SILVER NANOPARTICLES-DISK DIFFUSION TEST AGAINST Escherichia coli ISOLATES

Francisco Afrânio CUNHA Kamila Rocha MAIA Eduardo José Jucá MALLMAN Maria da Conceição dos Santos Oliveira CUNHA Antonio Auberson Martins MACIEL Ieda Pereira de SOUZA Everardo Albuquerque MENEZES Pierre Basílio Almeida FECHINE About the authors

SUMMARY

Nanotechnology can be a valuable ally in the treatment of infections. Silver nanoparticles (AgNPs) are structures that have antimicrobial activity. The aim of this study was to produce AgNPs by green methods, characterize these structures, and assess their antimicrobial activity against Escherichia coli associated with the antibiotic ciprofloxacin. AgNPs were characterized by spectroscopic and microscopic techniques. Antimicrobial activity was evaluated by the disk diffusion method against 10 strains of E. coli. The synthesized AgNPs showed a spherical shape and a size of 85.07 ± 12.86 nm (mean ± SD). AgNPs increased the activity of ciprofloxacin by 40% and may represent a new therapeutic option for the treatment of bacterial infections.

KEYWORDS:
Silver nanoparticles; Antimicrobial activity; Escherichia coli

INTRODUCTION

Nanotechnology involves the study of bodies that have dimensions of up to 100 nm in any direction and possess properties that differ from bulk material11. Chiu CY, Ruan L, Huang Y. Biomolecular specificity controlled nanomaterial synthesis. Chem Soc Rev. 2013;42:2512-27.. This peculiarity represents a new area of knowledge that can open a technological frontier, with the possibility of developing new compounds that could help to improve people's lives11. Chiu CY, Ruan L, Huang Y. Biomolecular specificity controlled nanomaterial synthesis. Chem Soc Rev. 2013;42:2512-27.,22. Chernousova S, Epple M. Silver as antibacterial agent: ion, nanoparticle, and metal. Angew Chem Int Ed Eng. 2013;52:1636-53..

There is an immense perspective of the use of nanoparticles in the diagnosis and treatment of human and animal diseases33. Doane TL, Burda C. The unique role of nanoparticles in nanomedicine: imaging, drug delivery and therapy. Chem Soc Rev . 2012;41:2885-911.. The main products available are those for personal hygiene, such as toothpaste, shaving creams, and deodorants22. Chernousova S, Epple M. Silver as antibacterial agent: ion, nanoparticle, and metal. Angew Chem Int Ed Eng. 2013;52:1636-53.. These products mainly contain silver nanoparticles (AgNPs), due to their known antiseptic action22. Chernousova S, Epple M. Silver as antibacterial agent: ion, nanoparticle, and metal. Angew Chem Int Ed Eng. 2013;52:1636-53.. AgNPs have various applications, including antiseptic, antibacterial, and antifungal properties. Silver is a cheaper metal than gold, which makes it attractive for research. Moreover, silver in the nanoparticle state has peculiar optical properties, allowing its use in technological products44. Arvizo RR, Bhattacharyya S, Kudgus RA, Giri K, Bhattacharya R, Mukherjee P. Intrinsic therapeutic applications of noble metal nanoparticles: past, present and future. Chem Soc Rev . 2012;41:2943-70.,55. Hervés P, Pérez-Lorenzo M, Liz-Marzán LM, Dzubiella J, Yan L, Ballauff M. Catalysis by metallic nanoparticles in aqueous solution: model reactions. Chem Soc Rev . 2012;41:5577-87.. The use of medical catheters covered with a nanometer-thin layer of AgNPs can prevent microorganisms colonization and thus decrease the length of hospital stay as it prevents infections22. Chernousova S, Epple M. Silver as antibacterial agent: ion, nanoparticle, and metal. Angew Chem Int Ed Eng. 2013;52:1636-53.,66. Jha RK, Jha PK, Chaudhury K, Rana SV, Guha SK. An emerging interface between life science and nanotechnology: present status and prospects of reproductive healthcare aided by nano-biotechnology. Nano Rev. 2014;5..

