Original Article

Biologically formed silver nanoparticles and in vitro study of their antimicrobial activities on resistant pathogens


Background and Objectives: Silver nanoparticles (AgNPs) have been found to have multiple uses as antibacterial, antifungal and anti-biofilm agents because of their biological activities and safety. The present study was aimed to analyze the antimicrobial and anti-biofilm activities as well as the cytotoxic effect of AgNPs against different human pathogens.
Materials and Methods: AgNPs were synthesized using cell free supernatants of Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 19433), Pseudomonas aeruginosa (ATCC 27856), Enterobacter cloacae (ATCC 13047) and Penicillium oxalicum strain, then were analyzed using UV/Vis Spectral Analysis, Transmission electron microscopy (TEM). Scanning Electron Microscope (SEM) and Energy Dispersive-X-ray Spectroscopy (EDX) analysis. Antimicrobial activities of biosynthesized AgNPs were assessed with selected antimicrobial agents against multidrug resistant bacteria and candida. Anti-biofilm and cytotoxicity assays of these biosynthesized AgNPs were also done.
Results: The synthesis of AgNPs were confirmed through observed color change and monitoring UV-Vis spectrum which showed homogeneous (little agglomeration) distribution of silver nanoparticles. TEM and SEM have shown that the particle size ranged from 13 to 34 (nm) with spherical shape and a high signal with EDX analysis. Antibacterial and antifungal efficacy of antibiotics and fluconazole were increased in combination with biosynthesized AgNPs against resistant bacteria and candida. Significant reduction in biofilm formation was found better with Penicillium oxalicum AgNPs against biofilm forming bacteria.
Conclusion: Penicillium oxalicum has the best effect towards synthesizing AgNPs, for antimicrobial activities against resistant bacteria and candida, in addition to anti-biofilm activities against biofilm forming Staphylococcus aureus and E. coli and the safest cytotoxicity effect on (MRC-5) cell line.

1. Luepke KH, Suda KJ, Boucher H, Russo RL, Bonney MW, Hunt TD, et al. Past, present, and future of antibacterial economics: increasing bacterial resistance, limited antibiotic pipeline and societal implications. Pharmacotherapy 2017; 37: 71-84.
2. Rai M, Kon K, Ingle A, Duran N, Galdiero S, Galdiero M. Broad-spectrum bioactivities of silver nanoparticles: the emerging trends and future prospects. Appl Microbiol Biotechnol 2014; 98: 1951-1961.
3. Amini N, Amin G, Jafari Azar Z. Green synthesis of silver nanoparticles using Avena sativa L. extract. Nanomed Res J 2017; 2: 57-63.
4. Wang L, Hu C, Shao L. The antimicrobial activity of nanoparticles: present situation and prospects for the future. Int J Nanomedicine 2017; 12: 1227-1249.
5. Cheesbrough M (2006). District laboratory practice in tropical countries. Part 2. 2nd ed. Cambridge University Press. The Edinburgh Building, Cambridge CB2 8RU, UK.
6. The Clinical Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing. 29th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2019.
7. The Clinical Laboratory Standards Institute (CLSI). Performance standards for antifungal susceptibility testing of yeast. 1st ed. CLSI supplement M60. Wayne, PA: Clinical and Laboratory Standards Institute; 2017.
8. Saeb AT, Alshammari AS, Al-Brahim H, Al-Rubeaan KA. Production of silver nanoparticles with strong and stable antimicrobial activity against highly pathogenic and multidrug resistant bacteria. ScientificWorldJournal 2014; 2014: 704708.
9. Ottoni CA, Simões MF, Fernandes S, Dos Santos JG, da Silva ES, de Souza RFB, et al. Screening of filamentous fungi for antimicrobial silver nanoparticles synthesis. AMB Express 2017; 7: 31.
10. Suriati G, Mariatti M, Azizan A. Synthesis of silver nanoparticles by chemical reduction method: effect of reducing agent and surfactant concentration. Int J Automot Mech Eng 2014; 10: 1920-1927.
