In vitro evaluation of the synergetic interaction between antibiotics tetracyclin and penicillin with (+)-usnic acid isolated from Usnea steineri against multiresistant bacteria

In recent decades, many antibiotics have shown little effectiveness in combating infectious diseases due to the high incidence of infections caused by multiresistant bacteria, being estimated that only in 2007 approximately 25 000 patients died from infection due the antibiotic-resistant bacteria in the European Union, Iceland and Norway [1]. Evaluations of synergism between antimicrobial agents have been used as a new strategy against the advances of antimicrobial resistance, where these combinations have allowed the development of new antimicrobial agents [2]. Recently, our group reported a strong antimycobacterial activity of (+)-usnic acid, isolated from the lichen Usnea steineri [3]. The aim of this study was to evaluate the in vitro interaction of (+)-usnic acid in combination with tetracycline and penicillin antibiotics against four clinically isolated multiresistant bacteria, in view of the good results previously obtained in the determination of minimum inhibitory concentration (MIC). The assays were performed according to the checkerboard protocol described by White et al. [4]. The following formulas were used to calculate the fractional inhibitory concentration FIC index: FIC of usnic acid = MIC of usnic acid in combination/MIC of usnic acid alone, FIC of antibiotic = MIC of antibiotic in combination/MIC of antibiotic alone, and FIC index = FIC of usnic acid + FIC of antibiotic, where a synergistic effect was defined as an FIC index ≤0.5, indifference as an FIC index > 0.5 but ≤4, and antagonism effect as an FIC index > 4 [4]. The results of the evaluation showed in Table 1 indicated that combinations of penicillin and tetracycline with (+)-usnic acid did not show synergistic properties, but antagonistic against Staphylococcus haemolyticus. In summary, our results showed that regarding the respective MIC, usnic acid is not really promising.

Tab. 1: FIG and FICI of usnic acid in combination with the antibiotics penicillin G and tetracydine.

Strains	Agent	MICa		FIGb	FICIc	Outcome
		Alone	Combination
Staphylococcus haemolyticus (243 HCRPd)	(+)-Usnic acid	25	12.5	0.5	4.5	Antagonism
	Penicillin G	625	2500	4.0
Staphylococcus aureus (220 HCRPd)	(+)-Usnic acid	50	12.5	0.25	2.25	Indifference
	Penicillin G	312.5	625	2
Staphylococcus epidermidis (218 HCRPd)	M-Usnic acid	6.25	3.12	0.5	1.5	Indifference
	Penicillin G	312.5	312.5	1
Enterococcus faecalis (216 HCRPd)	(+)-Usnic acid	6.25	6.25	1	2.0	Indifference
	Tetracycline	19.5	19.5	1
aMIC (µmg ml-1), bThe fractional inhibitory concentration (FIC), cThe fractional inhibitory concentration index (FICI), dHospital das Clinicas de Ribeirão Preto(HCRP)

Acknowledgements: FAPESP (grants # 2012/22636 – 1, 2012/1618208), CAPES and CNPq.

Keywords: usnic acid, Usnea steineri, multiresistant bacteria.

References:

1. ECDC/EMEA. The bacterial challenge: time to react. http://www.ecdc.europa.eu/en/publications/Publications/0909_TER_The_Bacterial_Challenge_Time_to_React.pdf, September 2009; 1 – 42.

2. Vuuren S van, Viljoen A. Plant-Based Antimicrobial Studies – Methods and Approaches to Study the Interaction between Natural Products. Planta Med. 2011; 77: 1168 – 1282.

3. Lucarini R, Tozatti MG, Salloum AI, Crotti AE, Silva ML, Gimenez VM, Groppo M, Januário AH, Martins CHG, Cunha WR. Antimycobacterial activity of Usnea steineri and its major constituent (+)-usnic acid. Afr. J. Biotechnol. 2012; 11: 4636 – 4639.

4. White RL, Burgess DS, Manduru M, Bosso JA. Comparison of three different in vitro methods of detecting synergy: timekill, checkerboard, and E test. Antimicrob. Agents Chemother. 1996; 40: 1914 – 1918.