Potential Bactericidal Effect of Silver Nanoparticles Synthesised from Enterococcus Species

The present study proposed a synthesis of silver nanoparticles (AgNPs) from Enterococcus species. Silver has a strong antimicrobial potential, which has been used since the ancient times. The present work is carried out to screen bactericidal potential of silver nanoparticles against clinically isolated multidrug resistant bacteria’s. Biosynthesized silver nanoparticles were characterized by analytical techniques including as UV-Visible spectrophotometer, Field Emission Scanning Electron Microscopy (FeSEM), Energy dispersive x-ray spectroscopy (EDX), Nanoparticle Tracking Analyzer (NTA) analysis. Antimicrobial effect of silver nanoparticles against E. coli and K. pneumonia and Methicillin resistant Staphylococcus aureus (MRSA) respectively were investigated by Agar well diffusion method.

The bactericidal activity of silver nanoparticles against the pathogenic, MDR as well as multidrug susceptible strains of bacteria was studied by many scientists, and it was proved that the silver nanoparticles are the powerful weapons aga inst the MDR bacteria such as Pseudomonas aeruginosa, ampicillin-resistant Escherichia coli, erythromycin-resistant Streptococcus pyogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA) 6 . Bactericidal efficacy of silver nanoparticles was investigated by many researchers and their effective potential against broad range of microbes was proved, including antibiotic-resistant bacteria. Silver nanoparticles are also termed as new-generation of antimicrobials 4 . The group of researchers actively proved the bactericidal potential of silver nanoparticles. Feng et al., 6 reported the bactericidal potential of silver ions against S. aureus and E. coli.
Synthesis of the silver nanoparticles by Fusarium acuminatum and its bactericidal efficiency of silver nanoparticles against four human pathogenic bacteria, viz. E. coli, Salmonella typhi, Staphylococcus epidermidis and Staphylococcus aureus, were carried out by Ingle et al., 7 . Biosynthesis of silver nanoparticles using bacteria, fungi, and plants are already well-documented (8,9). Jeevan et al., 10 reported the biosynthesis of silver nanoparticles from P. aeruginosa, and its inhibitory effect on important human pathogens, E. coli and Vibrio cholerae. It is also clear that the bacterium P. aeruginosa can be used to synthesize bioactive silver nanoparticles efficiently using inexpensive substances in an eco-friendly and non toxic manner. The synthesis of silver nanoparticles was achieved using Bacillus megaterium supernatant and 1 mM silver nitrate 11 .
In the current study, silver nanoparticles were synthesized from Enterococcus sps. And their antibacterial activity was performed against different clinical pathogens.

Bacterial culture
The bacterial strain of Enterococcus faecalis (Non pathogenic) were collected from Medical Biotechnology and Phage Therapy Laboratory (MBPT), Department of Biotechnology, Gulbarga University, Gulbarga. The culture was inoculated on Bile esculin azide agar medium and incubated at 37 0 C for 24hrs for obtained pure culture 12 .

Preparation of cell free microbial extract
Luria-Ber tani broth were prepared, sterilized and inoculated with fresh Enterococcus faecalis culture. The cultured flasks were incubated at 37 o C for 72hrs. After incubation the culture were centrifuged at 10,000 rpm for 10 minute and the supernatant were used for silver nanoparticle synthesis.

Synthesis of silver nanoparticles
The bacterial supernatant was added separately to the reaction vessel containing Silver nitrate (AgNO 3 ) at a concentration of 100 mM (v/v) and control (without the silver nitrate). The reaction was carried out in dark conditions for 24 hours, at 37 0 C, pH: 7 in rotary shaker with 120 rpm 13 .

Characterization of silver nanoparticles
Synthesized silver nanoparticles were characterized by means visual observation and analytical techniques. After 24 hours of incubation the reaction medium is collected and centrifuged at 10,000 rpm for 10 min, supernatant is further used for UV-Visible spectrum using T90+ UV-VIS spectrophotometer and for Nanoparticle Tracking Analyzer (NTA) pattern analysis, for UV-VIS spectrophotometer scanning the spectra between 300 to 600 nm at room temperature.
The size and shape of nanoparticles was analyzed using Field Emission Scanning Electron Microscopy (FeSEM), after centrifuging the reaction mixture, obtained pellet is washed twice with double distilled water and phosphate saline buffer (pH:7.2), air dry the pellet and then used for FeSEM and EDX analysis. The samples slide were prepared by simple drop coating of the suspension of silver nanoparticles (dissolved in buffer) on a carbon-coated copper grid, by simply dropping a very small amount of the sample on the grid, with excess solution removed using blotting paper. The film on the scanning electron microscopy (FeSEM from Carl Zeiss, UK) grid was then allowed to dry under mercury lamp for 5 minutes. FeSEM instrument is equipped with a Thermo energy dispersive x-ray spectroscopy (EDX) attachment.

