Antibacterial Activity of Silver Nanoparticles that extracted by using Staphylococcus aureus

 

Frial Gemeel Abd

Department of Biology, University of Babylon, Iraq

 

ABSTRACT:

Silver nanoparticles (AgNPs) was extracted by using clinical isolates for Staphylococcus aureus. The detection of AgNPsby qualitative method as Uv- visible. Antibacterial activity against (S .aureus, Pseudomonas, and Escherichia coli) isolates by plate diffusion method by using different concentrations (0.5, 0.25, 0.125, 0.062, 0.032) g/ml .the high significant differences among concentrations. Means of inhibition zones were higher with concentration at 0.5g/ml about (15.36 , 17.33and 13.5) mm while least inhibition zone with (0.065, 0.25 and 0.125) g/ml that appeared (11.27, 14.33 and 10.5) mm against S. aureus, Pseudomonas, and E. coli respectively. The AgNps mixed with some antibiotics and found that inhibition zone with mixed increased significantly than without silver nanoparticles, the conclusion from this study the AgNps was extracted from S.aureus and this nanoparticles had antibacterial activity against some bacteria and act synergistically with antibiotics

 

 

 

INTRODUCTION:

The green synthesis of nanoparticles has significant importance in recentyears for obtaining biocompatible, cost-effective, nontoxic, easily scaled up for large-scale synthesis, andecofriendly size-controlled nanoparticles (Dobrucka and Dlugaszewska, 2015). Various bacteria (Chaudhari et al., 2012; Saifuddin et al., 2009), fungi (Gade et al., 2008; Vigneshwaran et al., 2007) and plant sources (Masurkar et al., 2011; Raut et al., 2010) have been studied to achievethe green synthesis of silver nanoparticles. The development of resistant, or even multiresistant, pathogens has become a major problem (Schaller et al., 2004). The investigations on the antibacterial activity of AgNPs have increased (Durán et al., 2010; Gade et al., 2008). Soni and Prakash, have reported antimicrobial AgNPs can be synthesized by bacteria as agreen chemistry process [12].

 

It that referred above, AgNPs in conjunction with biopolymerscan originate nanocomposites. The nanomaterials also have important antibacterial activity forboth Gram-positive and Gram-negative bacteria,

 

Cao et al. described an antimicrobial activity with a broad spectrum of action of AgNPs with chitosanand xanthan gum [13]. In this case, the complete inhibition of Escherichia coli and Staphylococcus aureus growth was described [14].

 

Silver is a metal with a described antibacterial activity and significant efficacy against Gram-negative and Gram-positive bacteria [7]. The research area has a challenge for nanomedicine and biotechnology [8]. The bactericidal and bacteriostatic activities of AgNPs because its ability to lyse the bacterial cell wall, so allowing the exit of the cytoplasm content, inhibiting the respiratory chain, and effects on the DNA [9]. Escherichia coli, Staphylococcus aureus, , Salmonella typhimurium, Pseudomonas aeruginosa and Klebsiella pneumoniae are pathogenic bacteria sensitive to silver and its nanoparticles [10,11]. The major nosocomial pathogens (hospital outbreaks) are Acinetobacter baumannii  and  Pseudomonas aeruginosa, these bacteria multidrug resistance ( 2)

MATERIALS AND METHODS:

Bacterial isolate:

Staphylococcus aureus isolates, Escherichiae coli and Pseudomonas aeruginosa isolates were taken from advance lab of microbiology, these isolates were diagnosed by biochemical tests according (Mac Fadden, 2000). and these isolates were used to synthesis silver nanoparticle.

 

Media:

Muller-Hinton (MHA ) agar medium was ready for conferring to the manufacturing company and it was used in antimicrobial susceptibility testing. Nutrient Agar Medium, It was used for cultivation of the bacterial isolates when it was necessary. Nutrient Broth, This medium was used to grow and preserve the bacterial isolates. Brain Heart Infusion medium was prepared according to the manufacturing Instructions (Himedia, India) (MacFadden, 2000).

 

Biosynthesis of Silver Nanoparticles by Staphylococcus aureus:

All strains of S .aureus were initially grown at 37 ⁰C for 24 hr in a 500 ml Erlenmeyer flask that contained nutrient broth (100 ml) in a shaker incubator set at 200 rpm. Following bacterial growth, all the culture suspensions were incubated with aqueous 5 mM solutions of AgNO₃ at 37 ⁰C in a shaker incubator at 200 rpm in the dark, the reactions was carried out for up to 120 hr (5 days).The extracellular synthesis of AgNPs was initially detected by visual inspection of the culture flask for a change in the color of culture medium from clear light-yellow to brown/green. Extracellular AgNPs were separated from bacterial cells by centrifuging aliquots of culture supernatants at 3000 rpm for 6 min at 25˚C. UV-vis analysis, the AgNPs suspensions were diluted 10 times using deionized water at every time point and UV-vis spectra were obtained, the samples were prepared by precipitating AgNPs obtained after 20 hr of biosynthesis at 13,000 rpm for 20 min, followed by four washings with deionized water and drop casting the samples onto a glass substrate (Song and Kim et al., 2009).

