Skip to main content
SpringerLink
Log in

Australian propolis ethanol extract exerts antibacterial activity against methicillin-resistant Staphylococcus aureus by mechanisms of disrupting cell structure, reversing resistance, and resisting biofilm

  • Bacterial and Fungal Pathogenesis - Research Paper
  • Published:
Brazilian Journal of Microbiology Aims and scope Submit manuscript

Abstract

The antibacterial activity and mechanisms of Australian propolis ethanol extract (APEE) against methicillin-resistant Staphylococcus aureus (MRSA) were investigated herein. The diameter of inhibition zones (DIZ) of APEE was 19.7 mm, while the minimum inhibition concentration (MIC) and minimum bactericide concentration (MBC) of APEE were both 0.9 mg/mL against the tested strain of MRSA. Nucleic acid leakage and propidium iodide (PI) staining assays showed that APEE can stimulate the release of intracellular nucleic acids by disrupting the integrity of the cell wall and cytoplasmic membrane. Scanning electron microscopy (SEM) further confirmed that APEE could depress cellular activities via damaging the cell structure, including the cell wall and membrane. Western blot analysis and β-lactamase activity assay showed that APEE could inhibit the expression of PBP2a and reduce the activity of β-lactamase, suggesting that APEE is able to reverse the drug resistance of MRSA. XTT and crystal violet (CV) assays indicated that APEE had the capacity to prevent the formation of biofilms through decreasing cellular activities and biomass. Bacterial adhesion assay revealed that APEE could reduce the adhesive capacity of the strain, belonging to its antibiofilm mechanisms. Furthermore, nine main compounds of APEE were identified and quantified by HPLC–DAD/Q-TOF–MS. The results above all verified that the antibacterial activity of APEE against MRSA was mainly due to disrupting cell structure, reversing resistance, and resisting biofilm formation, which indicates that APEE is expected to be an efficient functional ingredient with great potential application in the field of medicine and food.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

APEE:

Australian propolis ethanol extract

CAPE:

Caffeic acid phenethyl ester

CV:

Crystal violet

DIZ:

Diameter of inhibition zone

MBC:

Minimum bactericide concentration

MIC:

Minimum inhibition concentration

MRSA:

Methicillin-resistant Staphylococcus aureus

PBS:

Phosphate-buffered saline

PI:

Propidium iodide

SEM:

Scanning electron microscopy

TSA:

Trypticase soy agar

TSB:

Trypticase soy broth

XTT:

2,3-Bis (2-methyloxy 4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

References

  1. Flora M, Perrotta F, Nicolai A, Maffucci R, Pratillo A, Mollica M et al (2019) Staphylococcus aureus in chronic airway diseases: an overview. Respir Med 155:66–71

    Article  PubMed  Google Scholar 

  2. Mera RM, Suaya JA, Amrine-Madsen H, Hogea CS, Miller LA, Lu EP et al (2011) Increasing role of Staphylococcus aureus and community-acquired methicillin-resistant Staphylococcus aureus infections in the United States: a 10-year trend of replacement and expansion. Microb Drug Resist 17(2):321–328

    Article  PubMed  Google Scholar 

  3. Huang C, Wang XL, Zhang L, Shen W (2008) Distribution and drug resistance of pathogenic bacteria in children with lower respiratory tract infection from Chengdu Children’s Hospital between 2001 and 2006. Chinese Journal of Contemporary Pediatrics 10(1):17–20

    CAS  PubMed  Google Scholar 

  4. Lister JL, Horswill AR (2014) Staphylococcus aureus biofilms: recent developments in biofilm dispersal. Front Cell Infect Microbiol 4:178

    Article  PubMed  PubMed Central  Google Scholar 

  5. Azmi K, Qrei W, Abdeen Z (2019) Screening of genes encoding adhesion factors and biofilm production in methicillin resistant strains of Staphylococcus aureus isolated from Palestinian patients. BMC Genomics 20(1):578

    Article  PubMed  PubMed Central  Google Scholar 

  6. Vancraeynest D, Herman K, Haesebrouck F (2004) Genotypic and phenotypic screening of high and low virulence Staphylococcus aureus isolates from rabbits for biofilm formation and MSCRAMMs. Vet Microbiol 103(3–4):241–247

