Synthesis of silver nanoparticles using the ...

Materials Science and Engineering C 45 (2014) 434–437

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Synthesis of silver nanoparticles using the Streptomyces coelicolor klmp33 pigment: An antimicrobial agent against extended-spectrum beta-lactamase (ESBL) producing Escherichia coli Deene Manikprabhu, K. Lingappa ⁎ Department of Microbiology, Gulbarga University, Gulbarga 585106, Karnataka, India

a r t i c l e

i n f o

Article history: Received 29 December 2013 Received in revised form 17 February 2014 Accepted 23 September 2014 Available online 28 September 2014 Keywords: Silver nanoparticles Antimicrobial activity Streptomyces coelicolor klmp33 Escherichia coli

a b s t r a c t The increasing emergence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli (E. coli) occurred mainly due to continuous persistent exposure to antibiotics causing high morbidity and mortality so studies in controlling this infection are required. In the present investigation, we developed a synthesis for silver nanoparticles employing a pigment produced by Streptomyces coelicolor klmp33, and assessed the antimicrobial activity of these nanoparticles against ESBL producing E. coli. The ESBL producing E. coli were isolated from urine samples collected from the Gulbarga region in India. As can been seen from our studies, the silver nanoparticles having irregular shapes and size of 28–50 nm showed remarkable antimicrobial activity and moreover the synthesis time is just 20 min and thus the same can be used for formulating pharmaceutical remedies. © 2014 Elsevier B.V. All rights reserved.

1. Introduction “Small is beautiful and small is powerful” [1], the important statement holds well when we talk about nanoparticles. Nanoparticles, generally considered as particles with a size of up to 100 nm, exhibit completely new or improved properties as compared to the larger particles of the bulk material that they are composed of [2]. Of the noble metal nanoparticles, silver nanoparticles are of great interest [3]. Since ancient times, silver in different forms has been widely used as a medicine for treatment of various diseases. Siddhars the great Indian ancient scientist practiced the use of Rajath Bhasma (Rajath is silver and Bhasma means fine powder) in medicine. With the present day understanding of nanoscience, one can clearly get enlightened that this formulation contained silver nanoparticles [4]. At present, the antimicrobial property of silver nanoparticles has been explored in various applications, like water purification, sterile coating for biomedical devices and packaging for food stuffs [5]. Recently increasing emergence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli (E. coli) occurred mainly due to continuous persistent exposure to b-lactam antibiotics in health care centers [6]. Beta-lactam antibiotics are the largest and most common group of antimicrobial agents used world-wide, distinguished by a chemical structure known as the beta-lactam ring. The beta-lactam antibiotics kill the bacteria, mainly by targeting transpeptidase enzymes that synthesize ⁎ Corresponding author. E-mail address: [email protected] (K. Lingappa).

http://dx.doi.org/10.1016/j.msec.2014.09.034 0928-4931/© 2014 Elsevier B.V. All rights reserved.

the bacterial cell wall forming a lethal covalent penicilloyl–enzyme complex that serves to block the normal transpeptidation reaction. This results in weakly cross-linked peptidoglycan, which makes the growing bacteria highly susceptible to cell lysis and death. However, the continuous use of beta-lactam antibiotics in health care systems made bacteria resistant to them, mainly due to the production of beta-lactamase enzymes, that hydrolyze beta-lactam antibiotics by breaking the nitrogen–carbonyl bond in the beta-lactam ring [7] causing the increase rate of morbidity and mortality, especially the infections caused due to extendedspectrum beta-lactamase (ESBL) producing E. coli [8] so studies in controlling these infections are gravely required if public health is the concern. In this study, we made an attempt to synthesize silver nanoparticles by using a pigment produced by Streptomyces coelicolor klmp33 (S. coelicolor klmp33) and to asses its antimicrobial activity against ESBL producing E. coli, isolated from the Gulbarga region in India.

