1. Introduction

Cleanrooms, where pharmaceutical medicaments were manufactured in pharmaceutical industries must be present in free of contamination by microorganisms, which may be inserted from exposure to air condition or contact with human body working there. Although the strict precautions some microorganisms allowed to be present in few amounts. During recent years resistance acquired by the pathogenic microorganisms to various available antibiotics has become a serious health issue ^[1]. Therefore, there is an urgent demand to find out new bactericidal materials, which counter act against bacterial resistance among them. Recent studies have shown that combining nanoparticles with antibiotics not only reduces the toxicity of both agents towards human cells by decreasing the requirement for high dosages but also enhances their bactericidal properties. Combining antibiotics with nanoparticles also restores their ability to destroy bacteria that have acquired resistance to them. Furthermore, nanoparticles tagged with antibiotics have been shown to increase the concentration of antibiotics at the site of bacterium–antibiotic interaction, and to facilitate binding of antibiotics to bacteria. Hence, nanoparticle-based antibacterial formulations could be effective bactericidal materials, as they will exhibit combined effects of silver and antibacterial agents. The enhanced activity of silver nanoparticles and antibiotics together has been reported earlier ^{[2, 3, 4, 5]}. Green nanotechnology is to utilize microbial resources for the fabrication of nanoparticles. In the recent past, nanoparticles have been synthesized using various bacterialike Bacillus licheniformis, Staphylococcus aureus, and Pseudomonas stutzeri ^{[6, 7, 8]}. Compared with chemical and physical methods, biological synthesis of nanoparticles has proved to be free of any limitations associated with production of hazardous by products, and it is simple and cost effective in nature ^{[9, 10]}. It can be expected that the high specific surface area and high fraction of surface atoms of silver nanoparticles will lead to high antimicrobial activity as compared with bulk silver metal. It was also interesting to note here that silver nanoparticles were able to exert inhibitory effect at a concentration that is below their cytotoxic limits,so they were regarded as safe to be used as antimicrobials ^[11]. In this paper, a strain Staphylococcus arlettae AUMC b-163 was identified on Phenotypic and Phylogenetic characterization, studied the antibacterial activity of biogenic Silver-nanoparticles, characterized by UV-vis spectrophotometer, FTIR, XRD, TEM and itssynergistic effect with a β-lactam antibiotic in the third-generation class of cephalosporin’s, (Cefotaxime sodium) against resistant strain Staphylococcus arlettae AUMC b-163 isolated fromT3A cleanroom, Assiut, Egypt.

2. Materials and Methods

Air samples were collected from pharmaceutical cleanroom, at T3A pharmaceutical company Assiut, Egypt, using air sampler (Reuter Centrifugal Sampler), model RCS high flow (Biotest Hicon) with a flow rate of 100 L/min for 10 minutes, after sampling agar strip were incubated for 48 hours at 32° ^[12] the isolated colony were subcultured and obtained in pure culture, characterized henotypically & Phylogenetically identified and maintained at 4°C at Assiut University Mycological Center as (AUMC b-163) and selected for further study.

Confirmation of the species, of the bacterial isolates was done by Gram staining and various biochemical tests.

The bacterial isolate of AUMC b-163was sent to Sol Gent Company (Daejeon, South Korea) for RNA gene sequencing. The sequence of the 16s rRNA gene has been widely used as a phylogenetic marker to study genetic relationships between different strains of bacteria. The analysis of this gene can therefore be considered as a standard method for the identification of bacteria at the family, genus and species levels, ^[13] bacterial DNA was extracted and isolated using SolGent purification bead. prior to sequencing, the ribosomal rRNA gene was amplified using the polymerase chain reaction (PCR) technique in which two universal bacterial primers 27F (forward) and 1492R (reverse) were incorporated in to the reaction mixture, primers used for gene amplification has the following composition: 27 F (5'- AGA GTT TGA TCM TGG CTC AG - 3’), and 1492R (5'- TAC GGY TAC CTT GTT ACG ACT T- 3').Sequence was further analyzed using BLAST from the national Center of biotechnology information (NCBI) website , phylogenetic analysis of sequences was done with the help of Meg Aligen (DNA star) software version 5.05.

