PHYTOTHERAPY RESEARCH Phytother. Res. 23, 1367–1370 (2009) Published online 17MODULATION February 2009 inOF Wiley InterScience DRUG RESISTANCE IN STAPHYLOCOCCUS AUREUS (www.interscience.wiley.com) DOI: 10.1002/ptr.2695
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Modulation of Drug Resistance in Staphylococcus aureus by a Kaempferol Glycoside from Herissantia tiubae (Malvaceae) Vivyanne S. Falcão-Silva1, Davi A. Silva2, Maria de Fátima V. Souza2 and José P. Siqueira-Junior1* 1
Laboratório de Genética de Microrganismos, Departamento de Biologia Molecular, Universidade Federal da Paraíba, João Pessoa (PB), Brazil 2 Laboratório de Tecnologia Farmacêutica ‘Delby Fernandes de Medeiros’, Universidade Federal da Paraíba, João Pessoa (PB), Brazil
In an ongoing project to evaluate natural compounds isolated from plants from the Brazilian biodiversity as modulators of antibiotic resistance, kaempferol-3-O-β-d-(6″″-E-p-coumaroyl) glucopyranoside (tiliroside), isolated from Herissantia tiubae (Malvaceae) was investigated using the strain SA-1199B of Staphylococcus aureus, which overexpresses the norA gene encoding the NorA efflux protein which extrudes hydrophilic fluorquinolones and some biocides, such as benzalkonium chloride, cetrimide, acriflavine and ethidium bromide. The minimum inhibitory concentrations (MICs) of the antibiotics and biocides were determined by the microdilution assay in the absence and in the presence of sub-inhibitory concentration of tiliroside. Although tiliroside did not display relevant antibacterial activity (MIC = 256 μg/mL), it modulated the activity of antibiotics, i.e. in combination with antibiotics a reduction in the MIC was observed for norfloxacin (16-fold), ciprofloxacin (16-fold), lomefloxacin (four-fold) and ofloxacin (two-fold), and an impressive reduction in the MICs for the biocides (up to 128-fold). The results presented here represent the first report of a kaempferol glycoside as a putative efflux pump inhibitor in bacteria. The present finding indicates that H. tiubae (and broadly Malvaceae) could serve as a source of plant-derived natural products that modulate bacterial resistance, i.e. a source of potential adjuvants of antibiotics. Copyright © 2009 John Wiley & Sons, Ltd. Keywords: tiliroside; kaempferol glycoside; Herissantia tiubae (Malvaceae); modulation of drug resistance; Staphylococcus aureus; efflux pump inhibitor.
INTRODUCTION Efflux pumps are integral proteins of bacterial membranes accounting for much of the bacterial resistance, since they extrude antibiotics and other antimicrobial agents from the cell (Piddock, 2006). Some of these pumps are specific for a given compound or class of compounds, whereas others may transport or are capable of removing a variety of structurally unrelated antimicrobial compounds. Resistance modifying agents/modulators of drugresistance are compounds that potentiate the activity of an antibiotic against resistant strains, and some of these agents may act as inhibitors of efflux pumps (EPIs), as in the case of phenothiazines and other synthetic compounds (Kaatz et al., 2003; Marquez, 2005). Plants provide a rich source of EPIs and several compounds have been identified as potent inhibitors (Gibbons, 2004, 2005; Stavri et al., 2007). The small shrub Herissantia tiubae (K. Shum) Brizicky (Malvaceae) is a native plant of Northeast Brazil where it is popularly known as ‘mela-bode’ or ‘lava-prato’ and is used in folk medicine against influenza and fever * Correspondence to: José P. Siqueira-Junior, Caixa Postal 5007 (UFPB), 58051-970 João Pessoa (PB), Brazil. E-mail:
[email protected] Contract/grant sponsor: CNPq (PIBIC/UFPb); CAPES; FAPESQ-PB. Copyright © 2009 John Wiley & Sons, Ltd. Copyright © 2009 John Wiley & Sons, Ltd.
(Albuquerque et al., 2007). The first phytochemical investigations of H. tiubae resulted in the isolation of several classes of compounds, including kaempferol glycosides (Silva et al., 2004), triterpenes (Silva et al., 2008) and polyoxygenated/polymethoxylated flavones (Silva et al., 2005, 2009). In an ongoing project to evaluate natural compounds isolated from plants of the Brazilian biodiversity, mainly of the Malvaceae family, as modulators of antibiotic resistance, the modulatory activity of a pentamethoxyflavone isolated from H. tiubae (Silva et al., 2008) has been demonstrated. This work evaluated kaempferol3-O-β-D-(6″-E-p-coumaroyl) glucopyranoside (tiliroside), also isolated from H. tiubae (Silva et al., 2005), for its effect on drug resistance using an effluxing strain of Staphylococcus aureus. For comparison, the phenothiazines chlorpromazine and trifluoperazine were used.
