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Inhibition of development, swarming differentiation and virulence factors in Proteus mirabilis by an extract of Lithrea molleoides and its active principle (Z,Z)-5-(trideca-4',7'-dienyl)-resorcinol.

doi: 10.1016/j.phymed.201 1.03.003

ABSTRACT

Antibacterial activity of Lithrea moileoides extract against Proteus mirabilis has been previously reported by our group. In the present study, the compound (Z, Z)-5-(trideca-4', 7'-dienyI)-resorcinol (1) was isolated dts its responsible active principle. The effects of the compound obtained and of L moileoides extract on P. mirabilis growth and virulence factors were evaluated.

Compound 1 showed MIC and MBC values of 4000 fxg/ml. It was found that the extract, at four times the MIC, produced complete killing of the uropathogen at 2 h from the beginning of the experiment, while the alkylresorcinol, at four times the MIC, produced the same effect after 24 h. Hemolysis was adversely affected in treatments with both products at 8 [micro]g/ml, while hemagglutination was not altered. The whole extract induced complete autoaggregation of P. mirabilis at 2000 [micro]g/ml, while compound 1 at the same concentration did not show this property. Swarming motility was delayed in treatments with the extract and withl at 1000 and 8 [micro]g/ml, respectively, at 8 h from the beginning of the assay. Complete inhibition of the phenomenon was still observed after 24 h when compound 1 was added at 125 [micro]g/ml.

These findings offer the possibility of new classes of antimicrobial medicines to tackle infections caused by P. mirabilis.

[c] 2011 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Antibacterial compounds Lithrea moileoides (Z,Z)-5-(trideca-4', 7'-dienyl)-resordnol Proteus mirabilis

Introduction

Proteus mirabilis is the second most common cause of urinary tract infections (UTIs), especially in individuals with complicated urinary tracts and catheterized patients (Coker et al. 2000). Its virulence, which is crucial for successful colonization and the establishment of the infection, has been related to several potential factors including hemolysin production, adherence to the uroep-ithelium due to fimbriae and swarming motility mediated by flagella(Rozalskyetal. 1997).

Like many other microorganisms, P. mirabilis has acquired resistance to many antibiotics including [beta]-lactams (Aragon et al. 2008), aminoglycosides (Wachino et al. 2006) and fluoroquinolones (Saito et al. 2006) with a progressive increase in the number of resistant isolates over recent years. In this context, and continuing the search for bioactive compounds of plant-origin (Carpinella et al. 2005; Palacios et al. 2007), we have recently reported the antibacterial activity of ethanoiic extracts derived from 51 native plants of central Argentina (Joray et al. 2011). From this screening, Lithrea molleoides (Veil.) Engl. (Anacardiaceae) extract showed itself to be one of the most effective with notable bacteriostatic and bactericide activity against gram-positive and negative bacteria. Its effect on P. mirabilis showed MIC and MBC values of 2000 and 8000 [micro]g/ml, respectively (Jorayetal. 2011). No active principle had been isolated as responsible for this effect.

The goal of this study was to isolate and identify the active ingredient able to interfere with the growth, virulence factors and swarming motility of P. mirabilis.

Materials and methods

Plant material

L molleoides was collected in the Province of Cordoba, Argentina in February-March 2008. A voucher specimen has been deposited in the "Marcelino Sayago" Herbarium of the School of Agricultural Science, UCC: UCCOR 183. Plant material was extracted by 48 h maceration with ethanol. Yield of the extract, expressed as percentage weight of air-dried plant material, was 10.9%.

Bacterial strain

The uropathogenic tetracycline, erythromycin and polymyxintt-resistant P. mirabilis strain Pr2921 was originally isolated from a patient with symptomatic UTIs (Zunino et al. 2003, 2007). Bacterial suspensions were prepared on PBS (0.1 ML pH 7.2) from an overnight-grown organism in Nutrient Agar (NA) at 37 [degrees]C. Turbidity was spectrophotometrically adjusted to 0.5 McFarland standard.

Media, chemicals and apparatus

All media were from Difco Laboratories (Detroit, Ml, USA) and Oxoid Ltd. (Basingstoke, Hampshire, UK). Chemicals were purchased from Sigma-Aldrich Corporation (St Louis, MO). Silica gel grade 70-230 mesh, 60 A, for column chromatography was used and all solvents were HPLC grade. 1H and 13C NMR spectra were recorded in Chloroform-dwithBrukerAVANCE II400 spectrometer (Bruker Corporation, Ettlingen, Germany) operated at 400 MHz for 1H and at 100 MHz for the 13C nucleus. MS spectra were measured with a ZAB SEQ. (BeqQ) instrument (VG Analytical, Manchester). HPLC was performed on a Phenomenex Prodigy 5 [micro]ODS (4.6 mm i.d. x 250 mm) reversed-phase column eluting with 90% acetoni-trile in water with 1% trifluoracetic acid (TFA) and UV detection at 280 nm. For electron microscopy, a Jeol JEM 1010 electron microscope was used. Light microscopy was performed in an Olympus BX61 equipped with a graticule.