For silver nanoparticles biosynthesis studies, 250-ml Erlenmeyer conical flasks containing 100 ml nutrient broth were prepared, autoclaved and inoculated with 1 ml of the test strain. The inoculated flasks were incubated in a rotating shaker set at 200 rpm for 48 h at 35°C. After the end of incubation period, the cultures were centrifuged at 12,000 rpm for 10 min. Supernatant of the organism added to reaction vessels containing filter-sterilized AgNO3 solution at 0.1M final concentration. At the same time, experimental control containing supernatant without silver nitrate was prepared. All the reaction mixtures were incubated on rotating shaker (200 rpm) at 35°C for a period of 72 h in light.

The formation of AgNPs was followed by visual observation of color change into reddish color that indicated the reduction reaction ^[14].

The reduction of pure Ag⁺ was further confirmed by the sharp peak given by scanning the AgNPs of the reacting solution using Perkin-Elmer Lamda-45 spectrophotometer, in a 1cm path quartz cell at a resolution of 1 nm from 300 to 800 nm ^[15].

FTIR spectra of dried synthesized AgNPs were recorded using FTIR Nicolet Avatar 660 FTIR spectrometer ^[16].

The synthesized AgNPs were dried and the powder form the sample was subjected for XRD analysis using X-ray diffractometer (Model PW 1710 control unit Philips Anode material Cu, 40 KV, 30 M.A, optics: Automatic divergence slit) with Cu Kα radiation λ=1.540562 °A ^[17].

The morphology of the AgNPs were investigated by TEM using JEOL- JEM-100 CXII instrument in electron microscope unit in Assiut University. By drying a drop of the washed colloidal dispersion onto a copper grid covered with a conductive polymer

Silver nanoparticles (AgNPs) synthesized from test strain Staphylococcus arlettae AUMC b-163, and bacterial supernatant were tested for antimicrobial activity by agarwell - diffusion methods against various pathogenic organisms. The test organisms used were related to gram-negative bacteria (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027) and gram-positive bacteria (Staphylococcus aureus ATCC 6538P), and the fungal strains (Candida albicans ATCC 10231, and Aspergillus niger ATCC 16404). Each strain was swabbed on the surface of individual plates using sterile cotton swab, wells of size 6 mm diameter were made on nutrient agar plates, with the help of a sterilized corkborer. Different concentrations (100, 50, 25, 15 μL) of the AgNPs and bacterial supernatant was loaded separately into wells and all the plates were incubated at 35°C for 24h in case of bacterial strains and at 28°C for 72 h for fungal strains. The zones of inhibition were measured and the lowest concentration of AgNPs and bacterial supernatant that inhibited the growth of the test organisms was recorded as the minimum inhibitory concentration (MIC) ^{[16, 18]}

In this experiment, AgNPssynthesized from resistant strain Staphylococcus arlettae AUMC b-163 was used for studying the synergism between synthesized AgNPs andβ. lactam cefotaxime sodium antibiotic. Well-diffusion method on nutrient agar plates was used to assess the synergistic effect. Different concentrations of the tested antibacterial (200, 150, 100, 50, 25 and 12.5 μg /ml) were prepared to determine MIC. A 25 μL of freshly prepared AgNPs and 25 μL of each concentration of the antibacterial were loaded into each well as the final content of 50 μL of AgNPs and antibacterial per well. After incubation at 35°C for 24 hours, the zones of inhibition were measured, the assays were performed in triplicates ^[3]

Results were compared statistically to understand the level of significance using Graphpad Prism v5.00 statistical software. The results are presented as mean ± standard deviation (S.D.). Statistical significance was set at p < 0.05.

3. Results

Staphylococcus arlettae AUMC b-163 is a Gram-positive coccus, 0.5–1.5 μm, non-motile, nonspore forming and aerobic growth. Colonies beige or yellow, opaque, with entire margins, 6–8 mm in diameter after 2 days’ incubation at 37°C on brain heart infusion agar. Biochemical characterization,give positive results for catalase, alkaline phosphate, and acid production from L-arabinose, arbutin, beta-gentibiose, fructose, D-fucose (weak), glucose, lactose, trehalose, mannitol, raffinose, xylose, melezitose, turanose, ribose & sucrose. Negative results for nitrates reduction, oxidase, hyaluronidase, staphylokinase, Tween 80 hydrolysis, betaglucosidase, urease, acid production from: xylitol, cellobiose, rhamnose, sorbitol & salicin.

The selected strain was isolated from pharmaceutical cleanroom was further subjected to molecular identification method. The sequence data of the bacterium was obtained and compared with the available gene sequences from organisms in the GenBank database. The AUMC b-163 strain was showed a very high percentage of similarity (99%) with the sequence of staphylococcus sp. mainly Staphylococcus arlettae (Figure 1). and it was maintained at Assiut University Mycological Center as Staphylococcus arlettae AUMC b-163. The GenBank accession numbers are represented in parentheses.