    Article  CAS  PubMed  Google Scholar 

  7. Resch A, Rosenstein R, Nerz C, Gotz F (2005) Differential gene expression profiling of Staphylococcus aureus cultivated under biofilm and planktonic conditions. Appl Environ Microbiol 71(5):2663–2676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Back KT, Grundling A, Mogensen RG, Thegersen L, Petersen A, Paulander W et al (2014) Beta-Lactam resistance in methicillin-resistant Staphylococcus aureus USA300 is increased by inactivation of the ClpXP protease. Antimicrob Agents Cemother 58(8):4593–4603

    Article  Google Scholar 

  9. Liao XY, Cullen PJ, Liu DH, Muhammad AI, Chen SG, Ye XQ et al (2018) Combating Staphylococcus aureus and its methicillin resistance gene (mecA) with cold plasma. Sci Total Environ 645:1287–1295

    Article  CAS  PubMed  Google Scholar 

  10. Qin N, Tan XJ, Jiao YM, Liu L, Zhao WS, Yang S et al (2014) RNA-Seq-based transcriptome analysis of methicillin-resistant Staphylococcus aureus biofilm inhibition by ursolic acid and resveratrol. Sci Rep 4:5467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yuan WW, Chang HS, Liu XY, Wang SQ, Liu H, Xuan HZ (2019) Brazilian green propolis inhibits Ox-LDL-stimulated oxidative stress in human umbilical vein endothelial cells partly through PI3K/Akt/mTOR-mediated Nrf2/HO-1 pathway. Evidence-based Complementary and Alternative Medicine 2019 5789574

  12. Huang S, Zhang CP, Wang K, Li GQ, Hu FL (2014) Recent advances in the chemical composition of propolis. Molecules 19(12):19610–19632

    Article  PubMed  PubMed Central  Google Scholar 

  13. Sforcin JM, Bankova V (2011) Propolis: is there a potential for the development of new drugs? J Ethnopharmacol 133(2):253–260

    Article  CAS  PubMed  Google Scholar 

  14. Hu FL, Hepburn HR, Xuan HZ, Chen ML, Daya S, Radloff SE (2005) Effects of propolis on blood glucose, blood lipid and free radicals in rats with diabetes mellitus. Pharmacol Res 51(2):147–152

    Article  Google Scholar 

  15. Braakhuis A (2019) Evidence on the health benefits of supplemental propolis. Nutrients 11(11):2705

    Article  CAS  PubMed Central  Google Scholar 

  16. Gezgin Y, Kazan A, Ulucan F, Yesil-Celiktas O (2019) Antimicrobial activity of propolis and gentamycin against methicillin-resistant Staphylococcus aureus in a 3D thermo-sensitive hydrogel. Industrial Crops and Products 139:111588

    Article  CAS  Google Scholar 

  17. Ambi A, Vera C, Parikh N, Perez N, Rojas AL, Kumar S et al (2019) Plasma-initiated graft polymerization as an immobilization platform for metal free Russian propolis ethanol extracts designed specifically for biomaterials. Biofouling 34(5):557–568

    Article  Google Scholar 

  18. Lee JH, Kim YG, Khadke SK, Yamano A, Woo JT, Lee J (2019) Antimicrobial and antibiofilm activities of prenylated flavanones from Macaranga tanarius. Phytomedicine 63:153033

    Article  CAS  PubMed  Google Scholar 

  19. Chen YW, Ye SR, Ting C, Yu YH (2018) Antibacterial activity of propolins from Taiwanese green propolis. J Food Drug Anal 26(2):761–768

    Article  CAS  PubMed  Google Scholar 

  20. Xuan HZ, Yuan WW, Chang HS, Liu MM, Hu FL (2019) Antiinflammatory effects of Chinese propolis in lipopolysaccharide-stimulated human umbilical vein endothelial cells by suppressing autophagy and MAPK/NF-κB signaling pathway. Inflammopharmacology 27(3):561–571

    Article  CAS  PubMed  Google Scholar 

  21. Chen ZF, He B, Zhou J, He DH, Deng JD, Zeng RH (2016) Chemical compositions and antibacterial activities of essential oils extracted from Alpinia guilinensis against selected foodborne pathogens. Ind Crops Prod 83:607–613

    Article  CAS  Google Scholar 

  22. Liu YC, Xu YJ, Song QH, Wang F, Sun LG, Liu L et al (2017) Anti-biofilm activities from Bergenia crassifolia leaves against Streptococcus mutans. Front Microbiol 8:1738