The antibacterial susceptibility test using AgNPs disk diffusion is well known77. Kharissova OV, Dias HV, Kharisov BI, Pérez BO, Pérez VM. The greener synthesis of nanoparticles. Trends Biotechnol. 2013;31:240-8.. These particles act by causing damage to membranes and to DNA, therefore preventing reproduction of microorganisms, leading to death88. Rizzello L, Pompa PP. Nanosilver-based antibacterial drugs and devices: mechanisms, methodological drawbacks, and guidelines. Chem Soc Rev . 2014;43:1501-18.. In addition to its intrinsic bactericidal property, the microorganism-killing power of AgNPs can be increased when associated with antibiotics88. Rizzello L, Pompa PP. Nanosilver-based antibacterial drugs and devices: mechanisms, methodological drawbacks, and guidelines. Chem Soc Rev . 2014;43:1501-18.. This mechanism may be useful in the treatment of multidrug-resistant infections, and may represent an important therapeutic alternative99. Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R. Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria. Nanomedicine. 2010;6:103-9.. Ciprofloxacin is a very useful antibiotic that acts by inhibiting bacterial DNA synthesis, resulting in the death of microorganisms1010. Castro W, Navarro M, Biot C. Medicinal potential of ciprofloxacin and its derivatives. Future Med Chem. 2013;5:81-96..

The aim of this study was to synthesize, characterize and evaluate the activity of AgNPs produced by green synthesis and associated with the antibiotic ciprofloxacin against strains of Escherichia coli using the diffusion disk method.

AgNP synthesis was performed using glucose as a reducing agent and sodium dodecyl sulfate (SDS) as a stabilizing agent. Briefly, 1.0 g of glucose and 0.5 g of SDS were added to 500 mL of a AgNO3 solution (5 mM). The solution was continuously stirred and the temperature was maintained at 50 °C to favor the reaction. Then 1.0 mL of 0.2 M NaOH was added to the mixture. The reaction was maintained under these conditions for 30 min, then stirred, heated and suspended1111. Darroudi M, Ahmad MB, Abdullah AH, Ibrahim NA. Green synthesis and characterization of gelatin-based and sugar-reduced silver nanoparticles. Int J Nanomedicine . 2011;6:569-74.,1212. Mallmann EJ, Cunha FA, Castro BN, Maciel AM, Menezes EA, Fechine PB. Antifungal activity of silver nanoparticles obtained by green synthesis. Rev Inst Med Trop Sao Paulo. 2015;57:165-7.. The AgNPs were purified by ultracentrifugation at 8,500 xg for 20 min, and characterization was carried out using spectrophotometric reading at 300 to 700 nm (UV-Vis). The size and shape were determined by atomic force microscopy (AFM), and scanning electron microscopy (SEM)1313. Lin PC, Lin S, Wang PC, Sridhar R. Techniques for physicochemical characterization of nanomaterials. Biotechnol Adv. 2014;32:711-26., (National Institutes of Health, USA, version 1.48v).

This study used 10 strains of Escherichia coli; provided by the collection maintained at the Microbiology Laboratory of the Department of Clinical Analysis, School of Pharmacy, Federal University of Ceará, Brazil. The strains of E. coli were isolated from urine samples of patients and they are all sensitive to ciprofloxacin. The tests were performed by the disk diffusion (DD) method on Müller-Hinton agar. In this experiment we used three types of discs: (1) paper disc (10 mm) saturated with 10 µL of AgNPs; (2) ciprofloxacin discs with (Ciprofloxacin) 5 µg (Cecon(r)); and (3) ciprofloxacin disks saturated with 10 µL of AgNPs. E. coli strains were suspended in saline solution and plated in the culture medium, and then the discs were placed. The plates were incubated at 35 °C for 24 hours, after this time the inhibition zones were measured1212. Mallmann EJ, Cunha FA, Castro BN, Maciel AM, Menezes EA, Fechine PB. Antifungal activity of silver nanoparticles obtained by green synthesis. Rev Inst Med Trop Sao Paulo. 2015;57:165-7.. The synergism was evaluated by the formula {(C2 - B2) / B2} x 100, where, B = the inhibition zones of the ciprofloxacin alone and C = ciprofloxacin + AgNPs1414. Shahverdi AR, Fakhimi A, Shahverdi HR, Minaian S. Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli. Nanomedicine . 2007;3:168-71.. This formula allowed us to evaluate the increment of the inhibition zone around the bacteria caused by the antibiotic in association with AgNPs99. Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R. Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria. Nanomedicine. 2010;6:103-9.,1414. Shahverdi AR, Fakhimi A, Shahverdi HR, Minaian S. Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli. Nanomedicine . 2007;3:168-71..

