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23, 1367–1370 (2009) Published online 17MODULATION OF DRUG RESISTANCE IN STAPHYLOCOCCUS AUREUS February 2009 in Wiley InterScience ( DOI: 10.1002/ptr.2695


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*

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: 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 2008 Phytother. Res. 23, 1367–1370 (2009) Revised 17 July 2008 DOI: 10.1002/ptr Accepted 22 July 2008

Brazil. along with an impressive reduction in the MICs for the NAB compounds (Table 1). 2004).. and this quality. NAB compounds. Lipophilicity is a common feature of several putative EPIs. causing inhibition of drug removal. Phytother. Copyright © 2009 John Wiley & Sons. 2007) cannot be ruled out. ciprofloxacin. FALCÃO-SILVA ET AL. i. ofloxacin.. 1) modulated the activities of the drugs by reducing the concentration needed to inhibit the growth of the effluxing bacteria.A. ciprofloxacin. The stock solution of the NAB compounds benzalkonium chloride. Table 1. All experiments were carried out at least twice with consistent results. For the evaluation of tiliroside as a modulator of drug resistance. (2005) and a voucher specimen (no. DISCUSSION The amphipathic kaempferol glycoside tiliroside (Fig. The strain. Fluorquinolones. Other amphipathic compounds have been reported as putative EPIs against strain SA-1199B.125 (≥8) <0.g. 23. NOR. benzalkonium chloride.. NAB compounds. LOM. lomefloxacin. RESULTS Tiliroside showed no antibacterial activity at 128 μg/mL against the strain of S. CIP. Ltd. The minimum inhibitory concentrations (MICs) of the antibiotics. 105 cfu/mL and a drug concentration range of 256–0.1368 V. was maintained in blood agar base (Laboratórios Difco Ltda. the cells were grown overnight at 37 °C in brain heart infusion broth (BHI – Laboratórios Difco Ltda.1002/ptr . ethidium bromide. phenothiazines and tiliroside were determined in BHI by the microdilution assay using a suspension of ca. acriflavine and ethidium bromide). ACR.. tiliroside.. lomefloxacin and ofloxacin were prepared according to CLSI Guidelines (2005). a reduction in the MIC of at least two-fold was observed for the fluorquinolones. CET.5 μg/mL (two-fold serial dilutions). BAC. TFP.g. CPZ.125 (≥8) <0. Res. OFX. and of the phenothiazines chlorpromazine and trifluoperazine were prepared in distilled water. 2434) was placed in the Herbarium ‘Lauro Pires Xavier-JPB’ at the Universidade Federal da Paraíba. Ciprofloxacin was from Bayer S.125 (≥16) + CPZ (16 μg/mL) – – – 16 (4) 4 (4) 4 (4) 2 (2) 8 (8) 32 (4) <0. such as a Figure 1.125 (≥8) <0. 2007).125 (≥16) + TIL (32 μg/mL) – – – 8 (8) 2 (8) 4 (4) 2 (2) 1 (64) 1 (128) <0. benzalkonium chloride and cetrimide) and intercalating dyes (e. although other means of efflux pump inhibition such as an effect on transcription/translation of the pump (Smith et al.125 (≥16) TIL. compounds (e. Brazil) slants. tiubae as previously described by Silva et al. the MICs of the antibiotics and NAB compounds were determined in the presence of the tiliroside at a sub-inhibitory concentration. Drug susceptibility testing and modulation assay. The MIC is defined as the lowest concentration at which no growth is observed. 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) Alone TIL CPZ TFP NOR CIP LOM OFX ETB ACR BAC CET 256 64 32 64 16 16 4 64 128 1 2 + TIL (64 μg/mL) – – – 4 (16)a 1 (16) 4 (4) 2 (2) 1 (64) 1 (128) <0. phenothiazines. acriflavine and ethidium bromide. ETB. kindly provided by Professor Simon Gibbons (University of London).. 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. is probably important for its solubility in the bacterial membrane and binding to the efflux proteins. USA. The stock solution of the fluorquinolones norfloxacin. Structure of kaempferol-3-O-β-D-(6″-E-p-coumaroyl) glucopyranoside (tiliroside). as pointed out by Gibbons (2004).125 (≥8) <0. a (fold reduction in MIC). trifluoperazine.125 (≥16) + TFP (8 μg/mL) – – – 32 (2) 4 (4) 8 (2) 2 (2) 16 (4) 64 (2) <0. acriflavine. norfloxacin..e. This activity may be related to the lipophilicity of the flavon moiety of tiliroside. 1367–1370 (2009) DOI: 10. a method that has been widely applied to identify potential EPIs (Stavri et al. Tiliroside. cetrimide. The phenothiazines were also used at a sub-inhibitory concentration. the ‘modulation assay’ was used. or maybe binding to the pump substrates (Zloh et al. 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). Brazil). chlorpromazine. S. Brazil and all the other drugs were from Sigma Chemical Co. Tiliroside was obtained from the aerial parts of H. cetrimide. and prior to use.

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