2. Materials and methods 2.1. Media, chemicals and antibiotic disk Eosin Methylene Blue (EMB) agar, Mueller Hinton agar (MHA), Mueller Hinton broth (MHB), AgNO3, cefotaxime disk (CTX, 30 μg) and cefotaxime/clavulanate disk (CEC, 30 μg/10 μg) were purchased from Hi-media, India.

D. Manikprabhu, K. Lingappa / Materials Science and Engineering C 45 (2014) 434–437

435

Fig. 1. Characterization of silver nanoparticles. (a): UV–visible spectrum of silver nanoparticles, (b) XRD pattern of silver nanoparticles and (c) TEM image of silver nanoparticles.

2.2. Silver nanoparticle synthesis and characterization Silver nanoparticles were synthesized according to our earlier standard protocol. 15 mL of AgNO3 (10−3 M) solution was treated with 1 mL pigment produced by S. coelicolor klmp33 and exposed to direct sunlight for 20 min. A color change from colorless to brown took place within few minutes indicating the formation of silver nanoparticles. Further, the synthesized nanoparticles were characterized by using UV–visible spectroscopy, X-ray diffraction (XRD) and transmission electron microscopy (TEM) [9].

2.4. Antibacterial activity of silver nanoparticles against ESBL producing E. coli To evaluate the antimicrobial property of silver nanoparticles against ESBL producing E. coli, we determined the minimum inhibitory concentration (MIC). To determine the MIC, different volumes of synthesized silver nanoparticles (5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μL) and ESBL producing E. coli culture (maintained at 106 CFU/mL) were added into MHB and incubated at 37 °C for 18 h. The MIC was determined by measuring the optical density at 625 nm. 3. Result and discussion

2.3. Isolation and identification of ESBL producing E. coli E. coli have been isolated from urine samples collected from different hospitals and health care centers of the Gulbarga region in India. The preliminary identification of E. coli isolates was done using EMB agar. Further the E. coli isolates were identified by microscopic, morphological and biochemical characters. Among isolated E. coli, ESBL producing E. coli isolates were identified by antibiotic susceptibility test as per the guidelines recommended by the Clinical and Laboratory Standards Institute (CLSI-2012) [10]. The CLSI advocates the use of cefotaxime (30 μg) or ceftazidime disks (30 μg) with or without clavulanate (10 μg), and a difference of ≥ 5 mm between the zone diameters of the cephalosporin disks and their respective cephalosporin/clavulanate disk was taken to be phenotypic confirmation of ESBL production performed on MHA [11].

The introduction of b-lactam antibiotics in health care systems started during World War II, which represents one of the most important contributions to medical history owing to their comparatively high effectiveness, low cost, ease of delivery and minimal side effects [1]. However, over the last 15 years numerous outbreaks of infection with organisms producing ESBL have been observed worldwide due to bacteria resistant to b-lactam antibiotics, and their presence will surely create significant therapeutic problems in the future [12] if their prevalence is not controlled, so studies finding new drugs for controlling this infection are required. A substitute for antibiotics is the silver nanoparticles. Their antimicrobial properties were known from antiquity, having the history with manhood dating back to 4000 BC. At present they are being used in different pharmaceutical formulations such as antibacterial clothing, burn ointments, and coating for medical devices due to their mutation resistant antimicrobial activities [13]. Compared to bulk silver, extensive research on silver nanoparticles is ongoing due to the unique property possessed by these particles [9,14]. The antimicrobial activity of silver nanoparticles against various bacteria like Table 1 Morphological, microscopic and biochemical tests of E. coli. Morphological characters Size Color

Fig. 2. E. coli on EMB agar. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Medium a) White color on nutrient agar b) Green metallic sheen on Eosin Methylene Blue Agar

Microscopic characters Gram staining Motility Sporulation

Gram negative (rod shape) Motile Non-sporulating

Biochemical tests Indole Methyl red Voges–Proskauer test Citrate utilization Reduction of nitrate Catalase Starch hydrolysis

Positive Positive Negative Negative Positive Positive Negative

436


Fig. 3. Phenotypic confirmation of ESBL producing E. coli. a) Cefotaxime disk (CTX) and cefotaxime/clavulanate disk (CEC).