11. Gurunathan S, Jeong JK, Han JW, Zhang XF, Park JH, Kim JH. Multidimensional effects of biologically synthesized silver nanoparticles in Helicobacter pylori, Helicobacter felis, and human lung (L132) and lung carcinoma A549 cells. Nanoscale Res Lett 2015; 10: 35.
12. Saravanan M, Vemu AK, Barik SK. Rapid biosynthesis of silver nanoparticles from Bacillus megaterium (NCIM 2326) and their antibacterial activity on multi drug resistant clinical pathogens. Colloids Surf B Biointerfaces 2011; 88: 325-331.
13. Wojnicki M, Luty-Błocho M, Kotańska M, Wytrwal M, Tokarski T, Krupa A, et al. Novel and effective synthesis protocol of AgNPs functionalized using L-cysteine as a potential drug carrier. Naunyn Schmiedebergs Arch Pharmacol 2018; 391: 123-130.
14. Wu Z, Tsumura Y, Blomquist G, Wang XR. 18S rRNA gene variation among common airborne fungi and development of specific oligonucleotide probes for the detection of fungal isolates. Appl Environ Microbiol 2003; 69: 5389-5397.
15. Hinrikson HP, Hurst SF, De Aguirre L, Morrison CJ. Molecular methods for the identification of Aspergillus species. Med Mycol 2005; 43 Suppl 1: S129-137.
16. Scott AC (1989). Laboratory control of antimicrobial therapy. In: Mackie and McCartney Practical Medical Microbiology. Eds, JG Collee, JP Duguid, AG Fraser, BP Marmion. Churchill Livingstone, 13th ed. Edinburgh, pp. 161-181.
17. 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: 103-109.
18. Ishida K, Cipriano TF, Rocha GM, Weissmüller G, Gomes F, Miranda K, et al. Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticles characterization and analysis of antifungal activity against pathogenic yeasts. Mem Inst Oswaldo Cruz 2014; 109: 220-228.
19. The Clinical Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; twenty-second informational supplement. CLSI supplement M100-S22. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.
20. Regev-Shoshani G, Ko M, Chris M, Av-Gay Y. Slow release of nitric oxide from charged catheters and its effect on biofilm formation by Escherichia coli. Antimicrob Agents Chemother 2010; 54: 273-279.
21. Gomha SM, Riyadh SM, Mahmmoud EA, Elaaser MM. Synthesis and anticancer activities of thiazoles, 1,3-thiazines, and thiazolidine using chitosan-grafted-poly (vinylpyridine) as basic catalyst. Heterocycles 2015; 91: 1227-1243.
22. Kalimuthu K, Suresh Babu R, Venkataraman D, Bilal M, Gurunathan S. Biosynthesis of silver nanocrystals by Bacillus licheniformis. Colloids Surf B Biointerfaces 2008; 65:150-153.
23. Kumar CM, Yugandhar P, Savithramma N. Biological synthesis of silver nanoparticles from Adansonia digitata L. fruit pulp extract, characterization, and its antimicrobial properties. J Intercult Ethnopharmacol 2016; 5: 79-85.
24. Pulcrano, G, Pignanelli S, Vollaro A, Esposito M, Iula VD, Roscetto E, et al. Isolation of Enterobacter aerogenes carrying blaTEM-1 and blaKPC-3 genes recovered from a hospital intensive care unit. APMIS 2016; 124: 516-521.
25. Ansari MA, Khan HM, Khan AA, Cameotra SS, Saquib Q, Musarrat J. Gum arabic capped-silver nanoparticles inhibit biofilm formation by multi-drug resistant strains of Pseudomonas aeruginosa. J Basic Microbiol 2014; 54: 688-699.
26. Adebayo-Tayo BC, Popoola AO. Biogenic synthesis and antimicrobial activity of silver nanoparticle using exopolysaccharides from lactic acid bacteria. Int J Nano Dimens 2017; 8: 61-69.