Antimicrobial activity of silver nanoparticles against MDR pathogens Antimicrobial activity by Agar well diffusion method
According to Clinical and Laboratory Standards Institute (CLSI) (14) guidelines the antimicrobial activity of Ag nanoparticles was evaluated against E. coli (Clinical MDR pathogen) Klebsiella pneumoniae (Clinical MDR pathogen) and clinical pathogen Methicillin resistant Staphylococcus aureus (MRSA) by modification of the agar well diffusion method. Clinically isolated pathogens were inoculated in Luria-Bertani broth and incubate at 37 0 C for 6hrs. Approximately 10 8 colonyforming units of 100µl of each microorganism were inoculated on Muller Hinton agar (MHA) plates; Agar wells of 5 mm diameter were prepared with the help of a sterilized stainless steel cork borer. Different concentrations (5, 10, 15, 20, 25 µg/ml) of the nanoparticles and maintain water as control in another well, were loaded on marked wells with the help of micropipette under aseptic conditions and plates were incubated at 37ºC for 18 and 24 hrs. The zone of inhibition was measured using a ruler and expressed in mm 15 .

Minimum Inhibitory Concentration (MIC)
T h e b a c t e r i c i d a l a c t i v i t y o f s i l ve r nanoparticles were determining by Broth dilution method according to CLSI 2000 (14). Bacterial cells were grown in 10 ml LB broth by inoculating 10 µl of 18 hrs culture, supplemented with 2, 4, 6, 8, 10, 12, 14, and 16µg ml -1 of silver nanoparticles. All tubes were incubated at 37°C for 18 hrs and 24 hrs and measure the absorbance at 600 nm. Antimicrobial test compound below the MIC cannot inhibit microbial growth.