    Article  PubMed  PubMed Central  Google Scholar 

  23. Wu YP, Bai JR, Zhong K, Huang YN, Gao H (2017) A dual antibacterial mechanism involved in membrane disruption and DNA binding of 2R, 3R-dihydromyricetin from pine needles of Cedrus deodara against Staphylococcus aureus. Food Chem 218:463–470

    Article  CAS  PubMed  Google Scholar 

  24. Zhang YB, Liu XY, Wang YF, Jiang PP, Quek SY (2016) Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Food Control 59:282–289

    Article  CAS  Google Scholar 

  25. Yan FL, Dang QF, Liu CS, Yan JQ, Wang T, Fan B et al (2016) 3,6-O-[N-(2-Aminoethyl)-acetamide-yl]-chitosan exerts antibacterial activity by a membrane damage mechanism. Carbohydrate Polymer 149:102–111

    Article  CAS  Google Scholar 

  26. Wang F, Wei FY, Song CX, Jiang B, Tian SY, Yi JW et al (2017) Dodartia orientalis L. essential oil exerts antibacterial activity by mechanisms of disrupting cell structure and resisting biofilm. Ind Crops Prod 109:358–366

    Article  CAS  Google Scholar 

  27. Catteau L, Reichmann NT, Olson J, Pinho MG, Nizet V, Bambeke FV et al (2017) Synergy between ursolic and oleanolic acids from Vitellaria paradoxa leaf extract and–lactams against methicillin-resistant Staphylococcus aureus: in vitro and in vivo activity and underlying mechanisms. Molecules 22(12):2245

    Article  PubMed Central  Google Scholar 

  28. Liu J, Li W, Zhu XY, Zhao HZ, Lu YJ, Zhang C et al (2019) Surfactin effectively inhibits Staphylococcus aureus adhesion and biofilm formation on surfaces. Appl Microbiol Biotechnol 103(11):4565–4574

    Article  CAS  PubMed  Google Scholar 

  29. Novo DJ, Perlmutter NG, Hunt RH, Shapiro HM (2000) Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus. Antimicrob Agents Chemother 44(4):827–834

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. El-Guendouz S, Aazza S, Lyoussi B, Bankova V, Popova M, Neto L et al (2018) Moroccan propolis: a natural antioxidant, antibacterial, and antibiofilm against Staphylococcus aureus with no induction of resistance after continuous exposure. Evidence-based Complementary and Alternative Medicine 2018:9759240

    Article  PubMed  PubMed Central  Google Scholar 

  31. Salton MR (1953) Studies of the bacterial cell wall: IV. The composition of the cell walls of some gram-positive and gram-negative bacteria. Biochimica et Biophysica Acta 10(4) 512-523

  32. Sikkema J, De Bont JA, Poolman B (1995) Mechanisms of membrane toxicity of hydrocarbons. Microbiol Mol Biol Rev 59(2):201–222

    CAS  Google Scholar 

  33. Hsouna AB, Trigui M, Mansour RB, Jarraya RM, Damak M, Jaoua S (2011) Chemical composition, cytotoxicity effect and antimicrobial activity of Ceratonia siliqua essential oil with preservative effects against Listeria inoculated in minced beef meat. Int J Food Microbiol 148(1):66–72

    Article  PubMed  Google Scholar 

  34. Chen CZ, Cooper SL (2002) Interactions between dendrimer biocides and bacterial membranes. Biomaterials 23(16):3359–3368

    Article  CAS  PubMed  Google Scholar 

  35. Diao WR, Hu QP, Zhang H, Xu JG (2014) Chemical composition, antibacterial activity and mechanism of action of essential oil from seeds of fennel (Foeniculum vulgare Mill). Food Control 35:109–116

    Article  CAS  Google Scholar 

  36. Cox SD, Mann CM, Markham JL, Gustafson JE, Warmington JR, Wyllie SG (2001) Determining the antimicrobial actions of tea tree oil. Molecules 6(2):87–91

    Article  CAS  PubMed Central  Google Scholar 

  37. Sharma A, Bajpai VK, Baek KH (2013) Determination of antibacterial mode of action of allium sativum essential oil against foodborne pathogens using membrane permeability and surface characteristic parameters. J Food Saf 33(2):197–208

    Article  Google Scholar 

  38. Kohanski MA, Dwyer DJ, Collins JJ (2010) How antibiotics kill bacteria: from targets to networks. Nat Rev Microbiol 8(6):423–435

