Download PDF
Planta Med 2007; 73(6): 545-551
DOI: 10.1055/s-2007-967179
Pharmacology
Original Paper
© Georg Thieme Verlag KG Stuttgart · New York

Salvipisone and Aethiopinone from Salvia sclarea Hairy Roots Modulate Staphylococcal Antibiotic Resistance and Express Anti-Biofilm Activity

Elzbieta Walencka1 , Sylwia Rozalska2 , Halina Wysokinska3 , Marek Rozalski4 , Lukasz Kuzma3 , Barbara Rozalska1
  • 1Department of Immunology and Infectious Biology, Institute of Microbiology and Immunology, University of Łodz, Łodz, Poland,
  • 2Department of Industrial Microbiology and Biotechnology, Institute of Microbiology and Immunology, University of Łodz, Łodz, Poland,
  • 3Department of Biology and Pharmaceutical Botany, Medical University of Łodz, Łodz, Poland
  • 4Department of Pharmaceutical Biochemistry, Medical University of Łodz, Łodz, Poland
Further Information

Publication History

Received: October 15, 2006 Revised: March 15, 2007

Accepted: March 18, 2007

Publication Date:
19 April 2007 (online)

    Permissions and Reprints

Abstract

Two diterpenoids, salvipisone (Salv) and aethiopinone (Aeth), isolated from hairy roots of Salvia sclarea, were tested with respect to their activity against methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis (MRSE) strains, cultured as planktonic cells or as adherent biofilms. The standard CLSI method, MTT reduction assay or confocal laser scanning microscopy (CLSM) were used for this purpose and also applied for testing the susceptibility to oxacillin, vancomycin, linezolid and their potential synergy with diterpenoids (evaluated as a fractional inhibitory concentration (FIC) index). Salv and Aeth were shown to be bactericidal or bacteriostatic against S. aureus and S. epidermidis planktonic cultures. Both diterpenoids, at the concentrations of œ MIC, showed synergy with antibiotics representing the β-lactam, glycopeptide and oxazolidinone groups. None of the antibiotics used at a high concentration killed the staphylococcal biofilms. On the contrary, Salv and Aeth decreased the number of live biofilm cells by 45.7 - 77.1 % and slightly reduced the biofilm inhibitory concentration of oxacillin. Diterpenoids also changed the parameters of biofilm morphology, as shown by the CLSM image processing package (PHLIP). It was concluded that salvipisone and aethiopinone (relatively highly lipophilic, log P respectively = 3.4; 4.8) synergized the action of β-lactam antibiotics towards MRSA and MRSE probably by alteration of cell surface hydrophobicity and cell wall/membrane permeability, but not by changing penicillin-binding protein, PBP2a expression and penicillinase production or by direct binding to the cell wall peptidoglycan and teichoic acids.

Abbreviations

Aeth:aethiopinone

ATCC:American Type Culture Collection

CAM:calcein-AM

CLSI:Clinical and Laboratory Standards Institute

CLSM:confocal laser scanning microscopy

CSH:cell surface hydrophobicity

DMSO:dimethyl sulfoxide

FIC:fractional inhibitory concentration

MHB:Mueller-Hinton broth

MIC:minimal inhibitory concentration

MRSA/MSSA:methicillin resistant/susceptible Staphylococcus aureus

MRSE/MSSE:methicillin resistant/susceptible S. epidermidis

MTT:3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

OD:optical density

PBP:penicillin binding protein

PG:peptidoglycan

PI:propidium iodide

Salv:salvipisone

SAT:salt aggregation test

TA:teichoic acids

TSB:tryptic soy broth

Key words

Salvia sclarea - Lamiaceae - hairy roots - salvipisone - aethiopinone - biofilm - Staphylococcus