MATERIALS AND METHODS Bacteria. The strain of S. aureus used was SA-1199B which overexpresses the norA gene encoding the NorA efflux protein which extrudes not only norfloxacin and other hydrophilic fluorquinolones but also biocides (Kaatz et al., 1993; Kaatz and Seo, 1995), including those referred to as nucleic-acid binding (NAB) compounds (Enslie et al., 1995), such as quaternary ammonium Received 22 April(2009) 2008 Phytother. Res. 23, 1367–1370 Revised July 2008 DOI:1710.1002/ptr Accepted 22 July 2008
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compounds (e.g. benzalkonium chloride and cetrimide) and intercalating dyes (e.g. acriflavine and ethidium bromide). The strain, kindly provided by Professor Simon Gibbons (University of London), was maintained in blood agar base (Laboratórios Difco Ltda., Brazil) slants, and prior to use, the cells were grown overnight at 37 °C in brain heart infusion broth (BHI – Laboratórios Difco Ltda., Brazil). Fluorquinolones, NAB compounds, phenothiazines. The stock solution of the fluorquinolones norfloxacin, ciprofloxacin, lomefloxacin and ofloxacin were prepared according to CLSI Guidelines (2005). The stock solution of the NAB compounds benzalkonium chloride, cetrimide, acriflavine and ethidium bromide, and of the phenothiazines chlorpromazine and trifluoperazine were prepared in distilled water. Ciprofloxacin was from Bayer S.A., Brazil and all the other drugs were from Sigma Chemical Co., USA. Tiliroside. Tiliroside was obtained from the aerial parts of H. tiubae as previously described by Silva et al. (2005) and a voucher specimen (no. 2434) was placed in the Herbarium ‘Lauro Pires Xavier-JPB’ at the Universidade Federal da Paraíba, Brazil. The stock solution of tiliroside was prepared in DMSO which at its highest final concentration after dilution in the broth (4%) caused no inhibition of bacterial growth. Drug susceptibility testing and modulation assay. The minimum inhibitory concentrations (MICs) of the antibiotics, NAB compounds, phenothiazines and tiliroside were determined in BHI by the microdilution assay using a suspension of ca. 105 cfu/mL and a drug concentration range of 256–0.5 μg/mL (two-fold serial dilutions). The MIC is defined as the lowest concentration at which no growth is observed. For the evaluation of tiliroside as a modulator of drug resistance, the ‘modulation assay’ was used, a method that has been widely applied to identify potential EPIs (Stavri et al., 2007), i.e. the MICs of the antibiotics and NAB compounds were determined in the presence of the tiliroside at a sub-inhibitory concentration. The phenothiazines were also used at a sub-inhibitory concentration.
RESULTS Tiliroside showed no antibacterial activity at 128 μg/mL against the strain of S. aureus used (MIC = 256 μg/mL). When the compound was incorporated in the growth medium at 64 μg/mL (1/4 MIC) or at 32 μg/mL (1/8 MIC), a reduction in the MIC of at least two-fold was observed for the fluorquinolones, along with an impressive reduction in the MICs for the NAB compounds (Table 1). All experiments were carried out at least twice with consistent results.
DISCUSSION The amphipathic kaempferol glycoside tiliroside (Fig. 1) modulated the activities of the drugs by reducing the concentration needed to inhibit the growth of the effluxing bacteria. This activity may be related to the lipophilicity of the flavon moiety of tiliroside. Lipophilicity is a common feature of several putative EPIs, and this quality, as pointed out by Gibbons (2004), is probably important for its solubility in the bacterial membrane and binding to the efflux proteins, or maybe binding to the pump substrates (Zloh et al., 2004), causing inhibition of drug removal, although other means of efflux pump inhibition such as an effect on transcription/translation of the pump (Smith et al., 2007) cannot be ruled out. Other amphipathic compounds have been reported as putative EPIs against strain SA-1199B, such as a
Figure 1. Structure of kaempferol-3-O-β-D-(6″-E-p-coumaroyl) glucopyranoside (tiliroside).
Table 1. Minimum inhibitory concentrations (MICs) of antibiotics and biocides in the absence and presence of tiliroside or phenothiazines against Staphylococcus aureus strain SA-1199B MIC (μg/mL)
TIL CPZ TFP NOR CIP LOM OFX ETB ACR BAC CET
Alone
+ TIL (64 μg/mL)
+ TIL (32 μg/mL)
+ CPZ (16 μg/mL)
+ TFP (8 μg/mL)
256 64 32 64 16 16 4 64 128 1 2
– – – 4 (16)a 1 (16) 4 (4) 2 (2) 1 (64) 1 (128)