Determination of MIC and MBC

MIC and MBC assays were carried out as previously described (Joray et al. 2011). Positive controls with gentamicin sulfate (potency: 550-590 [micro]g/mg; Montreal, SA) and erythromycin (potency: 863 [micro]g/mg; Unifarma) were simultaneously carried out.

Bioguided isolation of the antibacterial compound

L molleoides extract was subjected to a column chromatography with hexane/Et20/MeOH gradient. Antibacterial fractions were re-chromatographed to finally obtain an active fraction which was further purified in radial preparative chromatography (solvent gradient C[H.sub.2][C.sub.12]/[Et.sub.2]O) furnishing a yellowish oil (yield 0.78g/100gof crushed plant material, 92% purity, by HPLC). This substance was identified as (Z, Z)-5-(trideca-4', 7'-dienyl)-resorcinol (1) according to (1)H NMR, (13)C NMR and MS spectra, and to the comparison with previous reports (Chiari et al. 2010; Valcic et al. 2002).

Time kill curves

These assays were conducted in Luria Bertoni broth (LB) containing tested products according to Joray et al. (2011). An aliquot of the P. mirabilis suspension was added to each tube to finally reach 5 x [10.sup.5] CFU/ml. Each tube was incubated at 37 [degrees] C with agitation.

Electron microscopy

For TEM, fresh cultures of bacteria grown for 24 h in the absence or presence of 8000 [micro]g/ml of extract or compound 1 were stained with 1% uranyl acetate.

Hemagglutination and hemolysin inhibition assay

The inhibition on hemagglutination and hemolysis was determined according to Mobley and Chippendale (1990). Controls of bacterial growth with or without ethanol were run simultaneously. Negative controls with no addition of P. mirabilis were also performed. For the hemolysin inhibition assay, L molleoides extract or compound 1 (previously dissolved in ethanol) was added to brain-heart-infusion broth, then the adjusted inoculum of the test organism was added to finally obtain 1 x [10.sup.7] CFU/ml.

For hemagglutination, LB containing the appropriate amounts of extract or 1 dissolved in ethanol was inoculated with Pr2921 inoculum reaching a final concentration of 1 x [10.sup.7] CFU/ml.

[FIGURE 1 OMITTED]

Autoaggregation assay

An assay in order to determine bacterial settling kinetics over time was carried out according to Sosa and Zunino (2009).

Inhibition of swarming differentiation

The effect of the extract or 1 on swarming behavior was determined as described by Liaw et al. (2000).

Statistical analysis

The results were analyzed statistically by the Kruskal-Wallis test and Dunn's post test at 0.05 significance level using InfoStat software (Crupo InfoStat, FCA, Universidad Nacional de Cordoba, Cordoba, Argentina).

Results and discussion

After submitting complete L molleoides extract to antibacterial bioguided isolation, one active compound identified as (Z, Z)-5-(trideca-4, 7-dienyl)-resorcinol (1) (Fig. 1) was obtained. The chromatographic fingerprints of the extract and of the alkyl-resorcinol are shown in Fig. 2. This is the first time that a compound showing antibacterial properties has been isolated from L molleoides.

The inhibitory effect of 1 on P. mirabilis was determined, showing a MIC and MBC of 4000^ig/rnl. Gentamicin and erythromycin showed MIC and MBC values of 10 and 10, and 500 and > 4000 [micro]g/ml, respectively. The results obtained agree with previous data where other 5-alkylresorcinols are described as possessing antibacterial properties (Zarnowski and Suzuki 2004; Jin and Zjawiony 2006).

In time kill assays, the L molleoides extract added at four times the MIC produced a reduction of-3.5 logio in the viable cell count at the first hour from the beginning of the experiment and complete killing at 2 h (Fig. 3). This short time effect may be associated with membrane disruption (Kubo and Fujita 2001), which was further confirmed through the electron microscopy assay. Compound 1 added at four times the MIC, gave rise to a decrease of 2.2-2.9 logio in the viability of the uropathogen from 1 to 5 h, respectively, producing bactericidal effect after 24 h.

TEM revealed that cells exposed to 8000[xg/ml of L molleoides extract showed undefined edges, were agglutinated and were markedly smaller in size than microorganisms from the control group. Similar results were observed in treatments with the same concentration of 1. The alterations observed in the bacterial membrane could be associated to the amphipathic structure of 1. Hydrophilic groups, such as OH present in the resorci-nol moiety, associate with the membrane phospholipids through hydrogen bonds, thereby creating disorder in the lipid bilayer and in membrane-bound respiratory enzymes (Bitkov et al. 1992; Kozubeketal. 1988; Kuboetal.2009). In addition, the hydrophobic hydrocarbon chain disrupts hydrogen bonding in the lipid-protein interface (Kubo et al. 2009).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

The minimum concentration of extract or 1 in which hemolysis was inhibited was 8 [micro]g/ml. Since hemolysin has been shown to be correlated with the ability of bacteria to invade cells (Peerbooms et al. 1984), its inhibition suggests that the products tested are promising therapeutic agents for preventing cell invasion.

