World Journal of Microbiology and Biotechnology

, Volume 28, Issue 2, pp 485–491 | Cite as

Bactericidal activity of ethanolic extracts of propolis against Staphylococcus aureus isolated from mastitic cows

  • Henrique Freitas Santana
  • Ana Andréa Teixeira Barbosa
  • Sukarno Olavo Ferreira
  • Hilário Cuquetto MantovaniEmail author
Original Paper


Staphylococcus aureus is an important pathogen for both humans and animals, and it has been an ubiquitous etiological agent of bovine mastitis in dairy farms worldwide. Elimination of S. aureus with classic antibiotics is difficult, and the current study aimed to evaluate the efficacy of ethanolic extracts of propolis (EEP) against S. aureus cultivated in complex media or milk. EEP (0–0.5 mg ml−1) decreased growth of S. aureus in BHI media and 1 mg ml−1 was bactericidal against washed cell suspensions (107 CFU ml−1). Propolis extracts also killed S. aureus cells resuspended in milk, but the bactericidal dose was at least 20-fold greater. Cultures that were transferred for at least 60 generations with sub-lethal doses of propolis did not change much their sensibility to EEP. Atomic force microscopy images revealed changes in morphology and cell size of S. aureus cells exposed to EEP (0.5 mg ml−1). Our results indicate that propolis extracts might be effective against mastitis-causing S. aureus strains in vivo, but milk constituents affect the inhibitory activity of propolis. Considering that propolis-resistance appears to be a phenotype not easily selected, the use of EEP combined or not with other antimicrobial agents might be useful for mastitis control in vivo.


Baccharis dracunculifolia Mastitis Udder infection AFM Dairy 



The authors thank the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Belo Horizonte, Brazil (Grant No. CBB-1318/06) for providing financial support for this research. HFS and AATC received fellowships from the Conselho de Desenvolvimento Científico e Tecnológico (CNPq). We thank Dr. Maximiliano Luis Munford, from the Physics Department at the Universidade Federal de Viçosa for technical assistance with the AFM analyses.


