PHYTOTHERAPY RESEARCH Phytother. Res.

23, 13671370 (2009) Published online 17MODULATION OF DRUG RESISTANCE IN STAPHYLOCOCCUS AUREUS February 2009 in Wiley InterScience (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. Falco-Silva1, Davi A. Silva2, Maria de Ftima V. Souza2 and Jos P. Siqueira-Junior1*
1

Laboratrio de Gentica de Microrganismos, Departamento de Biologia Molecular, Universidade Federal da Paraba, Joo Pessoa (PB), Brazil 2 Laboratrio de Tecnologia Farmacutica Delby Fernandes de Medeiros, Universidade Federal da Paraba, Joo 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--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 efux protein which extrudes hydrophilic uorquinolones and some biocides, such as benzalkonium chloride, cetrimide, acriavine 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 g/mL), it modulated the activity of antibiotics, i.e. in combination with antibiotics a reduction in the MIC was observed for noroxacin (16-fold), ciprooxacin (16-fold), lomeoxacin (four-fold) and ooxacin (two-fold), and an impressive reduction in the MICs for the biocides (up to 128-fold). The results presented here represent the rst report of a kaempferol glycoside as a putative efux pump inhibitor in bacteria. The present nding 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; efux pump inhibitor.

INTRODUCTION Efux 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 specic 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 efux 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 identied 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 inuenza and fever
* Correspondence to: Jos P. Siqueira-Junior, Caixa Postal 5007 (UFPB), 58051-970 Joo Pessoa (PB), Brazil. E-mail: jpsiq@pq.cnpq.br 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 rst 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 avones (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 pentamethoxyavone isolated from H. tiubae (Silva et al., 2008) has been demonstrated. This work evaluated kaempferol3-O--D-(6-E-p-coumaroyl) glucopyranoside (tiliroside), also isolated from H. tiubae (Silva et al., 2005), for its effect on drug resistance using an efuxing strain of Staphylococcus aureus. For comparison, the phenothiazines chlorpromazine and triuoperazine were used.

MATERIALS AND METHODS Bacteria. The strain of S. aureus used was SA-1199B which overexpresses the norA gene encoding the NorA efux protein which extrudes not only noroxacin and other hydrophilic uorquinolones 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, 13671370 (2009) Revised 17 July 2008 DOI: 10.1002/ptr Accepted 22 July 2008

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V. S. FALCO-SILVA ET AL.

compounds (e.g. benzalkonium chloride and cetrimide) and intercalating dyes (e.g. acriavine and ethidium bromide). The strain, kindly provided by Professor Simon Gibbons (University of London), was maintained in blood agar base (Laboratrios Difco Ltda., Brazil) slants, and prior to use, the cells were grown overnight at 37 C in brain heart infusion broth (BHI Laboratrios Difco Ltda., Brazil). Fluorquinolones, NAB compounds, phenothiazines. The stock solution of the uorquinolones noroxacin, ciprooxacin, lomeoxacin and ooxacin were prepared according to CLSI Guidelines (2005). The stock solution of the NAB compounds benzalkonium chloride, cetrimide, acriavine and ethidium bromide, and of the phenothiazines chlorpromazine and triuoperazine were prepared in distilled water. Ciprooxacin 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 Paraba, Brazil. The stock solution of tiliroside was prepared in DMSO which at its highest nal 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 2560.5 g/mL (two-fold serial dilutions). The MIC is dened 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 g/mL against the strain of S. aureus used (MIC = 256 g/mL). When the compound was incorporated in the growth medium at 64 g/mL (1/4 MIC) or at 32 g/mL (1/8 MIC), a reduction in the MIC of at least two-fold was observed for the uorquinolones, 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 efuxing bacteria. This activity may be related to the lipophilicity of the avon 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 efux proteins, or maybe binding to the pump substrates (Zloh et al., 2004), causing inhibition of drug removal, although other means of efux 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--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 (g/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 g/mL) 4 (16)a 1 (16) 4 (4) 2 (2) 1 (64) 1 (128) <0.125 (8) <0.125 (16) + TIL (32 g/mL) 8 (8) 2 (8) 4 (4) 2 (2) 1 (64) 1 (128) <0.125 (8) <0.125 (16) + CPZ (16 g/mL) 16 (4) 4 (4) 4 (4) 2 (2) 8 (8) 32 (4) <0.125 (8) <0.125 (16) + TFP (8 g/mL) 32 (2) 4 (4) 8 (2) 2 (2) 16 (4) 64 (2) <0.125 (8) <0.125 (16)