27. Magudapathy P, Gangopadhyay P, Panigrahi BK, Nair KGM, Dhara S. Electrical transport studies of Ag nanoclusters embedded in glass matrix. Physica B: Condensed Matter 2001; 299: 142-146.
28. Wisam JA, Haneen AJ. A novel study of pH influence on Ag nanoparticles size with antibacterial and antifungal activity using green synthesis. World Sci News 2018; 97: 139-152.
29. Deljou A, Goudarzi S. Green extracellular synthesis of the silver nanoparticles using thermophilic Bacillus sp. AZ1 and its antimicrobial activity against several human pathogenetic bacteria. Iran J Biotechnol 2016;14: 25-32.
30. Nanda A, Saravanan M. Biosynthesis of silver nanoparticles from Staphylococcus aureus and its antimicrobial activity against MRSA and MRSE. Nanomedicine 2009; 5: 452-456.
31. Longhi C, Santos JP, Morey AT, Marcato PD, Durán N, Pinge-Filho P, et al. Combination of fluconazole with silver nanoparticles produced by Fusarium oxysporum improves antifungal effect against planktonic cells and biofilm of drug-resistant Candida albicans. Med Mycol 2016; 54: 428-432.
32. Jalal M, Ansari MA, Alzohairy MA, Ali SG, Khan HM, Almatroudi A, et al. Anticandidal activity of biosynthesized silver nanoparticles: effect on growth, cell morphology, and key virulence attributes of Candida species. Int J Nanomedicine 2019; 14: 4667-4679.
33. Masurkar SA, Chaudhari PR, Shidore VB, Kamble SP. Effect of biologically synthesized silver nanoparticles on Staphylococcus aureus biofilm quenching and prevention of biofilm formation. IET Nanobiotechnol 2012; 6: 110-114.
34. Gupta K, Barua S, Hazarika SN, Manhar AK, Nath D, Karak N, et al. Green silver nanoparticles: enhanced antimicrobial and antibiofilm activity with effects on DNA replication and cell cytotoxicity. RSC Adv 2014; 4: 52845-52855.
35. Kalishwaralal K, BarathManiKanth S, Pandian SR, Deepak V, Gurunathan S. Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis. Colloids Surf B Biointerfaces 2010; 79: 340-344.
36. Chávez-Andrade GM, Tanomaru-Filho M, Basso Bernardi MI, de Toledo Leonardo R, Faria G, Guerreiro-Tanomaru JM. Antimicrobial and biofilm anti-adhesion activities of silver nanoparticles and farnesol against endodontic microorganisms for possible application in root canal treatment. Arch Oral Biol 2019; 107: 104481.
37. Mahmoudi M, Laurent S, Shokrgozar MA, Hosseinkhani M. Toxicity evaluations of superparamagnetic iron oxide nanoparticles: cell “vision” versus physicochemical properties of nanoparticles. ACS Nano 2011; 5: 7263-7276.
38. Fabrega J, Renshaw JC, Lead JR. Interactions of silver nanoparticles with Pseudomonas putida biofilms. Environ Sci Technol 2009; 43: 9004-9009.
39. Yen HJ, Hsu SH, Tsai CL. Cytotoxicity and immunological response of gold and silver nanoparticles of different sizes. Small 2009; 5: 1553-1561.
40. Moteriya P, Chanda S. Biosynthesis of silver nanoparticles formation from Caesalpinia pulcherrima stem metabolites and their broad spectrum biological activities. J Genet Eng Biotechnol 2018; 16:105-113.
IssueVol 13 No 6 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijm.v13i6.8090
Penicillium oxalicum; Sliver nanoparticles; Biofilm; Cytotoxicity

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Ali A, Anani H, Selim F. Biologically formed silver nanoparticles and in vitro study of their antimicrobial activities on resistant pathogens. Iran J Microbiol. 2021;13(6):848-861.