Processes that use sugars to obtain AgNPs are called green synthesis, because of the absence of toxic compounds and no formation of toxic waste77. Kharissova OV, Dias HV, Kharisov BI, Pérez BO, Pérez VM. The greener synthesis of nanoparticles. Trends Biotechnol. 2013;31:240-8.. Furthermore, sugars are cheap and affordable77. Kharissova OV, Dias HV, Kharisov BI, Pérez BO, Pérez VM. The greener synthesis of nanoparticles. Trends Biotechnol. 2013;31:240-8.. In our study, we used glucose for the synthesis and SDS for the stabilization. Nanoparticles were formed with an average size of 85.07 ± 12.86 nm (average ± SD) (Fig. 1a); UV-VIS spectra confirmed the efficient synthesis of AgNPs; these particles absorbed energy at 420 nm and exhibited a spherical shape, as can be seen in Fig. 1b and 1c. AgNP synthesis using glucose is a recurring theme in the literature77. Kharissova OV, Dias HV, Kharisov BI, Pérez BO, Pérez VM. The greener synthesis of nanoparticles. Trends Biotechnol. 2013;31:240-8.,1515. Sharma VK, Yngard RA, Lin Y. Silver nanoparticles: green synthesis and their antimicrobial activities. Adv Colloid Interface Sci. 2009;145:83-96.. The particles are spherical and stable, and SDS enhances the antimicrobial activity1616. Kora AJ, Manjusha R, Arunachalam J. Superior bactericidal activity of SDS capped silver nanoparticles: synthesis and characterization. Mater Sci Eng C. 2009;29:2104-9..

Fig. 1
Characterization of AgNPs: (a) UV-vis spectrum; (b) SEM and (c) AFM. (d) Microbial activity of AgNPs: (a) comparison of activities: cipro, cipro + AgNPs and AgNPs.

AgNPs stand out for their antimicrobial activity1717. Rai MK, Deshmukh SD, Ingle AP, Gade AK. Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria. J Appl Microbiol. 2012;112:841-52.. The mechanism of action involves the inactivation of enzymes and the DNA damage of microorganisms66. Jha RK, Jha PK, Chaudhury K, Rana SV, Guha SK. An emerging interface between life science and nanotechnology: present status and prospects of reproductive healthcare aided by nano-biotechnology. Nano Rev. 2014;5.. In our study, we evaluated the effect of AgNPs alone and associated with the antibiotic ciprofloxacin (cipro). Ciprofloxacin alone produced an inhibition zone of 45.9 ± 7.4 mm; AgNPs alone showed an inhibition zone of 17.1 ± 5.9 mm; and the combination of cipro + AgNPs showed an inhibition zone of 54.3 ± 8.6 mm (Fig.1d). The association of cipro + AgNPs produced a 40% increase in the inhibition zone when compared with the antibiotic alone (Table 1). The association AgNPs and antibiotics against bacteria and fungi have shown good results99. Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R. Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria. Nanomedicine. 2010;6:103-9.,1414. Shahverdi AR, Fakhimi A, Shahverdi HR, Minaian S. Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli. Nanomedicine . 2007;3:168-71.,1818. Gajbhiye M, Kesharwani J, Ingle A, Gade A, Rai M. Fungus-mediated synthesis of silver nanoparticles and their activity gainst pathogenic fungi in combination with fluconazole. Nanomedicine . 2009;5:382-6.. In Figure 1(d) we can observe the action of this association; the inhibition zone on the disc containing cipro + AgNPs is much larger than the disc with cipro alone and AgNPs alone. The AgNPs bind proteins and DNA of the bacteria and ciprofloxacin damages the DNA, and this combination enhances the effects on E. coli strains1010. Castro W, Navarro M, Biot C. Medicinal potential of ciprofloxacin and its derivatives. Future Med Chem. 2013;5:81-96..

Table 1
Antibacterial activity of AgNPs, cipro discs and cipro + AgNPs against E.coli

AgNPs were synthesized using glucose and SDS and were characterized through physical-chemical techniques: UV-VIS, SEM, and AFM; they showed a size of 85.07 ± 12.86 nm and a spherical shape. The AgNPs showed activity against E. coli isolates and an increased activity of the antibiotic ciprofloxacin. AgNPs associated with antimicrobial agents can be a therapeutic option for the treatment of bacterial infections. The disk diffusion method has limitations so that new tests using different methodologies should be used to confirm the results found in our study.

