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

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

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