References

  • 1 Lewis K. Riddle of biofilm resistance.  Antimicrob Agents Chemother. 2001;  45 999-1007.
    CrossrefPubMedGoogle Scholar
  • 2 Schierholz J M, Beuth J, Pulverer G. Adherent bacteria and activity of antibiotics.  J Antimicrob Chemother. 1999;  43 158-60.
    CrossrefPubMedGoogle Scholar
  • 3 Hall-Stoodley L, Costerton J W, Stoodley P. Bacterial biofilms: from the natural environment to infectious diseases.  Nat Rev Microbiol. 2004;  2 95-106.
    CrossrefPubMedGoogle Scholar
  • 4 Goetz F. Staphylococcus and biofilms.  Mol Microbiol. 2002;  43 1367-78.
    CrossrefPubMedGoogle Scholar
  • 5 Nascimento G GF, Locatelli J, Freitas P C, Silva G L. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria.  Braz J Microbiol. 2000;  31 1-13.
    PubMedGoogle Scholar
  • 6 Cowan M M. Plant products as antimicrobial agents.  Clin Microbiol Rev. 1999;  12 564-82.
    PubMedGoogle Scholar
  • 7 Jabra-Rizk M A, Meiller T F, James C E, Shirtliff M E. Effect of farnesol on Staphylococcus aureus biofilm formation and antimicrobial susceptibility.  Antimicrob Agents Chemother. 2006;  50 1463-9.
    CrossrefPubMedGoogle Scholar
  • 8 Giang P M, Son P T, Matsunami K, Otsuka H. Anti-staphylococcal activity of ent-kaurane-type diterpenoids from Croton tonkiensis .  J Nat Med. 2006;  60 93-5.
    CrossrefPubMedGoogle Scholar
  • 9 Chen X, Ding J, Ye Y M, Zhang J S. Bioactive abietane and seco-abietane diterpenoids from Salvia prionitis .  J Nat Prod. 2002;  65 1016-20.
    CrossrefPubMedGoogle Scholar
  • 10 Tatsimo S JN, Tane P, Srinivas P V, Sondengam B L, Melisa J, Okunij C O. et al . Novel antimicrobial diterpenoids from Turraeanthus africanus .  Planta Med. 2005;  71 1145-51.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 11 Ulubelen A. Chemical constituents. Terpenoids in the genus Salvia. In: Kintzios SE, editor. Sage. The genus Salvia .  Nuttall: Harwood Academic. Publisher;  2000 55-68.
    PubMedGoogle Scholar
  • 12 Kuzma L, Skrzypek Z, Wysokinska H. Diterpenoids and triterpenoids in hairy roots of Salvia sclarea .  Plant Cell Tiss Org Cult. 2006;  84 152-60.
    PubMedGoogle Scholar
  • 13 Kuzma L, Rozalski M, Walencka E, Rozalska B, Wysokinska H. Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L.: Salvipisone as a potential anti-biofilm agent active against antibiotic resistant staphylococci.  Phytomedicine. 2007;  14 31-5.
    CrossrefPubMedGoogle Scholar
  • 14 European committee for antimicrobial susceptibility testing ( Eucast). Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents.  Clin Microbiol Infect. 2000;  6 503-8.
    CrossrefPubMedGoogle Scholar
  • 15 Walencka E, Sadowska B, Rozalska S, Hryniewicz W, Rozalska B. Lysostaphin as a potential therapeutic agent for staphylococcal biofilm eradication.  Pol J Microbiol. 2005;  54 191-200.
    PubMedGoogle Scholar
  • 16 Mueller L N, de Brouwer J FC, Almeida J S, Stal L J, Xavier J B. Analysis of a marine phototrophic biofilm by confocal laser scanning microscopy using the new image quantification software PHLIP.  MMC Ecol. 2006;  6 1-15.
    PubMedGoogle Scholar
  • 17 Aqil F, Khan M SA, Owais M, Ahmad I. Effect of certain bioactive plant extracts on clinical isolates of β-lactamase producing methicillin resistant Staphylococcus aureus .  J Basic Microbiol. 2005;  45 106-14.