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Figure 1. Phylogenetic analysis of 16S rRNA gene of isolates AUMC b-163 and other related Staphylococcus arlettae, GenBank accession number given in parentheses

The color change from pale yellow to reddish after 24 h of incubation in the presence of light with Staphylococcus arlettae AUMC b-163 as shown in Figure 2. At the same time, experimental control containing supernatant without 0.1 M silver salt showed no color change when incubated at the same environmental condition (Figure 2).

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Figure 2. culture of Staphylococcus arlettae AUMC b-163 with and without silver salt (0.1M), Test: color change to reddish after 24 h of incubation in the presence of salver salt. Experimental control: showed no color change

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Figure 3. UV –Vis spectral analysis of AgNPs absorption band at 420 nm

Bioreduction of silver nitrate ions was confirmed by the sharp peaks given by the AgNPs in the visible region from UV–VS spectrum of the reacting solution, (Figure 3) showed that,the UV–Vis absorption spectrum of silver nanoparticles exhibiting a strong broad peak at 420 nm. The spectrum showed a strong surface Plasmon absorption band indicating the presence of AgNPs.

The nature of the biomolecules involved in the reduction and formation of AgNPs was studied by FTIR (Figure 4). The FTIR signals of AgNPs were observed at 1078, 1290, 1648, 2140, 2410, 2963 and 3269 cm^-1. FTIR measurement was carried out to identify the potential biomolecule in enzyme filtrate responsible for the reduction of silver ions and capping agent responsible for the stability of the bioreduced silver nanoparticles. The FTIR spectra in 1400-1700 cm^-1 region provides information about the presence of “C=O” and “N=H” groups which is responsible for the reduction of AgNO3 to Ag. The peaks in the region between 3269 to 2140 cm^-1 were assigned to O-H stretching of alcohols and phenol compounds and aldehyde -C-H- stretching of alkanes. The peaks in the region 1078 and 1290 to 1648 cm-1 corresponds to N-H of primary and secondary amides and –C-N- stretching vibrations of amines or –C-O- stretching of alcohols, ethers, carboxylic acids and anhydrides.

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Figure 4. FTIR spectra of synthesized AgNPs of Staphylococcus arlettae AUMC b-163

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Figure 5. XRD pattern of synthesized AgNPs of Staphylococcus arlettae AUMC b-163

The crystalline nature of the particles was confirmed using XRD. Figure 5 shows X-ray powder diffraction patterns of the synthesized AgNPs at 80 °C. The peak positions are consistent with metallic silver. The peaks assigned in the AgNPs sample were 110, 200, 206, 222 and 310. A° number of Bragg reflections with 2θ values of 27.26°, 31.77°, 46.23°, 38.33°, 46.91°, 57.06°, 57.41°, 67.48°, 74.80° and 75.65°.

Transmission electron microscopy was carried out to know the morphology and size of the biosynthesized silver nanoparticles. The TEM image of AgNPs Figure 6 shown that the particles are spherical in shape and the size of the AgNPs was found to be in the range from 8 to 35 nm.

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Figure 6. TEM micrograph of synthesized AgNPs showed that particles are spherical in shape and the size range from 8 to 35 nm.

The AgNPs exhibited good antibacterial activity against Gram-negative bacteria were E. coli ATCC 8739 7.66±0.58mm, P. aeruginosa ATCC 9027 8.33±1.53mm, gram-positive bacteria S. aureus ATCC 6538P 11.33±1.35 mm and resistantS. arlettea AUMC b-163 6.33±0.57. It also showed antifungal activity againstC. albicans ATCC 10231 8.00±2.00 and A. niger ATCC 16404 10.00±2.00 mm Figure 7.

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Figure 7. Inhibition zones of AgNPs against S. aureus ATCC 6538P (A), S. arlettae (R) AUMC b-163 (B), E. Coli ATCC 8739 (C), C. Albicans ATCC 10231(D) and A. niger ATCC 16404(E) culture plates were incubated at 35°C for 24 h in case of bacterial strains and at 28°C for 72 h for fungal strains

On the other hand, the combination of AgNPs with antibacterial agent’s cefotaxime sodium was also investigated the inhibition activity of antibiotics increased in the presence of AgNPs against test strains as compared to antibiotic alone. The maximum fold increase of antibacterial activity in combination with silver nanoparticles plus antibiotic was 85 % (Table 1).