ACKNOWLEDGMENTS

The authors are grateful to Central Analítica - UFC/CT-INFRA/MCTI-SISNANO/Pró-Equipamentos, UFC Department of Physics, CAPES, CNPq and Funcap, for the grant provided to support this research on nanoparticles.

REFERENCES

  • 1
    Chiu CY, Ruan L, Huang Y. Biomolecular specificity controlled nanomaterial synthesis. Chem Soc Rev. 2013;42:2512-27.
  • 2
    Chernousova S, Epple M. Silver as antibacterial agent: ion, nanoparticle, and metal. Angew Chem Int Ed Eng. 2013;52:1636-53.
  • 3
    Doane TL, Burda C. The unique role of nanoparticles in nanomedicine: imaging, drug delivery and therapy. Chem Soc Rev . 2012;41:2885-911.
  • 4
    Arvizo RR, Bhattacharyya S, Kudgus RA, Giri K, Bhattacharya R, Mukherjee P. Intrinsic therapeutic applications of noble metal nanoparticles: past, present and future. Chem Soc Rev . 2012;41:2943-70.
  • 5
    Hervés P, Pérez-Lorenzo M, Liz-Marzán LM, Dzubiella J, Yan L, Ballauff M. Catalysis by metallic nanoparticles in aqueous solution: model reactions. Chem Soc Rev . 2012;41:5577-87.
  • 6
    Jha RK, Jha PK, Chaudhury K, Rana SV, Guha SK. An emerging interface between life science and nanotechnology: present status and prospects of reproductive healthcare aided by nano-biotechnology. Nano Rev. 2014;5.
  • 7
    Kharissova OV, Dias HV, Kharisov BI, Pérez BO, Pérez VM. The greener synthesis of nanoparticles. Trends Biotechnol. 2013;31:240-8.
  • 8
    Rizzello L, Pompa PP. Nanosilver-based antibacterial drugs and devices: mechanisms, methodological drawbacks, and guidelines. Chem Soc Rev . 2014;43:1501-18.
  • 9
    Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R. Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria. Nanomedicine. 2010;6:103-9.
  • 10
    Castro W, Navarro M, Biot C. Medicinal potential of ciprofloxacin and its derivatives. Future Med Chem. 2013;5:81-96.
  • 11
    Darroudi M, Ahmad MB, Abdullah AH, Ibrahim NA. Green synthesis and characterization of gelatin-based and sugar-reduced silver nanoparticles. Int J Nanomedicine . 2011;6:569-74.
  • 12
    Mallmann EJ, Cunha FA, Castro BN, Maciel AM, Menezes EA, Fechine PB. Antifungal activity of silver nanoparticles obtained by green synthesis. Rev Inst Med Trop Sao Paulo. 2015;57:165-7.
  • 13
    Lin PC, Lin S, Wang PC, Sridhar R. Techniques for physicochemical characterization of nanomaterials. Biotechnol Adv. 2014;32:711-26.
  • 14
    Shahverdi AR, Fakhimi A, Shahverdi HR, Minaian S. Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli Nanomedicine . 2007;3:168-71.
  • 15
    Sharma VK, Yngard RA, Lin Y. Silver nanoparticles: green synthesis and their antimicrobial activities. Adv Colloid Interface Sci. 2009;145:83-96.
  • 16
    Kora AJ, Manjusha R, Arunachalam J. Superior bactericidal activity of SDS capped silver nanoparticles: synthesis and characterization. Mater Sci Eng C. 2009;29:2104-9.
  • 17
    Rai MK, Deshmukh SD, Ingle AP, Gade AK. Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria. J Appl Microbiol. 2012;112:841-52.
  • 18
    Gajbhiye M, Kesharwani J, Ingle A, Gade A, Rai M. Fungus-mediated synthesis of silver nanoparticles and their activity gainst pathogenic fungi in combination with fluconazole. Nanomedicine . 2009;5:382-6.

Publication Dates

  • Publication in this collection
    2016

History

  • Received
    15 Oct 2015
  • Accepted
    29 Mar 2016
Instituto de Medicina Tropical de São Paulo Av. Dr. Enéas de Carvalho Aguiar, 470, 05403-000 - São Paulo - SP - Brazil, Tel. +55 11 3061-7005 - São Paulo - SP - Brazil
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