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. de Campos JV, Garrido Assis OB, Bernardes-Filho R (2020) Atomic force microscopy evidences of bacterial cell damage caused by propolis extracts on E. coli and S. aureus. Food Science and Technology 40(1):55–61

    Article  Google Scholar 

  40. Paul S, Dubey RC, Maheswari DK, Kang SC (2011) Trachyspermum ammi (L.) fruit essential oil influencing on membrane permeability and surface characteristics in inhibiting food-borne pathogens. Food Control 22:725–731

    Article  CAS  Google Scholar 

  41. Liu C, Bayer A, Cosgrove SE, Daum RS, Fridkin SK, Gorwitz RJ et al (2011) Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary. Clin Infect Dis 52(3):285–292

    Article  PubMed  Google Scholar 

  42. Tazi A, Chapron J, Touak G, Longo M, Hubert D, Collobert G et al (2013) Rapid emergence of resistance to linezolid and mutator phenotypes in Staphylococcus aureus isolates from an adult cystic fibrosis patient. Antimicrob Agents Chemother 57(10):5186–5188

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Nannini E, Murray BE, Arias CA (2010) Resistance or decreased susceptibility to glycopeptides, daptomycin, and linezolid in methicillin-resistant Staphylococcus aureus. Curr Opin Pharmacol 10(5):516–521

    Article  CAS  PubMed  Google Scholar 

  44. Melchior MB, Vaarkamp H, Fink-Gremmels J (2006) Biofilms: a role in recurrent mastitis infections? Veterinary Journal 171:398–407

    Article  CAS  Google Scholar 

  45. Sandasi M, Leonard CM, Viljoen AM (2010) The in vitro antibiofilm activity of selected culinary herbs and medicinal plants against Listeria monocytogenes. Lett Appl Microbiol 50(1):30–35

    Article  CAS  PubMed  Google Scholar 

  46. Miladi H, Mili D, Slama RB, Zouari S, Ammar E, Bakhrouf A (2016) Antibiofilm formation and anti-adhesive property of three Mediterranean essential oils against a foodborne pathogen Salmonella strain. Microb Pathog 93:22–31

    Article  CAS  PubMed  Google Scholar 

  47. Lewis K (2001) Riddle of biofilm resistance. Antimicrob Agents Chemother 45(4):999–1007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Mah TC, O’Toole GA (2001) Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 9:34–39

    Article  CAS  PubMed  Google Scholar 

  49. Aiassa V, Zoppi A, Becerra MC, Albesa I, Longhi MR (2016) Enhanced inhibition of bacterial biofilm formation and reduced leukocyte toxicity by chloramphenicol: β-cyclodextrin:N-acetylcysteine complex. Carbohyd Polym 152:672–678

    Article  CAS  Google Scholar 

  50. Kot B, Sytykiewicz H, Sprawka L, Witeska M (2020) Effect of Manuka honey on biofilm-associated genes expression during methicillin-resistant Staphylococcus aureus biofilm formation. Sci Rep 10:13552

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by grants from the following foundations: the National Natural Science Foundation of China (No. 31672499), the Modern Agricultural Technology System of Shandong Province (SDAIT-24–05), and the Doctoral Research Foundation of Liaocheng University (No. 318051826).

Author information

Author notes

  1. Fei Wang and Hui Liu contributed equally to this work.

Authors and Affiliations

  1. School of Life Science, Liaocheng University, Liaocheng, 252059, China

    Fei Wang, Hui Liu, Junya Li & Hongzhuan Xuan

  2. Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing, 100093, China

    Wenwen Zhang

  3. College of Food Science and Engineering, Jilin Agricultural University, Changchun, 130118, China

    Bin Jiang

Authors
  1. Fei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hui Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Junya Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wenwen Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bin Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hongzhuan Xuan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Bin Jiang or Hongzhuan Xuan.

Additional information

Responsible Editor: Fernando R. Pavan

Publisher's Note

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, F., Liu, H., Li, J. et al. Australian propolis ethanol extract exerts antibacterial activity against methicillin-resistant Staphylococcus aureus by mechanisms of disrupting cell structure, reversing resistance, and resisting biofilm. Braz J Microbiol 52, 1651–1664 (2021). https://doi.org/10.1007/s42770-021-00547-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s42770-021-00547-7

Keywords

Search

Navigation