 

Antibacterial activity of silver nanoparticles:

A 0.1 ml volume of the standard inoculums (1.5 X 10⁸ cell/ ml) of the test bacterial isolated was streak on Mueller Hinton Agar with a sterile swab and permitted to dry. Then, 6mm. diameter wells were bored using cork borer in the MHA AgNPs (0.5, 0.25, 0.125, 0.065, 0.032gm/ml concentration) were introduced into each well and allowed to stand for 1 hr, at room temperature to diffuse the AgNPs into medium before incubation at 37⁰C for 24 hr. The Inhibition zone diameter (ISD) was measured by obvious ruler to nearest mm (Okoli and Iroegbu., 2004). Antimicrobial susceptibility test by agar disk method according (CLSI, 2012).

 

Drug mix with AgNps:

Four drugs (C: cefixime 400 mg manufacture company Siprax A: Acamoxil 250mg Aki company and T: tetracycline 250 mg Apcycline company AC: Ampicillin and cloxacillin 250 mg LDP company) were dissolved with sterile distill water ,and then added with 0.5 mg/ml from AgNPs. by using by agar disk method and used the S .aureus isolates

 

RESULTS AND DISCUSSION:

Determine AgNPs from seven isolates of S .aureus by using AgNO3, the ability of some bacteria to extracellular synthesis of silver nanoparticles this result agree with (Raheem et al, 2016, AbdAl Hussain et al, 2017) and the optical density for AgNPs that product from bacteria as table 1.

 

The AgNPs extract from S .aureus was detected by UV visible, it was appeared  That UV–Vis absorption band in the current visible light region 420–450 nm as figure 1.  It wasan evidence of the presence of surface plasmon resonance of AgNPs this agree with (Muthukrishnan et al. 2015). Various reports have established that the resonance peak of silver nanoparticles appears around this region, but the exact position depends on some factors such as particles size, and the surface-adsorbed species (Pal et al ., 2007).The absence of absorbance at wavelengths greater than 550 nm indicated their well-dispersed state in solution (Eliham et al., 2015). The optical density for isolates that used to silver nanoparticles extract was appeared different value, from this result the isolate for extraction was selected so isolate 2 was used to synthesis silver nanoparticles.

 

 

Figure 1 UV-Visible Spectra of Silver Nanoparticles Synthesis by Staphylococcus aureus

 

Antibacterial activity of AgNPs were studied on some clinical isolates from E.coli, P. aeruginosa and S.aureus. inhibition zone appeared significantly differences among each concentrations of AgNPs with each isolates this agree (Santos et. al, 2016). Antibacterial activity was studied on S. aureus isolates by using different concentrations from AgNPs extract (table 2) the result was appeared significant differences among the diameters of inhibition zones for each concentrate, the diameter of inhibition zone was higher in concentration 0.5, 0.032 as 15.36 and 15.09 mm respectively .this result was agreed with Nanda and Saravanan (2009).

 

In this study antibiotic were used alone and used with silver nanoparticles table 5 and figure 2 the results appeared the inhibition zones were increased significantly with silver nanoparticles with antibiotic compared with using antibiotic only, this results agreed with (Abdullah ,2016) who mixed TiO₂ nanoparticles with Cephalothin indicate highly efficiency toward all isolates except Pseudomonas aeruginosa.

 

Table 1. Optical density for silver nanoparticles synthesis of Staphylococcus aureus isolates

 

 

 

 

Table 2. The diameter of inhibition zone for Staphylococcus aureus with different concentrations of silver nanoparticle

0.5≥significant at p

 

Table 3. The diameters of inhibition zone for Escherichia coli at different concentrations form silver nanoparticles

Significant p≥.5

 

Table 4. Diameter of inhibition zone for Pseudomonas aeruginosa at different concentrations silver nanparticles

Significant at various concentrations at p≥.5

 

 

 

 

 

Table 5.Diameter of inhibitionzone by antibioticwere used as alone or with silver nanoparticle against Staphylococcus aureus

Significant at p≥.5

 

Figure 1: The effect of silver nanoparticles with tetracycline on the growth of Staphylococcus aureus A tetracycline alone B Tetracycline with silver nanoparticles

C:cefixime 400 mg siprax A:Acamoxil 250mg Aki ,and T: tetracycline 250 mg Apcycline AC: Ampicillin and cloxacillin 250 mg LDP

 

 

 

 

REFERENCES:

1.       Abdullah R.M.(2016) A study the effect of TiO2 nanoparticles combination with antibiotics and plant extracts against some Gram negative bacteria. Baghdad science journal, 13(3) in Arabic Abd AlhussainA.J.; Abd F.G. ;Alkaim A.F (2017) Biological synthesis and characterization of silver nanoparticles using Bacillus subtili . Journal of Global Pharma Technology. 09(9): 239-244

2.       Cao, X.; Cheng, C.; Ma, Y.; Zhao, C. (2010) Preparation of silver nanoparticles with antimicrobial activities and the researches of their biocompatibilities. J. Mater. Sci. Mater. Med., 21, 2861–2868.

