Silver Nanoparticles from Cow’s Milk to Combat Multidrug-Resistant Gram-Negative Bacteria from Clinical Isolates


The emergence of multidrug-resistant (MDR) bacteria is causing severe and lethal human infections and is posing a major threat in health care. Our previous study reports successful synthesis and characterization of silver nanoparticles (AgNPs) from pasteurized cow’s milk along with its potential antibacterial activity against non-MDR bacteria. The present research aims to study the antibacterial activity of AgNPs and its synergistic effects with five antibiotics (in regular clinical use) against the clinically isolated MDR bacteria such as Pseudomonas aeruginosa and Klebsiella pneumoniae. These bacteria were initially treated against 26 standard antibiotics to check the sensitivity using the Kirby–Bauer disc diffusion method. Results revealed that both organisms were resistant to all antibiotics tested and were MDR’s. Antibacterial studies indicated that cow’s milk AgNPs displayed effective inhibition zones ranging between 11–15 mm against P. aeruginosa and 8–10.5 mm against K. pneumoniae at 50 and 75 µl vis-à-vis control (source) and standard antibiotics where no inhibition zones were observed. Further, the synergistic effects of AgNPs with antibiotics revealed that maximum fold increase was obtained with piperacillin/tazobactam (4–5 folds) and co-trimoxazole (1.7–1.8 folds) against P. aeruginosa and K. pneumoniae respectively. Thus, AgNPs from pasteurised cow’s milk can be used as an effective antibacterial agent to combat the MDR’s and can be suggested as an alternative to antibiotics in the future.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. 1.

    Cillóniz C, Dominedò C, Torres A (2019) Multidrug resistant gram-negative bacteria in community-acquired pneumonia. Crit Care 23:79

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Basak S, Singh P, Rajurkar M (2016) Multidrug resistant and extensively drug resistant bacteria: a study. J Pathog Article ID 4065603

  3. 3.

    Liao S, Zhang Y, Pan X, Zhu F, Jiang C, Liu Q, Cheng Z, Dai G, Wu G, Wang L, Chen L (2019) Antibacterial activity and mechanism of silver nanoparticles against multidrug-resistant Pseudomonas aeruginosa. Int J Nanomed 14:1469–1487

    CAS  Article  Google Scholar 

  4. 4.

    Prasad R (2014) Synthesis of silver nanoparticles in photosynthetic plants. J Nanopart.

    Article  Google Scholar 

  5. 5.

    Siddiqi KS, Husen A, Rao RAK (2018) A review on biosynthesis of silver nanoparticles and their biocidal properties. J Nanobiotechnol 16:14

    Article  Google Scholar 

  6. 6.

    Roy A, BulutO SomeS, Mandal KA, Yilmaz MD (2019) Green synthesis of silver nanoparticles:biomolecule-nanoparticle organizations targeting antimicrobial activity. RSC Adv 9:2673

    CAS  Article  Google Scholar 

  7. 7.

    Barros CHN, Fulaz S, Stanisic D, Tasic L (2018) Biogenic nanosilver against multidrug-resistant bacteria (MDRB). Antibiotics 7:69.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Kui-Jae L, Sung HP, Muthusamy G, Pyoung H, Young SS, Min C, Lee Wang-Hyu, Jai-Young L, Seralathan KK (2013) Synthesis of silver nanoparticles using cow milk and their antifungal activity against phytopathogens. Mater Lett 105:128–131

    Article  Google Scholar 

  9. 9.

    Hegazi A, Ezzat HE, Amr MA, Allah FA, Eman H, Rahman A (2014) Potential antibacterial properties of silver nanoparticles conjugated with cow and camel milks. Glob Vet 12:745–749

    CAS  Google Scholar 

  10. 10.

    Athreya AG, Shareef MI, Gopinath SM (2018) Antibacterial activity of silver nanoparticles isolated from cow’s milk, hen’s egg white and lysozyme: a comparative study. Arab J Sci Eng.

    Article  Google Scholar 

  11. 11.