  1. Aires-de-Sousa M, Parente C, Vieira-da-Motta O, Bonna I, Silva D, de Lencastre H (2007) Characterization of Staphylococcus aureus isolates from buffalo, bovine, ovine, and caprine milk samples collected in Rio de Janeiro State, Brazil. Appl Environ Microbiol 73:3845–3849CrossRefGoogle Scholar
  2. Ali-Vehmas T, Westphalen P, Myllys V, Sandholm M (1997) Binding of Staphylococcus aureus to milk fat globules increases resistance to penicillin-G. J Dairy Res 64:253–260CrossRefGoogle Scholar
  3. Bonvehí J, Coll F, Jordà R (1994) The composition, active components and bacteriostatic activity of propolis in dietetics. J Am Oil Chem Soc 71:529–532CrossRefGoogle Scholar
  4. Brito MAVP, Brito JRF (1999) Diagnóstico microbiológico da mastite. Circular Técnica 55, Embrapa Gado de Leite, Juiz de ForaGoogle Scholar
  5. Capurro A, Aspán A, Ericsson UH, Persson WK, Artursson K (2010) Identification of potential sources of Staphylococcus aureus in herds with mastitis problems. J Dairy Sci 93:180–191CrossRefGoogle Scholar
  6. Cushnie T, Lamb A (2005) Detection of galangin-induced cytoplasmic membrane damage in Staphylococcus aureus by measuring potassium loss. J Ethnopharmacol 101:243–248CrossRefGoogle Scholar
  7. Denyer SP, Stewart GSAB (1998) Mechanisms of action of disinfectants. Int Biodeterior Biodegrad 41:261–268CrossRefGoogle Scholar
  8. Eaton P, Fernandes J, Pereira E, Pintado M, Xavier MF (2008) Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus. Ultramicroscopy 108:1128–1134CrossRefGoogle Scholar
  9. Erskine R, Wagner S, DeGraves F (2003) Mastitis therapy and pharmacology. Vet Clin N Am Food Anim Pract 19:109–138CrossRefGoogle Scholar
  10. Fernandes Júnior A, Balestrin E, Betoni J, Orsi R, Cunha M, Montelli A (2005) Propolis: anti-Staphylococcus aureus activity and synergism with antimicrobial drugs. Mem Inst Oswaldo Cruz 100:563–566CrossRefGoogle Scholar
  11. Fischer G, Paulino N, Marcucci M, Siedler B, Munhoz L, Finger P, Vargas G, Hübner S, Vidor T, Roehe P (2010) Green propolis phenolic compounds act as vaccine adjuvants, improving humoral and cellular responses in mice inoculated with inactivated vaccines. Mem Inst Oswaldo Cruz 105:908–913CrossRefGoogle Scholar
  12. Gehring R, Smith G (2006) An overview of factors affecting the disposition of intramammary preparations used to treat bovine mastitis. J Vet Pharmacol Ther 29:237–241CrossRefGoogle Scholar
  13. Gould S, Cuschieri P, Rollason J, Hilton A, Easmon S, Fielder M (2010) The need for continued monitoring of antibiotic resistance patterns in clinical isolates of Staphylococcus aureus from London and Malta. Ann Clin Microbiol Antimicrob 9:20CrossRefGoogle Scholar
  14. Gundogan N, Citak S, Yucel N, Devren A (2005) A note on the incidence and antibiotic resistance of Staphylococcus aureus isolated from meat and chicken samples. Meat Sci 69:807–810CrossRefGoogle Scholar
  15. Iio A, Ohguchi K, Inoue H, Maruyama H, Araki Y, Nozawa Y, Ito M (2010) Ethanolic extracts of Brazilian red propolis promote adipocyte differentiation through PPARγ activation. Phytomedicine 17:974–979CrossRefGoogle Scholar
  16. Juven B, Henis Y, Jacoby B (1972) Studies on the mechanism of the antimicrobial action of oleuropein. J Appl Bacteriol 35:559–567CrossRefGoogle Scholar
  17. Kuang Y, Jia H, Miyanaga K, Tanji Y (2009) Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis. Appl Microbiol Biotechnol 84:135–142CrossRefGoogle Scholar
  18. Lu L, Chen Y, Chou C (2005) Antibacterial activity of propolis against Staphylococcus aureus. Int J Food Microbiol 102:213–220CrossRefGoogle Scholar
  19. McDougall S, Parker K, Heuer C, Compton C (2009) A review of prevention and control of heifer mastitis via non-antibiotic strategies. Vet Microbiol 134:177–185CrossRefGoogle Scholar
  20. Messerli S, Ahn M, Kunimasa K, Yanagihara M, Tatefuji T, Hashimoto K, Mautner V, Uto Y, Hori H, Kumazawa S (2009) Artepillin C (ARC) in Brazilian green propolis selectively blocks oncogenic PAK1 signaling and suppresses the growth of NF tumors in mice. Phytother Res 23:423–427CrossRefGoogle Scholar
  21. Mirzoeva O, Grishanin R, Calder P (1997) Antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and motility of bacteria. Microbiol Res 152:239CrossRefGoogle Scholar
  22. National Committee for Clinical Laboratory Standards (2003) Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. Approved standard-sixth edition. Technical report M07-A6, Wayne, PAGoogle Scholar
  23. Nickerson S (2009) Control of heifer mastitis: antimicrobial treatment-an overview. Vet Microbiol 134:128–135CrossRefGoogle Scholar
  24. Owens W, Watts J (1987) Effects of milk on activity of antimicrobics against Staphylococcus aureus isolated from bovine udders. J Dairy Sci 70:1946–1951CrossRefGoogle Scholar
  25. Park Y, Fukuda I, Ashida H, Nishiumi S, Yoshida K, Daugsch A, Sato H, Pastore G (2005) Suppressive effects of ethanolic extracts from propolis and its main botanical origin on dioxin toxicity. J Agric Food Chem 53:10306–10309CrossRefGoogle Scholar
  26. Paulino N, Abreu S, Uto Y, Koyama D, Nagasawa H, Hori H, Dirsch V, Vollmar A, Scremin A, Bretz W (2008) Anti-inflammatory effects of a bioavailable compound, Artepillin C, in Brazilian propolis. Eur J Pharmacol 587:296–301CrossRefGoogle Scholar
  27. Pinto MS (2008) Atividade de própolis verde e bovicina HC5 sobre bactérias isoladas de mastite bovina. Thesis, Universidade Federal de ViçosaGoogle Scholar
  28. Raghukumar R, Vali L, Watson D, Fearnley J, Seidel V (2010) Antimethicillin resistant Staphylococcus aureus (MRSA) activity of ‘pacific propolis’ and isolated prenylflavanones. Phytother Res 24:1181–1187Google Scholar
  29. Salomao K, Pereira P, Campos L, Borba C, Cabello P, Marcucci M, De Castro S (2008) Brazilian propolis: correlation between chemical composition and antimicrobial activity. Evid Based Complement Alternat Med 5:317–324CrossRefGoogle Scholar
  30. Sayed S, Abou El-Ella G, Wahba N, El Nisr N, Raddad K, Abd El Rahman M, Abd El Hafeez M, Abd El Fattah Aamer A (2009) Immune defense of rats immunized with fennel honey, propolis, and bee venom against induced Staphylococcal infection. J Med Food 12:569–575CrossRefGoogle Scholar
  31. Sears P, McCarthy K (2003) Management and treatment of staphylococcal mastitis. Vet Clin N Am Food Anim Pract 19:171–185CrossRefGoogle Scholar
  32. Teixeira E, Negri G, Meira R (2005) Plant origin of green propolis: bee behavior, plant anatomy and chemistry. Evid Based Complement Alternat Med 2:85–92CrossRefGoogle Scholar
  33. Van Eenennaam A, Cullor J, Perani L, Gardner I, Smith W, Dellinger J, Guterbock W, Jensen L (1993) Evaluation of milk antibiotic residue screening tests in cattle with naturally occurring clinical mastitis. J Dairy Sci 76:3041–3053CrossRefGoogle Scholar
  34. Varella Coelho M, Santos Nascimento J, Fagundes P, Madureira D, Oliveira S, Vasconcelos de Paiva Brito M, Freire Bastos M (2007) Activity of staphylococcal bacteriocins against Staphylococcus aureus and Streptococcus agalactiae involved in bovine mastitis. Res Microbiol 158:625–630CrossRefGoogle Scholar
  35. Wu J, Hu S, Cao L (2007) Therapeutic effect of nisin Z on subclinical mastitis in lactating cows. Antimicrob Agents Chemother 51:3131–3135CrossRefGoogle Scholar
  36. Zoraghi R, See R, Gong H, Lian T, Swayze R, Finlay B, Brunham R, McMaster W, Reiner N (2010) Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from methicillin-resistant Staphylococcus aureus. Biochemistry 49:7733–7747CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Henrique Freitas Santana
    • 1
  • Ana Andréa Teixeira Barbosa
    • 1
  • Sukarno Olavo Ferreira
    • 2
  • Hilário Cuquetto Mantovani
    • 1
    Email author
  1. 1.Departamento de MicrobiologiaUniversidade Federal de ViçosaViçosa-MGBrazil
  2. 2.Departamento de FísicaUniversidade Federal de ViçosaViçosa-MGBrazil

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