3.       Chaudhari, P. R., Masurkar, S. A., Shidore, V. B., and Kamble, S. P. (2012) Effect of biosynthesized silver nanoparticles on Staphylococcus aureusbiofilm quenching and prevention of biofilm formation.Int. J. Pharm. Biol. Sci., 3, 222–229

4.       Dobrucka, R. and Dlugaszewska, J. (2015) Antimicrobial activities of silver nanoparticles synthesized by using water extract of Arinicaeanthodium. Ind. J. Microbiol., 55, 168–174.

5.       Durán, N., Marcato, P. D., Ingle, A., Gade, A., and Rai, M. (2010) Fungi mediated synthesis of silver nanoparticles, characterization processesand applications. Prog. Mycol., 425–449.

6.       Ferri, M.; Ranucci, E.; Romagnoli, P.; Giaccone, V.(2015) Antimicrobial resistance: A global emerging threat to public health systems. Crit. Rev. Food Sci. Nutr.

7.       Gade, A., Bonde, P., Ingle, A. P., Marcato, P. D., Durán, N. et al. (2008) Exploitation of Aspergillusniger for synthesis of silver

8.       nanoparticles. J. Biobased Mater. Bioenergy, 2, 243–247..

9.       Potron, A.; Poirel, L.; Nordmann, P. Emerging broad-spectrum resistance in Pseudomonas aeruginosa and

10.     Acinetobacterbaumannii: Mechanisms and epidemiology. Int. J. Antimicrob. Agents 2015, 45, 568–585.

11.     Rai, M.K.; Deshmukh, S.D.; Ingle, A.P.; Gade, A.K. (2012)Silver nanoparticles: The powerful nanoweapon agains tmultidrug-resistant bacteria. J. Appl. Microbiol., 112, 841–852

12.     Wilding, L.A.; Bassis, C.M.; Walacavage, K.; Hashway, S.; Leroueil, P.R.; Morishita, M.; Maynard, A.D.;

13.     Philbert, M.A.; Bergin, I.L. (2016,)Repeated dose (28-day) administration of silver nanoparticles of varied size andcoating does not significantly alter the indigenous murine gut microbiome. Nanotoxicology 10, 513–520.

14.     Theivasanthi, T.; Alagar, M. Anti-Bacterial Studies of Silver Nanoparticles. 2011. Available online: https:

15.     //arxiv.org/ftp/arxiv/papers/1101/1101.0348.pdf (accessed on 10 May 2016).

16.     Li,W.R.; Xie, X.B.; Shi, Q.S.; Duan, S.S.; Ouyang, Y.S.; Chen, Y.B. Antibacterial effect of silver nanoparticleson Staphylococcus aureus. Biometals2011, 24, 135–141. [CrossRef] [PubMed

17.     Soni, N.; Prakash, S. Antimicrobial and mosquitocidal activity of microbial synthesized silver nanoparticles.

18.     Parasitol. Res. 2015, 114, 1023–1030. [CrossRef] [PubMed]

19.     Xu,W.; Jin,W.; Lin, L.; Zhang, C.; Li, Z.; Li, Y.; Song, R.; Li, B. Green synthesis of xanthan conformation-based silver nanoparticles: Antibacterial and catalytic application. Carbohydr.Polym.2014, 101, 961–967. [CrossRef]

20.     Clinical and Laboratory Standards Institute.(2012). Performance Tandards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement . CLSI document M02-A10 and M07-A8. Clinical and Laboratory Standards Institute, Wayne, PA.

21.     Collee, J.; Fraser, A.G.; Marmian, B.P. and Simmon, S.A. (1996).Mackie and McCartney Practical Medical Microbiology.4th ed. Churchill Cancerand Livingstone, INC.USA.

22.     MacFaddin, J. F. (2000). Biochemical Test for Identification of Medical Bacteria.3rd ed., Williams and wilkins – baltimor., pp. 321-400.

23.     Song, J.Y. and Kim, B.S. (2009) .Rapid biological synthesis of silver nanoparticles using plant leaf extracts. BioprocBiosystEng 44:1133–1138.

24.     Nanda A.; Saravanan M. 2009 Biosynthesis of silver nanoparticles from Staphylococcus aureus and its antimicrobial activity against MRSA and MRSE Nanomedicine: Nanotechnology, Biology and Medicine

25.     Santos K.S.; Barbosa A. M.; Costa L.P.; Pinheiro M. S.; Oliveira M. P P.; Padilha F. F(2016) Silver nanocomposite biosynthesis: antibacterial activity against multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii. Molecules, 21, 1255

 

 

Accepted on 20.05.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(10): 4215-4218.