L molleoides extract and 1 were not able to inhibit hemagglutination at the maximum concentrations tested where these products did not provoke hemolysis per se (62 and 250 [micro]g/ml, respectively).

Autoaggregation assays showed a significant increase after lh in autoaggregation of Pr2921 in treatments with 1000 and 2000 [micro]g/ml of the extract in comparison to control (p >0.05). According to these results, complete extract increases the autoaggregation capacity of Pr2921, but this property was not due to the effect of the isolated alkylresorcinol, since treatments with 1 did not affect P. mirabilis autoaggregation.

When the Pr2921 culture was supplemented with the extract at 1000 (xg/ml, a delay in swarming migration was observed with the bacteria lying in the original colony place for the first 8 h (Table 1). Compound 1, however, showed a noticeable inhibition of swarming. A complete delay in swarming motility was observed up to 8 h from the beginning, even at 8 [micro]g/ml (Table 1). The activity of 1 was higher than that observed in the other plant-origin compound, resveratrol, which showed complete blocking of swarming at 60 [micro]g/ml at 8h (Wang et al. 2006). After 24h, total inhibition of the phenomena was observed at 125 [micro]g/ml, while at 62 and 16 [micro]g/ml, 90 and 70% inhibition was still observed.
Table 1
Swarming inhibition of Proteus mirabilis by Lithrea
molleoides extract and (Z. Z)-5-Ctrideca-4'.7'-dienyl)-resorcinol (1).

Time(h) Distance (mm) (a)

 Extract ([mu]g/ml)

 1000 500 250

6 -- 1.0 [+ or -] 0 1.7 [+ or -] 0.1
8 -- 2.0 [+ or -] 0 6.0 [+ or -] 0
24 7 [+ or -] 1.0 6.0 [+ or -] 0 9.0 [+ or -] 0
Time(h)
 Compound 1 ([micro]g/ml)

 125 125 62 31

6 2.0 [+ or -] 0 -- -- --
8 8.0 [+ or -] 0 -- -- --
24 8.0 [+ or -] 0 -- 1.0 [+ or -] 0 3.0 [+ or -] 0

 Control Control EtOH
 16 8

6 -- -- 1.7 [+ or -] 0.1 1.5 [+ or -] 0
8 -- -- 2.0 [+ or -] 0 2.0 [+ or -] 0
24 3.0 [+ or -] 0 6.0 [+ or -] 0 10.3 [+ or -]0.3 9.0 [+ or -] 0

(a.)1 Data represent the mean [+ or -] standard error of the parameter
evaluated. Control ETOH: control ethanol; -: no growth.


Even when compound 1 showed promising inhibitory activity against P. mirabilis development and its virulence factors, its effectiveness could be enhanced through chemical modifications. Different studies carried out with other 5-alkylresorcinols indicated that the antibacterial activity increases when the number of double bonds in the side chain increases Qin and Zjawiony 2006; Zjawiony 2009). Compound 1 could be thus considered as a lead for further chemical structure optimization in order to have an umbrella of new molecules which show inhibitory properties against P. mirabilis.

Acknowledgments

This work was supported by the UCC, FONCyT PICTO-CRUP 2005-6-31396, DICYT. Assistance for International Cooperation was supported by Argentina-Uruguay SECyT-MEC Grant FCE 064/2005, DICYT. We thank Joss Heywood for revising the English language.

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M.C. Carpinellaal (a), (1), L De Bellis1 (b), M.B. Joray (a). V. Sosab (b), P.M. Zunino (b), *, S.M. Palacios (b), **, (1)

(a.) Fine Chemicals and Natural Products Laboratory, School of Chemistry, Catholic University of Cordoba, Cordoba, Argentina

(b.) Department of Microbiology, Institute for Biological Research Clemente Estable, Avda. Italia 3318. Montevideo. Uruguay

* Corresponding author. Tel.: +59 824871616; fax: +59 824875548.

** Correspondingauthor.Tel.:+54 0351 4938000x611; fax: +54 0351 4938061.

E-mail addresses: pabio@iibce.edu.uy (P.M. Zunino), sarapaiacios@ucc.edti.ar (S.M. Palacios).

(1.) M.C. Carpinella and S.M. Palacios are members of the National Research Council of Argentina (CONICET).

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Author:Carpinella, M.C.; Bellis, De L.; Joray, M.B.; Sosa, V.; Zunino, P.M.; Palacios, S.M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3ARGE
Date:Aug 15, 2011
Words:2921
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