2014;Lateef et al., 2015a, b, d;Lateef et al., 2016a, e).In a similar work, Chandrakanth et al. (2014) reported inhibitory zones of 14-19 mm by AgNPs biosynthesized using Enterococcus faecium  In AgNPs-antibiotics synergistic studies, it was discovered that the presence of AgNPs led to the improved performance of antibiotics such as ampicillin, ciprofloxacin and cefuroxime against drug-resistant strains of K. pneumoniae.Improved performance of 13.6-71.4, 8.3-57.1 and 71.4-85.7% were obtained for ampicillin, ciprofloxacin and cefuroxime respectively when used in combination with AgNPs (Fig. 5).It is noteworthy that in most cases, the two levels of antibiotic concentrations of 0.5 and 1 mg/ml used alone did not inhibit the growth of the test isolates.Therefore, the inclusion of AgNPs might have rendered the isolates susceptible, either by acting as drug carrier or by enhancing entry of antibiotics as a result of damage to the bacterial cell wall.These results are similar to those previously reported (Devi and Joshi, 2012;Lateef et al., 2015a;Lateef et al., 2016b;Lateef et al., 2016e) on the synergistic antibacterial effects of AgNPs on some antibiotics against Gram positive and negative bacteria.The susceptibility of drug-resistant bacteria as offered by AgNPsantibiotics synergy could be a promising panacea to solving problems of curtailing the prevalent drug-resistance phenomenon among bacteria in clinical practice (Lateef et al., 2005).
Similarly, the incorporation of biosynthesized AgNPs into emulsion paint yielded pronounced antimicrobial efficacy, which has potential of protecting the paint against microbial attack and biodeterioration.Remarkable antibacterial and antifungal activities were noticed through the drastic reduction of growth of S. aureus, P. aeruginosa, A. niger and A. flavus leading to the appearance of a single or few colonies after 72 h of incubation as against dense growth that were obtained on the control plates (Fig. 6).The results are in agreement with those reported earlier (Lateef et al., 2015a;Lateef et al., 2016b;Lateef et al., 2016e).It is envisaged that the AgNPs can act as nanopreservative in paint to prevent microbial deterioration thereby extending the shelf life of paints and painted surfaces.

Conclusions
In the current study, green synthesis of AgNPs using the cell-free extracts of six strains of Enterococcus species was successfully carried out.The spherical biosynthesised AgNPs of 4-55 nm in size displayed good antibacterial activities against multi-drug resistant strains of bacteria in both single and synergistic studies.The antimicrobial properties of the particles were demonstrated in paint, resulting to elimination of S. aureus, P. aeruginosa, A. niger and A. flavus when the particles were used as additives.The study has shown the relevance of Enterococcus species as biotechnological tool in the green synthesis of AgNPs.

Fig. 5 .
Fig. 5.The synergistic activities of biosynthesized AgNPs with antibiotics on some clinical bacterial isolates

Table 1 .
Enterococcus species used in this study

Table 2 .
The antibacterial activities of biosynthesized AgNPs against some bacterial isolates W14, U8, T7 and IP11 are strains of Enterococcus species as defined in

Table 1 ;
A, B and C are concentrations of AgNPs of 60, 80 and 100 µg/ml respectively; each value is average of two readings.