RESULTS AND DISCUSSION
E. faecalis culture obtained from MBPT laboratory after inoculation onto the selective media Bile esculin azide agar they appears in black color colonies. In further biosynthesis of silver nanoparticles from test strain the primary conformation of synthesis of silver nanoparticles in the medium was characterized by the changes in color from yellowish white to brown (shown in the Fig:  1). The characteristic surface Plasmon resonance of silver nanoparticles ranges between 380 nm to 450 nm due to excitation of surface Plasmon vibrations and this is responsible for the striking yellow brown color of silver nanoparticles 16 .
Biological method of synthesis of silver nanoparticles exhibit strong absorption of electromagnetic waves in the visible range due to their optical resonant property, called Surface Plasmon Resonance (SPR). The characteristic surface plasmon resonance of silver nanoparticles ranges between 300 nm to 325 nm (shown in the Table: 1 and Fig: 2, 3) due to excitation of surface Plasmon vibrations. According to Mie theory, only a single SPR band is expected in the absorption spectra of spherical nanopar ticles whereas, the number of peaks increases as anisotropy increases 17 . According to Abd El-Raheem et al., 18   The diameters of particles were measured to obtain the particle size. Geethalakshmi et al., 19 were reported spherical silver nanoparticles with diameters of 36-94 nm are depicted in the Field emission scanning electron micrographs synthesized from Trianthema decandra (Aizoaceae) root extract and Deshpande et al., 20 also reported 90% of the silver nanoparticles synthesized from Carom Seed (Trachyspermum copticum) extract are in the range of 6 -50 nm in FeSEM studies. In our present study we are reporting FeSEM images of functionalized silver nanoparticles synthesized from E. faecalis bacterial biomass can be seen with core shell morphology of size 9 -130 nm and it is observed that there is a marginal variation in the particle size (Fig: 4). Maximum of the particles are in the range of 11-75 nm. The variation in particle size is possibly due to the fact that the nanoparticles are being formed at different times. Higher resolution image at 300 nm shows a group of particles in embedded in an organic moieties making a stable suspension. The particles appear to be polydispersed in nature and are roughly spherical in shape.
The study of metallic nature of these silver nanoparticles and reduction of silver into elemental silver and absence of other impurities has been confirmed and further strengthened by EDAX image. The optical absorption peak is observed approximately at 3 keV, which is typical for the absorption of metallic silver nanoparticles due to surface Plasmon resonance 21 . In the EDX spectrum of the bacterial mediated silver nanoparticles, the strongest peak detected was from silver with weaker peaks from carbon and oxygen 22 . In the current study energy dispersive x-ray spectroscopic profile of silver nanoparticles was confirmed by optical absorption peak at 3 keV, which is typical for silver nanocrystallite absorption. The spectrum shows the nanoparticles are primarily composed of silver with the only noticeable contaminant being phosphorus, sodium elements (Fig: 5).
Nanoparticle Tracking Analysis is a newly developed method for the direct and real-time visiualization on and analysis of nanoparticles in liquids. According to Pratik et al., 11 report NTA measurements revealed that the mean size of synthesized silver na noparticles was found to be 51 nm with concentration of 5.5 × 10 10 particles/ml in case of bacterial supernatant mediated synthesis. In our studies we revealed that the silver nano sample is quite polydisperse sample with particle size of 152nm and the concentration of sample after dilution comes to 5.6×10 11 particles/ml. It is observed that significant numbers of particles are observed from 30 to 300 nm with few aggregates being present at 400-500 nm (Fig: 6, 7 and 8).
The biologically synthesized silver nanopar ticles inhibited different pathogenic microorganisms. With the prevalence and increase of microorganisms resistant to multiple antibiotics, silver based antiseptics have been emphasized in recent years. Silver nanoparticles were biosynthesized using fungus Trichoderma viride 23 . Ag-NPs have a very broad range of antimicrobial activity and kill both Gram negative and Gram-positive bacteria, including Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans and Staphylococcus epidermidis [24][25][26][27][28][29][30][31] .
In the present work antimicrobial activity of biosynthesized silver nanoparticles studied against the multidrug resistant bacteria using standard zone of inhibition. MDR isolate E.coli showed maximum zone of inhibition of 18 mm for 10 µg/ml for K. pneumonia with maximum zone of inhibition of 18 mm for 25µg/ml and Staphylococcus aureus with maximum zone of inhibition of 19 mm for 25µg/ml of silver nanoparticle concentration. The highest antibacterial effect was found with zone of inhibition (19 mm) on Staphylococcus aureus and lowest antibacterial effect in Ag-NPs on Klebsiella pneumonia (14mm). The results were summarized in Table: 2 and Fig: 9, 10 and 11. Among antibiotic, the weakest activity was found to all three MDR pathogenic bacteria's. In comparison study between antibiotics and silver nanoparticles on E. coli (with antibiotics Rifampicin, Pipracillin, Ceftazidime), K. pneumonia (with antibiotics Ceftazidime, Cephalexin, Ceftriaxone) and Staphylococcus aureus (with antibiotics Ampicillin, Methicillin, Pencillin) showed weakest antibacterial effect as compare to silver nanaoparticles. Results were shown in Table: 3 and Fig: 12.
In Antibacterial activity the resulting zones of inhibition formed were mainly due to the destabilization of the outer membrane, collapse o f the plasma membrane, and depletion of intracellular ATP by the silver nanoparticles 32 . 12, 13 and 14 mm zone of inhibition was recorded in E.coli at the concentration of 20, 40, 60µg. In Staphylococcus aureus 11.5, 13.0 and 14.0 mm of zone of inhibition recorded at respective concentrations 33 .
The growth of the E. coli cells are inhibited at a concentration of 10 µg/ml of silver nanoparticles. This is the minimum concentration of the nanoparticles which inhibit the growth of the E. coli cells, i.e MIC of Ag nanoparticles 34 . Raffin et al., 35 observed that silver nanoparticles with mean sizes of 16 nm were completely cytotoxic for E. coli at a low concentration (60mg/ml). In the present work we report 11µg/ml, 3µg/ml and 12µg/ml of silver nanoparticles was recorded as the minimal inhibitory concentration (MIC) for K. pneumonia, E. coli, Staphylococcus aureus respectively by measuring absorbance at 600 nm shows 0.04 Optical Density indicates the complete inhibition of the growth. As compare to the recently published results, biosynthesized nanoparticles from Enterococcus fecalis showed complete inhibitory action on reported MDR at very low concentrations. Complete inhibitory action of biosynthesized nanoparticle is due to the size of the nanoparticles (9-130 nm), ultrafine particles (size range 10-100 nm) exhibited higher antimicrobial activity than big particles. The antibacterial properties were related to the total surface area of the nanoparticles (36). Smaller particles with larger surface to volume ratios have greater antibacterial activity. Similar results were published by Choi and Hu (37).

CONCLUSION
In the present scenario, pharmaceutical and biomedical sectors are facing the challenges of continuous increase in the multidrug-resistant (MDR) human pathogenic microbes. Diabetic patients were infected by the MDR pathogens leading to the high infection rates. Hence the researchers give more interest on nanotechnology and they framing the work to use nanoweapon against clinical multidrug resistant pathogens. Our present research interest is on nanoparticles synthesized from E. faecalis, and its efficacy against MDR pathogens act as a therapeutic agent to overcome of the antibiotic resistance especially in nosocomial infection pathogens. The biosynthesized silver nanoparticles using E. faecalis proved excellent antimicrobial activity. The antimicrobial activity is well demonstrated with agar well diffusion and MIC methods. Later silver nanoparticles are characterized with UV-Visible spectrum, FeSEM, EDX and NTA analysis to conclude the shape, size and their concentration. Thus, it is proven from this study that the Ag-NPs synthesized from E. faecalis seems to be promising and effective antibacterial agent against the multidrug resistant strains of bacteria. InternationalJournal of Nanomedicine, 2012, 7, 5375-5384. Malarkodi C, Rajeshkumar S, Paulkumar K, Gnanajobitha G, Vanaja M, Annadurai G, Nanoscience and Nanotechnology: An