    CrossrefPubMedGoogle Scholar
  • 18 Gibbons S. Plants as a source of bacterial resistance modulators and anti-infective agents.  Phytochem Rev. 2005;  4 63-78.
    CrossrefPubMedGoogle Scholar
  • 19 Fujita M, Shiota S, Kuroda T, Hatano T, Yoshida T, Mizushima T. et al . Remarkable synergies between baicalein and tetracycline, and baicalein and β-lactams against methicillin-resistant Staphylococcus aureus .  Microbiol Immunol. 2005;  49 391-6.
    PubMedGoogle Scholar
  • 20 Kondo K, Takaishi Y, Shibata H, Higuti T. ILSMRs (intensifier of β-lactam-susceptibility in methicillin-resistant Staphylococcus aureus) from Tara [Caesalpinia spinosa (Molina) Kuntze].  Phytomedicine. 2006;  13 209-12.
    CrossrefPubMedGoogle Scholar
  • 21 El-Azizi M, Rao S, Kanchanapoom T, Khardori N. In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci.  Ann Clin Microbiol Antimicrob. 2005;  4 1-9.
    CrossrefPubMedGoogle Scholar
  • 22 Moujir L, Gutiérrez-Navarro A M, San Andrés L, Luis J G. Structure-antimicrobial activity relationships of abietane diterpenes from Salvia species.  Phytochemistry. 1993;  34 1493-5.
    CrossrefPubMedGoogle Scholar
  • 23 Yang Z, Yoshikazu K, Kazuhiro C, Naohiro S, Hiroshi K, Yohei D. et al . Synthesis of variously oxidized abietane diterpenes and their antibacterial activities against MRSA and VRE.  Bioorg Med Chem. 2001;  9 347-56.
    CrossrefPubMedGoogle Scholar
  • 24 Doyle R J. Contribution of the hydrophobic effect to microbial infection.  Microbes Infect. 2000;  2 391-400.
    CrossrefPubMedGoogle Scholar
  • 25 Mannhold R, van de Waterbeemd H. Substructure and whole molecule approaches for calculating log P.  J Comput Aided Mol Des. 2001;  15 337-54.
    CrossrefPubMedGoogle Scholar
  • 26 Shibata H, Shirakata C, Kawasaki H, Sato Y, Kuwahara T, Ohnishi Y. et al . Flavone markedly affects phenotypic expression of β-lactam resistance in methicillin-resistant Staphylococcus aureus strains isolated clinically.  Biol Pharm Bull. 2003;  26 1478-83.
    CrossrefPubMedGoogle Scholar
  • 27 Zhao W -H, Hu Z -Q, Okubo S, Hara Y, Shimamura T. Mechanizm of synergy between epigallcatechin gallate and of β-lactams against methicillin-resistant Staphylococcus aureus .  Antimicrob Agents Chemother. 2001;  45 1737-42.
    CrossrefPubMedGoogle Scholar
  • 28 Nicolson K, Evans G O, Toole P W. Potentiation of methicillin activity against methicillin-resistant Staphylococcus aureus by diterpenes.  FEMS Microbiol Lett. 1999;  179 233-9.
    CrossrefPubMedGoogle Scholar
  • 29 Stermitz F R, Beeson T D, Mueller P J, Hsiang J -F, Lewis K. Staphylococcus aureus MDR efflux pump inhibitors from a Berberis and a Mahonia (sensu stricto) species.  Biochem Syst Ecol. 2001;  29 793-8.
    PubMedGoogle Scholar
  • 30 Kaatz G W, Moudgal V V, Seo S M, Kristiansen J E. Phenothiazines and thioxanthenes inhibit multidrug efflux pump activity in Staphylococcus aureus .  Antimicrob Agents Chemother. 2003;  47 719-26.
    CrossrefPubMedGoogle Scholar

Prof. Dr. hab. Barbara Rozalska

Department of Immunology and Infectious Biology

Institute of Microbiology and Immunology

University of Łodz

Banacha 12/16

90-237 Łodz

Poland

Phone: +48-42-635-4472

Fax: +48-42-665-5818

Email: rozab@biol.uni.lodz.pl

      >