Journal of Bioscience and Biotechnology Discovery

Volume 3. Page 59-64
Published 17th April, 2018
ISSN: 2536-7064

Full Length Research

Preparation, characterization, and well diffusion assay of Silver and Selenium nanoparticles against Biofilm forming Staphylococcus aureus

Poonam Verma1* and Sanjiv Kumar Maheshwari2

1School of Biotechnology, IFTM University, Moradabad, India.
2Institute of Bio-science and Technology, Shri Ramswaroop Memorial University, Lucknow-Deva Road, India.

Received 1st February, 2018; Accepted 15th March, 2018

*Correspondence: Dr. Poonam Verma, School of Biotechnology, IFTM University, Moradabad, India. Email:

Copyright © 2018 Verma and Maheshwari. This article remains permanently open access under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


In this study, biofilm-forming isolates from 36 clinical samples (Urine, Blood and Pus) were analyzed phenotypically via well-diffusion of nanoparticles, using Congo Red Agar, Tube and Tissue Culture Plate methods. Silver (Ag) and Selenium (Se) nanoparticles were synthesized by chemical reduction method. Antimicrobial activity was performed by well diffusion test of Ag and Se nanoparticles against biofilm forming Staphylococcus aureus strains. Distribution pattern showed the highest isolation rate from pus 30 (83.33%) followed by blood 4 (11.11%), and urine 2 (5.56%). All strains 36 (100%) were found to be non-biofilm forming S. aureus using CRA method. Another method for biofilm formation was performed by TM, 5 (13.89%) was strongly positive while 20 (55.56%) isolates unable to produced biofilm producer, whereas TCP method showed 12 (33.33%) high biofilm producer while 16 (44.44%) were non biofilm producer. Ag-NPs showed morphology average size and shape with scanning electron microscopy (SEM) reveals spherical particles with the size of 80.32 nm whereas, Se-NPs showed the size of 74.29 nm with SEM. In well diffusion assay, 50 μl of the nanoparticles (silver and selenium) and 50 μl antibiotic (amoxicillin as a positive control) were used. In this study, silver nanoparticles were found to have the active inhibitory effects against biofilm forming S. aureus strains (SA12, SA15, SA32), whereas selenium nanoparticles demonstrated low inhibitory effects (SA23).

Key words: Biofilm-forming, nanoparticles, Selenium, Silver, Staphylococcus aureus, Well-diffusion.


Staphylococcus aureus (S. aureus) is a gram positive cocci bacterium. It is an either commensal that colonizes healthy nasal mucosa (Williams, 1963) or pathogen of humans. The coagulase-positive species S. aureus is well known as a human pathogen, which can produce serious and sometimes life-threatening infections. Species of the S. aureus is identified on the basis of a variety of conventional physiological or biochemical tests. The key characters for S. aureus are colony pigment, heat-stable nuclease, clumping factor, free coagulase, and acid production from mannitol (Murray et al., 2003).

Clinical isolates of S. aureus species have different capacity to form biofilm. This may be due to the differences in the expression of biofilm related genes, genetic make-up and physiological conditions (Verma and Maheshwari, 2017). S. aureus biofilm mode of growth is tightly regulated by complex genetic factors. However, in recent decades some latest studies have taken biofilm development into account in terms of elucidating host immunity toward infection and may lead to the development of efficacious anti-biofilm S. aureus therapies (Verma and Maheshwari, 2017). A biofilm can be defined as a microbial derivative sessile community, typified by cells that are attached to a substratum, interface, and embedded in a matrix of extracellular polymeric substance. Biofilm exhibit an altered phenotype with regard to growth, gene expression and protein production (Donlan and Costerton, 2002). Biofilm thickness can range from a single cell layer to a substantial community encased by a viscous polymeric milieu (Costerton et al., 1995).

S. aureus isolated via clinical samples can express the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). The icaADBC dependent and independent pathways will be stimulated using different chemicals and level of biofilm formation as well as PIA/PNAG level will be assayed (Verma et al., 2013). In addition, proteomics and transcriptomics analysis will be performed to get insights into the interaction between the various factors of the pathways involved in the biofilm formation in wild type as well as mutant strains (Verma et al., 2013). The biofilm development in the Methicillin-resistant S. aureus (MRSA) is ica independent and involves a protein adhesin(s) regulated by SarA and agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development in ica dependent pathway (Verma et al., 2013).