TIL, tiliroside; CPZ, chlorpromazine; TFP, triuoperazine; NOR, noroxacin; CIP, ciprooxacin; LOM, lomeoxacin; OFX, ooxacin; ETB, ethidium bromide; ACR, acriavine; BAC, benzalkonium chloride; CET, cetrimide. a (fold reduction in MIC).
Copyright 2009 John Wiley & Sons, Ltd. Phytother. Res. 23, 13671370 (2009) DOI: 10.1002/ptr

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piperidine alkaloid (Gibbons, 2005), acylated oligosaccharides of the orizabin series (Pereda-Miranda et al., 2006) of the murucoidin series and stoloniferin I (Chrigo et al., 2008) and the phenolic diterpene totarol (Smith et al., 2007). However, lespedine, also an amphipathic kaempferol glycoside, was not shown to modulate drug resistance in the efuxing strain SA-1199B (V.S. Falco-Silva, unpublished results). Regarding totarol, it is slightly amphipathic, can dissociate and is also quite lipophilic. The lipophilicity may be a key factor for an inhibitor of an efux pump of Gram-positive bacteria (Stavri et al., 2007), but other factors, such as structural features of the compound, cannot be ruled out. Reductions in MICs of the test compounds when combined with chlorpromazine or triuoperazine were also observed, but tiliroside, even at 1/8 MIC, compared favourably with the phenothiazines. Chlorpromazine (at 25 g/mL) has been shown to inhibit MDR efux systems in S. aureus (Kaatz et al., 2003). The results for noroxacin and ethidium bromide in combination with chlorpromazine against strain SA-1199B were consistent with those reported by Kaatz et al. (2003); therefore chlorpromazine was used as the positive control. Triuoparazine is a well-known inhibitor of the mammalian multidrug resistance (MDR) transporter P-glycoprotein (Beck, 1990) and an inhibitor of yeast MDR transporters (Kolaczkowski et al., 2003), but, as far as is known, this is the rst report of triuoperazine as a putative EPI in bacteria. The lack of signicant activity of tiliroside on the MIC of ooxacin may be related, at least in part, to hydrophobicity. Among the uorquinolones used here, ooxacin is the most hydrophobic (Kaatz et al., 1993; Takenouchi et al., 1996), and as pointed out by Gibbons et al. (2003), hydrophobicity is a factor that may reduce recognition and transport by an efux pump (see also: Piddock et al., 2001). Regarding the impressive results obtained with NAB compounds, it is possible that the effect of tiliroside

on strain SA-1199B is related to an inhibitory effect also on a pump(s) other than NorA for which NAB compounds are a substrate (Oluwatuyi et al., 2004). It should be noted that sequence analysis of the S. aureus genome suggests that more than 30 ORFs encoding putative efux pumps are present in the chromosome of this bacterium, but only a few have been described to date (Yamada et al., 2006; Truong-Bolduc et al., 2006; DeMarco et al., 2007). Several known bacterial efux pump inhibitors (e.g. the alkaloid reserpine) do not full certain requisites of clinical relevance (e.g. serum concentration, toxicity, immunosuppression, and stability and solubility concerns) (Gibbons et al., 2003; Piddock, 2006), and therefore, the search for candidate EPIs among plant derived natural products is warranted. Besides, bacterial EPIs can be very useful tools for the study of efux pumps in eukaryotes (e.g. cancer cells with MDR phenotype), since bacterial and eukaryotic pumps have shown similar pharmacological characteristics, sharing similar inhibitors (van Veen et al., 1998; Gibbons et al., 2003). Some kaempferol glycosides were recently reported as modulators of MDR in human cancer cells (Chung et al., 2007), but the results presented here represent the rst report of a kaempferol glycoside as a putative EPI in bacteria. Along with a previous work from our laboratory (Silva et al., 2008), the present nding indicates that H. tiubae (and broadly Malvaceae) could serve as a source of plant-derived natural products that modulate bacterial multidrug resistance, i.e. a source of a potential adjuvant of antibiotics. Acknowledgements
We are very grateful to Dr Simon Gibbons (University of London) for his valuable and kind cooperation and to Dr L. F. MarquesSantos (UFPb, Brazil) for helpful discussion during the course of this work. We thank Dr A. Leyva for English editing of the manuscript. This work was supported by the following Brazilian agencies: CNPq (PIBIC/UFPb), CAPES and FAPESQ-PB.

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Phytother. Res. 23, 13671370 (2009) DOI: 10.1002/ptr