Streptococcus mutans [15], Staphylococcus aureus, Salmonella typhi, Providencia alcalifaciens and Proteus mirabilis [16] was reported. Recently we reported the antimicrobial activity of silver nanoparticles synthesized using a pigment produced by S. coelicolor klmp33 against methicillin resistant S. aureus [9]. In the view of the advantages of our silver nanoparticle synthesis process and understanding the future problem that will be caused by ESBL producing microbes, in the present work we report the antimicrobial activity of silver nanoparticles against ESBL producing E. coli. 3.1. Silver nanoparticle synthesis and characterization The pigment produced by S. coelicolor klmp33 was used for the synthesis of silver nanoparticles. Our earlier standard silver nanoparticle synthesis protocol is mentioned below. 15 mL of AgNO3 (10−3 M) solution was treated with the 1 mL pigment and exposed to direct sunlight for 20 min. A color change from colorless to brown took place within few minutes indicating the formation of silver nanoparticles. The synthesis was further confirmed by using UV–visible spectroscopy, X-ray diffraction (XRD) and transmission electron microscopy TEM. The UV–visible absorption maximum between 400 and 450 nm (Fig. 1 a) and intense peaks corresponding to (111), (200), (220) and (311) in X-ray diffraction (Fig. 1 b) clearly confirm the silver nanoparticle synthesis. The TEM results showed that the nanoparticles were 28–50 nm in size and irregular in shape (Fig. 1c).

A yield of about 0.14 g/100 mL was obtained from the above process and the same silver nanoparticles were used for further antimicrobial studies. Moreover, the advantages of the above synthesis process are as follows: a) The process is green, as only the microbial pigment was used for the synthesis rather than toxic chemicals. b) Since only the pigment not the microbes was used for the synthesis, there is no chance of contamination and the process is easy to handle. c) The synthesis time is very less (20 min) so it can be used for bulk synthesis for industrial purposes [9]. 3.2. Isolation and identification of ESBL producing E. coli The preliminary identification of E. coli was done using EMB medium. The E. coli were detected by the presence of green metallic sheen (Fig. 2). Further the E. coli isolates were identified based on morphological, microscopic and biochemical tests (Table 1). Among isolated E. coli the ESBL producing E. coli isolates were phenotypically identified according to CLSI guidelines [10]; Fig. 3 shows a difference of N5 mm between the zone diameters of cefotaxime and cefotaxime/clavulanate which confirms the ESBL production in E. coli. The antimicrobial activity of silver nanoparticles against isolated ESBL producing E. coli was evaluated.

Fig. 4. MIC of silver nanoparticles against ESBL producing E. coli.


3.3. Antibacterial activity of silver nanoparticles against ESBL producing E. coli To evaluate the antibacterial effect of silver nanoparticles against ESBL producing E. coli, we determined the MIC. The MIC of silver nanoparticles against ESBL producing E. coli was estimated (40 μL) (Fig. 4). The mechanism of the bactericidal effect of silver nanoparticles remains to be elucidated. Several studies have proposed that silver nanoparticles bind to the surface of the cell membrane, disrupting cellular permeability and the respiratory functions of the cell. Smaller silver nanoparticles have a large surface area available for interaction and greater bactericidal effect than larger silver nanoparticles [17]. It may also be possible that silver nanoparticles not only interact with the surface of the membrane, but also penetrate inside the bacteria and inactivate their DNA replicating ability [18] causing cell death. Another interesting result that we observed in our study is that the pigment from which silver nanoparticles were synthesized did not show any antimicrobial activity against E. coli [19] but when the same pigment was used for the synthesis of silver nanoparticles, the synthesized silver nanoparticles exhibited excellent antimicrobial activity against ESBL producing E. coli. 4. Conclusion ESBL producing E. coli infection is increasing remarkably so controlling this infection is very essential. In the present work, we described the antimicrobial activity of silver nanoparticles against ESBL producing E. coli. The silver nanoparticles were synthesized using a pigment produced by S. coelicolor klmp33 by photo-irradiation method for 20 min. The silver nanoparticles having irregular shapes and size between 28 and 50 nm showed remarkable antimicrobial activity against ESBL producing E. coli isolated from urine samples collected from the Gulbarga region, India. The MIC of silver nanoparticles against ESBL producing E. coli was 40 μL so this could be a new drug of choice. Acknowledgment The authors gratefully acknowledge the financial support from the University Grants Commission (UGC), New Delhi [F. No. 42-487/2013 (SR)] to carry out this work.