In human population, approximately 20 to 25% has become persistently colonized and 75 to 80% intermittently or never colonized (Kluytmans et al., 1997; Dall’Antonia et al., 2005). Invading staphylococci are then either removed by the host innate immune response or attach to host extracellular matrix proteins and form a biofilm.

Nanoparticles have been widely used in various fields to synthesized by the chemical processes are toxic in nature, hence there is a growing need to develop environment friendly, cost effective and conveniently reproducible methods of nanoparticle synthesis. Furthermore, nanoparticles have increased surface area and therefore increasing the area of interaction with the pathogenic bacteria. They are also more likely to enter the bacterial surfaces than micron particles due to smaller size, exerting stronger effects on bacterial targets (Tran and Webstar, 2011).

Microbes are diverse in nature and can be exploited usefully for the formation and harvesting of nanoparticles. These microorganisms when confronted with high concentrations of metal ions (such as silver, selenium) reduce them to their elemental state. The enzyme nitrate reductase reduces silver ion to the metallic silver. NADH dependant nitrate reductase enzyme is confirmed to be a significant factor in the biosynthesis of metal and metalloid nanoparticles, reducing silver metal ions to elemental silver nanoparticles. The possible mode of action can be the activity of nitrate reductase enzyme upon silver ion, when it is taken up by the cell and converted it to elemental silver (Kalimuthu et al., 2008). Many biosynthesized metal nanoparticles such as silver are being used as antimicrobial compounds (Malarkodi et al., 2013).

Silver nanoparticles are a non-toxic and safe antibacterial agent (Verma, 2015; Verma and Maheshwari, 2017) for the human body. In addition, Silver nanoparticles are also reported to possess antifungal activity, anti-inflammatory properties, antiviral activity, and anti-angiogenic activity. Nano-Ags can be applied safely in therapy when the effective concentrations against various types of organisms have been determined.

The application of silver and Selenium nanoparticles as antimicrobials are gaining relevance in the medical field. Silver nanoparticles, due to their unique properties have many potential applications. The major uses of silver nanoparticles in the clinical and medical fields consist of investigative and curative applications. Selenium metal is an essential micronutrient for human beings and animals (Verma and Maheshwari, 2017).

In this context, selenium nanoparticles (SeNPs) possess antibacterial, antiviral and antioxidant properties, suggesting they could be suitable as therapeutic agents to combat infectious diseases. In particular, nanostructured particles can be synthesized using bacterial and fungal cells as biological catalysts, providing a non-toxic and environmentally beneficial approach for the production of nanoparticles, including SeNPs (Xiangqian et al., 2011).

Research findings have shown that Selenium nanoparticles had the highest bactericidal and antimicrobial properties. In this study, antibacterial effects of silver and selenium nanoparticles were evaluated with respect to biofilm forming S. aureus strains (Verma and Maheshwari, 2017). Experiments for the Ag and Se nanoparticles surfaces effectiveness with the drugs are important, which will open new passages in medical biology. Also, the quality of Ag and Se nanoparticles as a catalyst, and targeted drug-delivery vehicles is requisite (Verma and Maheshwari, 2017).

Metal and metalloid nanoparticles have been frequently useful in almost every field of science, and technology including biomedical science, due to their small size and shape and physic-chemical properties. In the last few decades, applications of Ag and Se based nanoparticles were discussed in field of infectious diseases treatment like Biofilms on medical devices (Verma et al., 2013; Verma and Singh, 2015), and as an antimicrobial (Verma, 2015; Verma and Maheshwari, 2017). Some researchers (Haris and Khan, 2017) have discovered that selenium nanoparticle-enhanced photodynamic therapy of toluidine blue O against biofilm forming Streptococcus mutans bacterial isolates. Haris and Ahmad (2017) studied the impact of metal oxide (ZnO and TiO2) nanoparticles on beneficial soil microorganisms and their secondary metabolites. The antibacterial potential of nanoparticles was determined by growth kinetics of P. aeruginosa, P. fluorescens and B. amyloliquefaciens. Soni (2017) discussed the Biodegradable Nanoparticles for Delivering Drugs and Silencing Multiple Genes or Gene activation in Diabetic Nephropathy. Chauhan et al. (2017) also discussed the applications of Nanotechnology in Forensic investigation and revealed the hidden evidences, which can prove to be helpful for the forensic scientists to give an outcome to their investigation. Thus, the objective of this research is to examine the preparation, characterization, and well diffusion assay of Silver and Selenium nanoparticles against Biofilm forming Staphylococcus aureus


Collection of samples

Thirty-six coagulase positive strains of S. aureus isolated from clinical specimens (urine, blood, and pus) from different hospitals in Dehradun, India were used in this study. The research was carried out at the Department of Biotechnology, IFTM University, Moradabad, India.