Table 1. Antimicrobial activity of AgNPs synthesized from S. arlettae AUMC b-163 and its synergistic effect plus (Cefotaxime sodium antibiotic) expressed as (Zone of inhibition mm ±SD) against pathogenic microorganisms

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4. Discussion

The scope of the present study was based upon exploring the capabilities of bacterial strain S. arlettae AUMC b-163 to synthesize AgNPs along with their biomedical application in controlling infection. This research showed a great deal of capability towards synthesizing AgNPs. In addition, these nanoparticles alone and in conjugation with Cefotaxime antibiotic proved to be effective as antimicrobial agents.Change in color of the reaction mixture containing Ag salt and fungal cultural filtrate proved to be the first indication of AgNPs synthesis. When the reaction mixture was incubated under light conditions, the color of the liquid mixture changed to reddish after 24 hours of reaction (Figure 2). The change in color intensity (absorbance) was also monitored through ultraviolet-visible spectroscopy; peaks of the reaction mixture were obtained around 420 nm (Figure 3). It was reported that the reduction of Ag+ to atomic silver Ago corresponds to absorption at 420 nm ^{[3, 19, 20]}. Synthesis of AgNPs was previously linked with release of functional proteins in reaction mixture by bacterial supernatant, which might have helped reduction of the metal ions into nanoparticles. Various reports have provided evidence of extracellular generation of AgNPs by XRD and TEM images based upon these analytical ^{[15, 17, 21]}. In this study, S. arlettae AUMC b-163 was found to be an effective biological tool for the biosynthesis of stable AgNPs. Based on the results from this study, we can deduce that biologically synthesized AgNPs can be efficiently used as antimicrobial agents.AgNPs have been reported to be an effective as bactericidal agent ^{[11, 22, 23, 24]}. Inpresent study the antimicrobial effect of AgNPs prepared from strains S. arlettae AUMC b-163 showed a great deal of capability towards synthesizing AgNPs. In addition, these nanoparticles alone and also in conjugation with antibiotics proved to be effective in controlling pathogenic organisms. The mechanism ofaction of AgNPs is still not well defined. Many proposed mechanisms about antibacterial effect of silver ions have been presented by researchers ^{[3, 19, 20, 24, 25]}. It was hypothesized that AgNPs have large surface area, which allows them to closely interact with antibiotic. The antibiotic molecules contain active groups like hydroxyl and amido groups, which can easily react with AgNPs and disrupt peptidoglycan in the cell wall ^[3]. Being positively charged, they attack negative charges of transmembrane proteins and can destroy the cell membrane and block the transport channels ^[20]. It might be possible that they penetrate inside the bacteria and disrupt cellular activities like transportation, protein synthesis, and nucleic acid functioning ^[26]. Likewise, cefotaxime in combination with positively charged AgNPs both inhibited and disrupted cell-wall synthesis. It has also been proposed that silver ions penetrate the cell intercalate themselves between pyrimidine and purine, and denature the DNA molecule ^[24].In synergism, the bactericidal effect is enhanced by interaction between antibiotics with AgNPs. As a result, antibiotic–AgNP conjugate is formed in which an AgNP core is surrounded by antibiotic molecules. Thus, the antimicrobial concentration is increased, which leads to increased destruction of bacteria ^[19]. More studies need to be conducted to find out the exact mechanism of action to develop a novel antimicrobial drug against multidrug-resistant bacteria

5. Conclusion

Antibiotic resistant bacteria have been continuously increasing over the past decade; hence, there is a need to detect another method to overcome this problem. In the present scenario, AgNPs have appeared as a promising antibacterial candidate in the medical field. Hence it could be potentially applied in the fabrication of silver impregnated antimicrobial materials for biomedical applications. We have demonstrated green process for the synthesis of silver nanoparticles from S. arlettae AUMC b-163 Within 24 hours of time, spherical shaped nanoparticles from 8 -35 nm size were formed. The method described is highly efficient and cost effective to produce silver nanoparticle. The antibacterial activity of antibacterial antibiotic cefotaxime against resistant S. arlettae AUMC b-163 was augmented when impregnated with AgNPs. Based on the results from this study, we can deduce that biologically synthesized AgNPs may function by binding to Thiol (SH) groups of membrane proteins, enzymes, and phosphate groups of DNA, and can be efficiently used as antimicrobial agents.

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