MECHANISM OF ANTIMICROBIAL ACTION OF CARNOSIC ACID: INHIBITION OF EFFLUX PUMPS IN ENTEROCOCCUS FAECALIS AND STAPHYLOCOCCUS AUREUS

OJEDA SANA A.M; MORENO S

Mar del PLATA

Congreso; VIII CONGRESO DE MICROBIOLOGÍA GENERAL; 2012

Sociedad Argentina de Microbiología General

There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal and plant origins. Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria as stress responses, virulence and cell communication and development of efflux pump inhibitors, therefore the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria. Over the past four decades, there were only a few novel classes of antibacterials developed. Thus, development of novel antibacterial drugs has been challenged by the rapid emergence of bacterial resistance. Given the clinical significance of drug efflux pumps in pathogenic bacteria, exploration of efflux pump inhibitor has been under way. Natural compounds as carnosic acid are able to potentiate the antimicrobial activity of several common antibiotics used to combat infections. In particular, carnosic acid potentiates the antimicrobial activity of several antibiotics in Enterococcus faecalis and Staphylococcus aureus, although the underlying molecular mechanism of its action remains unclear. In this work the effect of carnosic acid on the efflux and uptake of drugs in comparison with the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone, the ATPase inhibitor vanadate and a general inhibitor of bacterial efflux pumps verapamil, were evaluated in E. faecalis and S. aureus. Fluorescent probes as SYTOX Green and 3??,3??-dipropylthiadicarbocyanine [DiSC3(5)] were assayed to investigate the effect of carnosic acid on membrane permeability and membrane potential. Results showed that carnosic acid is an effective modulator of the ethidium bromide uptake and efflux in E. faecalis, without significant effect on the cell membrane permeability. In addition, the proton uncoupler inhibitor, the effect of carnosic acid on the accumulation rates of ethidium bromide was blocked, while a notably positive effect on EtBr uptake was observed in the presence of the diterpene and the well-known inhibitor of efflux pumps dependent of ATP, vanadate. Moreover, results showed that carnosic acid modified the cell membrane potential gradient in E. faecalis and S. aureus. All these findings suggested the existence of an active efflux pump inhibition activity of carnosic acid, which is independent of the activity of ATPases but relies on active efflux of secondary transporters energized by trans-membrane electrochemical depending on the membrane potential. Other experiments showed slight toxicological effects of carnosic acid on eukaryotic cells. In conclusion, all finding supported a high potential use in the clinic of carnosic acid as a resistance-modulating agent for the treatment of some drug-resistant infections. These encouraging possibilities are now under investigation in our laboratory testing carnosic acid against different multidrug-resistant clinical isolates.