Isolation and Identification of Staphylococcus aureus

S. aureus strains were cultured in Mannitol salt agar (MSA) at 37°C for 24 to 48 hours. Mannitol salt agar (MSA) is both a selective and differential medium used for the isolation of Staphylococcus aureus (coagulase-positive Staphylococci). The bacterium produces yellow colonies with yellow zones in the medium. Each sample was sub-cultured and maintained into Nutrient agar medium. They tend to be white, circular, entire, convex colonies after incubating aerobically at 37ºC for 24 to 48 hours. The isolates were obtained from petri-plates identified on the basis of their cultural, morphological and biochemical characteristics according to Bergey’s Manual of Systemic Bacteriology (Holt et al., 1984).

Phenotypic Analysis of biofilm formation of Staphylococcus aureus strains

Congo Red Agar method

Brain heart infusion broth (BHI) medium was composed of BHI (37 g/l), sucrose (50 g/l), agar (10 g/l) and congo red stain (0.8 g/l). Congo red was prepared by concentrated aqueous solution and autoclaved at 121°C for 15 minutes, separately from other medium components and then mixed, when the agar had cooled to 55°C. Plates were inoculated and aerobically incubated for 24 to 48 hours at 37°C. Positive result was indicated by black colonies with a dry crystalline consistency. Weak slime (non-biofilm) producers generally remained in pink, though occasional darkening at the centers of colonies was observed. A darkening of the black colonies with the lack of a dry crystalline colonial morphology indicated an intermediate result (Freeman et al., 1989).

Tube method

Ten (10) ml of Trypticase soy broth with 1% glucose was inoculated with loopful of microorganisms from overnight culture plates and incubated for 24 hours at 37°C. The tubes were decanted and washed with PBS (pH 7.3) and dried test-tubes were stained with crystal violet (0.1%).

Excess stain was removed and tubes were washed with deionized water. Test-tubes were dried in inverted position and analyzed for biofilm formation. Biofilm formation was considered positive when a visible film lined the wall and bottom of the tube. Ring formation at the liquid interface was not indicative of biofilm development. Test-tubes were observed and the amount of biofilm formation was recorded as a non-biofilm, moderate or high biofilm (Christensen et al., 1982).

Tissue culture plate method

Ten (10) ml of Trypticase soy broth with 1% glucose was inoculated with a loopful of test organism from overnight culture on nutrient agar media. The broth was incubated for 24 hours at 370C. The culture was further diluted 1:100 with fresh medium. A 96-wells flat bottom tissue culture plate was filled with 0.2 ml of diluted cultures individually. The plate was incubated at 370C for 24 hours. After incubation, gentle tapping of the plate was done. The wells were washed with 0.2 ml of phosphate buffer saline solution (pH 7.2) four times to remove free floating bacteria. Biofilms remained enthusiast to the walls and the bottoms of the wells were fixed with 2% sodium acetate and stained with 0.1% crystal violet. Excess stain was rinsed with deionize water and plate was dried properly (Christensen et al., 1985).

Preparation and characterization of Silver and Selenium nanoparticles via SEM

Nanoparticles of the silver colloid were prepared by using chemical reduction method (Sileikaite et al., 2009) and Selenium nanoparticles (SeNPs) were synthesized (Tran and Webster, 2011) by the reduction of sodium selenite by glutathione (reduced form) and stabilized by bovine serum albumin (BSA). Nanoparticles (Ag and Se) were sterilized by ultra-violet light in laminar air flow. The sterilized Nanoparticles were carefully mounted on SEM stubs by using adhesive tape and uniformly coated with gold/palladium. The sample was placed in a sample chamber of SEM JEOL JSM-6490LV, Japan and scanning were performed under different magnifications ranging from 15,000x to 35,000x and voltage 20 to 30 kV (Sileikaite et al., 2009; Razi et al., 2011). Scanning electron microscopy (SEM) revealed spherical shape and particle size of 80.32 nm for AgNPs whereas, rods shape with particle size of 74.29 nm was revealed for SeNPs.