    Holt JG (1977) The shorter Bergey’s manual of determinative bacteriology. The shorter Bergey’s manual of determinative bacteriology, 8th edn

  12. 12.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Wayne PA (2017) CLSI. Performance standards of antibiotic disc susceptibility testing, 27th edn.CLSI Supplement M100. Vol 37 No.1

  14. 14.

    Bauer AW, Kirby WM, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45:493–496

    CAS  Article  Google Scholar 

  15. 15.

    Jyoti K, Baunthiyal M, Singh A (2016) Characterization of silver nanoparticles synthesized using Urtica dioica Linn. leaves and their synergistic effects with antibiotics. J Radiat Res Appl Sci 9:217–227

    CAS  Article  Google Scholar 

  16. 16.

    Singh K, Panghal M, Kadyan S, Chaudhary U, Yadav JP (2014) Green silver nanoparticles of Phyllanthus amarus: as an antibacterial agent against multi drug resistant clinical isolates of Pseudomonas aeruginosa. J Nanobiotechnol 12:40

    Article  Google Scholar 

  17. 17.

    Garg M, Devi B, Devi R (2017) In vitro antibacterial activity of biosynthesized silver nanoparticles from ethyl acetate extract of Hydrocotyle sibthorpioides against multidrug resistant microbes. Asian J Pharm Clin Res 10:63–266

    Google Scholar 

  18. 18.

    Naqvi SZH, Kiran U, Ali MI, Jamal A, Hameed A, Ahmed S, Ali N (2013) Combined efficacy of biologically synthesized silver nanoparticles and different antibiotics against multidrug-resistant bacteria. Int J Nanomed 8:3187–3195

    Article  Google Scholar 

  19. 19.

    Salomoni R, Léo P, Montemor AF, Rinaldi BG, Rodrigues MFA (2017) Antibacterial effect of silver nanoparticles in Pseudomonas aeruginosa. Nanotechnol Sci Appl 10:115–121

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Kumar N, Das S, Jyoti A, Kaushik S (2016) Synergistic effect of silver nanoparticles with doxycycline against Klebsiella pneumoniae. Int J Pharm Pharm Sci 8:183–186

    CAS  Article  Google Scholar 

  21. 21.

    Jeeva K, Thiyagarajan M, Elangovan V, Geetha N, Venkatachalam P (2014) Caesalpinia coriaria leaf extracts mediated biosynthesis of metallic silver nanoparticles and their antibacterial activity against clinically isolated pathogens. Ind Crops Prod 52:714–720

    CAS  Article  Google Scholar 

  22. 22.

    Kora AJ, Sashidhar RB (2018) Biogenic silver nanoparticles synthesized with rhamnogalacturonan gum: antibacterial activity, cytotoxicity and its mode of action. Arab J Chem 11:313–323

    CAS  Article  Google Scholar 

  23. 23.

    Catalina MJ, Hoek EMV (2010) A review of the antibacterial effects of silver nano-materials and potential implications for human health and the environment. J Nanopart Res 12:1531–1551

    Article  Google Scholar 

Download references


The authors would like to acknowledge V.T.U Research Centre, Muddenahalli, Aristogene Biosciences Pvt. Ltd., Kaade Hospital Bangalore and STIC, Cochin for providing required facilities to carry out the work.

Author information



Corresponding author

Correspondence to M. Ismail Shareef.

Ethics declarations

Conflict of interest

The authors declare that there is no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Significance statement The AgNPs from pasteurized cow’s milk have been tested on MDR bacteria for the first time in this study unlike other studies on antifungal and antibacterial activity against non-MDR pathogens.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 18 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Athreya, A.G., Shareef, M.I. & Gopinath, S.M. Silver Nanoparticles from Cow’s Milk to Combat Multidrug-Resistant Gram-Negative Bacteria from Clinical Isolates. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 90, 863–871 (2020).

Download citation


  • AgNPs
  • Antibacterial
  • Multidrug-resistant
  • Cow’s milk
  • Antibiotics