437

References [1] A.S. Khanna, Asian J. Exp. Sc. 21 (2008) 25–32. [2] Jae Yong Song, Hyeon-Kyeong Jang, Beom Soo Kim, Process Biochem. 44 (2009) 1133–1138. [3] T.C. Prathna, N. Chandrasekarana, M. Ashok, Amitava Mukherjee Raichur, Colloids Surf. B: Biointerfaces 82 (2011) 152–159. [4] Kalimuthu Kalishwaralal, Venkataraman Deepak, SureshBabu Ram Kumar Pandian, Muniasamy Kottaisamy, Selvaraj BarathManiKanth, Bose Kartikeyan, Sangiliyandi Gurunathan, Colloids Surf. B: Biointerfaces 77 (2010) 257–262. [5] S. Ghosh, R. Kaushik, K. Nagalakshmi, S.L. Hoti, G.A. Menezes, B.N. Harish, H.N. Vasan, Carbohydr. Res. 345 (2010) 2220–2227. [6] S.W. Mark, L.L. Andrew, C.J.S. Natalie, Curr. Opin. Microbiol. 8 (2005) 525–533. [7] Maria Tarnberg, Extended-spectrum Beta-lactamase Producing Enterobacteriaceae: Aspects on Detection, Epidemiology and Multi-drug Resistance, Linkoping University, 2012. [8] Mark Melzer, Irene Petersen, J. Infect. 55 (2007) 254–259. [9] D. Manikprabhu, K. Lingappa, J. Pharm. Res. 6 (2013) 255–260. [10] CLSI, Performing standards for antimicrobial susceptibility testing: twenty-second informational supplement, CLSI Document M100-S22, Clinical and Laboratory Standards Institute, Wayne, PA, 2012. [11] L.P. David, A.B. Robert, Clin. Microbiol. Rev. 18 (2005) 657–696. [12] L.P. David, Wen-Chien Ko, Anne Von Gottberg, Jose Maria Casellas, Lutfiye Mulazimoglu, Keith P. Klugman, A.B. Robert, B.R. Louis, Joseph G. McCormack, L.Y. Victor, J. Clin. Microbiol. 39 (2001) 2206–2212. [13] X. Chen, H.J. Schluesener, Toxicol. Lett. 176 (2008) 1–12. [14] N.T. Kaushik, S.M. Snehit, Y.P. Rasesh, Nanomed. Nanotechnol. Biol. Med. 6 (2010) 257–262. [15] L.F. Espinosa-Cristobal, G.A. Martinez-Castanon, R.E. Martinez-Martinez, J.P. LoyolaRodriguez, N. Patino-Marin, J.F. Reyes-Macias, Facundo Ruiz, Mater. Lett. 63 (2009) 2603–2606. [16] R. Bhat, R. Deshpande, V.G. Sharanabasava, Do Sung Huh, A. Venkataraman, Bioinorganic chemistry and applications (2011), http://dx.doi.org/10.1155/2011/ 650979. [17] Libor Kvitek, Ales Panacek, Jana Soukupova, Milan Kolar, Renata Vecerova, Robert Prucek, Mirka Holecova, Radek Zboril, J. Phys. Chem. 112 (2008) 5825–5834. [18] J.R. Morones, J.L. Elechiguerra, A. Camacho, J. Nanotechnol. 16 (2005) 2346–2353. [19] D. Manikprabhu, K. Lingappa, Saudi J. Biol. Sci. 20 (2013) 163–168.