Antibacterial assay of Nanoparticles (AgNPs and SeNPs) via Agar well diffusion against Biofilm forming Staphylococcus aureus strains

After the preparation and characterization of the silver and selenium nanoparticles, their antimicrobial activity was tested against the clinical pathogens (Staphylococcus aureus). Petri plates containing 20 ml Nutrient agar medium were seeded with 24 hours old culture of Biofilm forming S. aureus strains. Wells were cut and 50 µl of the nanoparticles each (aqueous solution of silver and selenium) and 50 µl antibiotic (aqueous solution of amoxicillin as a positive control) were added. The plates were incubated at 37°C for 24 hours. The nanoparticles (silver and selenium) activity was assayed by measuring the diameter of the inhibition zone formed around the well of Petri plates (NCCLS, 1993).


The antibacterial activity of silver and Selenium nanoparticles were tested against the Biofilm forming Staphylococcus aureus strains using the well diffusion assay. Findings showed that the measured diameter of the zone of inhibition (mm) is directly proportional to the potency of inhibition. For the antibacterial activity of these two Ag and Se nanoparticles were varied among the S. aureus species tested and showed an expected gradual result with same NPs concentrations (50 µl).

Out of the total 36 strains, only 9 strains (SA12, SA15, SA20, SA22, SA23, SA24, SA25, SA26, and SA32) of S. aureus species were sensitive to Ag and Se nanoparticles with zones of inhibition ranging from 13 to 28 mm (Figure 1) while the remaining 27 strains (SA1, SA2, SA3, SA4, SA5, SA6, SA7, SA8, SA9, SA10, SA11, SA13, SA14, SA16, SA17, SA18, SA19, SA21, SA27, SA28, SA29, SA30, SA31, SA33, SA34, SA35, and SA36) were resistant to Ag and Se nanoparticles, so, further study was done with the 9 strains (SA12, SA15, SA20, SA22, SA23, SA24, SA25, SA26, and SA32) of S. aureus.

Figure 1

CLSI Standard for an antibiotic (Amoxicillin, AMX), Diameter of Zone of inhibition (mm) for resistant (≤19 mm), intermediate (Not mentioned), and sensitive (≥ 20 mm). Among the 36 strains of S. aureus, only 8 strains (SA12, SA15, SA20, SA22, SA23, SA24, SA26, and SA32) showed sensitivity to AgNPs and only 8 strains (SA15, SA20, SA22, SA23, SA24, SA25, SA26, and SA32) were also sensitive to SeNPs (Table 1).

Table 1

Amongst the two nanoparticles (Ag and Se) tested, silver nanoparticles were found to exhibit the most active inhibitory effects against three biofilm forming S. aureus strains (SA12, SA15, SA32), whereas selenium nanoparticles demonstrated the highest activity only for one biofilm forming S. aureus strain (SA23). Some studies have documented a similar pattern of sensitivity of AgNPs among S. aureus strains (Salomoni et al., 2015) so that the antibacterial effect of the strains (SA12, SA15, and SA32) greater zone inhibition than Amoxicillin antibiotic for silver nanoparticles and S. aureus strain (SA23) showed the greater zone inhibition than Amoxicillin antibiotic for selenium nanoparticles (El-Kheshen and El-Rab, 2012).


In coming years, there will be increase in multi drug resistance against many types of antibiotics. Due to emergence of multi-drug resistant bacteria pathogens, it is becoming very difficult to control infectious diseases and cure patients, resulting in serious morbidity and mortality. Metal and metalloid nanoparticles are a viable alternative to antibiotics, antiseptics and appear to have more potential to solve the problem of the emergence of Multi Drug Resistant bacteria. Antibacterial activities of nanoparticles depend on two main factors: (i) Type of microbes (ii) Physicochemical properties of NPs. Smaller sized Silver and Selenium nanoparticles have numerous positive attributes. For instance, antibacterial activity, would make them suitable for many practical applications. Although there are excellent trends of correlation in a small number of aspects of the antibacterial activity of nanoparticles (for biofilms), individual studies are difficult to generalize. Silver nanoparticles are one of the more attractive nanomaterials in commercial applications. They have been usually used for antimicrobial, electronic and biomedical products. It is therefore suggested that silver nanoparticles should be used as an effective way to prevent biofilm forming S. aureus infections.


The authors declare that they have no conflict of interest.


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