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Antimicrobial and anti-biofilm properties of new taxodione derivative from hairy roots of Salvia Austriaca.



Salvia austriaca

Diterpenoids Taxodione






The aim of the present report was to evaluate antimicrobial/anti-biofilm activity of 7-(2-oxohexyl)-taxoclione, a novel taxodione derivative isolated from n-hexane extract of Salvia austriaca hairy roots. Antimicrobial assays showed that 7-(2-oxohexyl)-taxodione was at least 4 times more active than tax-odione against methicillin-susceptible as well against methicillin-resistant staphylococci with MIC of 1.25-2.5 vg [ml.sup.-1]. This compound was less active against vancomycin-resistant enterococci (VRE), on the same level as taxodione (MIC ranged 10.0-20.0 [micro]g [m1.sup.-1]). The presence of 7-(2-oxohexyl)-taxodione in the culture medium (at MIC, 1/2 MIC or 1/4 MIC) decreased adhesion of staphylococci to abiotic surfaces, which in turn caused a reduction in biofilrn formation during 24 h, by approximately 25-30%. Also, the extent of established biofilm eradication was found to be significant, although it required an increased concentration of the compound. This is the first report on the antimicrobial activity of this, up to now not known compound, isolated from transformed roots of S. austriaca.

[c] 2012 Elsevier GmbH. All rights reserved.


Pathogens that are able to form biofilms are responsible for serious infections which are usually very difficult to treat. This is due to their extremely high resistance (100-1000 times higher than for a suspension culture) to antibiotics and the host defense mechanisms (Floiby et al. 2010). This necessitates the search for new therapeutic strategies. Plant-derived products are currently in the spotlight as a promising source or template for new drugs, active against biofilm population (Gibbons 2008: Durig et al. 2010). Our previously published data showed potent activity of some abietane diterpenes (salvipisone and aethiopinone) isolated from transformed Salvia sclarea roots (Kuzma et al. 2007: Walencka et al. 2007) against biofilm of Staphylococcus aureus (MRSA-methicillin-resistant strains). A novel diterpenoid, structurally related to taxodione, was isolated by our group from roots of Salvia austriaca Jacq. transformed with Agrobacterium rhizogenes, and characterized as 7-(2-oxohexyl)-11-hydroxy-6, 12-dioxo-7,9( 11 ),13-abietatriene [=7-(2-oxohexyl)-taxod io (Kuzma et al. 2012). Based on a literature data, we suspected that this compound could be potentially active against microorganisms, similarly to taxalione (Yang et al. 2001; Tada et al. 2010). Moreover, we have shown that this compound exhibited high cytotoxic activity against cancer cell lines (Kuzma et al. 2012). In an initial screening, antimicrobial effect of the unfractionated n-hexane extract as well as its constituents 7-(2-oxohexyl)-taxodione and taxodione was evaluated against a basic panel of reference strains. The susceptibility of S. aureus and Enterococcus faecalis/E. faecium clinical isolates was subsequently examined. Then, the compounds were tested for the time-dependent killing activity and anti-biofilm potency against S. aureus.

Materials and methods

Extraction and isolation of diterpenes

Hairy roots were obtained by S. austriaca transformation with A. rhizogenes A4 strain as described previously (Kuzma et at., 2011). From the liquid culture of hairy roots, lyophilized and powdered biomass was obtained and then extracted with n-hexane. The extract was fractionated as previously described (Kuzma et al. 2012). From fractions 1-3 three known abietane-type diterpenoids, namely royleanone, 15-deoxyfuerstione and taxodione, were isolated and from fraction 5-a new taxodione derivative was obtained. Its chemical structure was determined on the basis of spectroscopic methods as 7-(2-oxohexyl)-taxodione (Kuzma et al., 2011 and Kuzma et al., 2012).

Evaluation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the compounds

The reference S. aureus ATCC 29213, E. faecalis ATCC 29212, Escherichia coli NCTC 8196, Pseudomonas aeruginosa NCTC 6749, Candida albicans ATCC 10231,5. aureus (A3, A7, D5) and Enterococcus spp. clinical strains (138/09, 988/09, 203/06) were used. Stock solutions of a crude extract, 7-(2-oxohexyl)-taxodione and taxo-dione were prepared in DMSO (20.0 mg [m1.sup.-1]). The concentration range of the compounds used in the tests was 0.625-160.0 [micro]g [ml.sup.-1]. Minimum inhibitory concentration (MIC) values were specified by a microdilution broth assay, according to CLSI recommendations (CLSI 2009). The concentrations of the compounds bactericidal to [greater than or equal to]99.9% of the inoculum (MBC) were also determined. MIC/MBC tests were performed and results were evaluated as previously described (Budzynska et al. 2011). In the reference experiments, standard antibiotics were used: oxacillin, vancomycin (Mast Diagnostic, UK). The experiments were run in quadruplicate and repeated twice.

Determination of bacterial growth rate in the presence of tested compounds

The growth rate of S. aureus ATCC 29213 cultured in TSB for 4,8 and 24h at 37[degrees]C in a 96-well microplate in the presence of 7-(2-oxohexyl)-taxodione or taxodione, both at MIC and 1/2 MIC, was evaluated and compared to the bacterial growth rate in the presence of oxacillin. Compound/antibiotic concentrations causing a reduction of microbial growth at each time point were determined by comparison of the mean values (from four replications) measurements of [A.sub.600] [+ or -] S.D. (calculated using program Statistica 5.0 PL).

S. aureus biofilm formation and eradication under the influence of 7-(2-oxohexyl)-taxodione

The suspension of S. aureus ATCC 29213 prepared from fresh overnight culture in tryptic-soy broth (TSB) supplemented with 0.25% D-(+)-glucose (TSBGIc), was added to the wells of a 96-well tissue culture-treated polystyrene plate (Nunc, Denmark). Bacterial cultures were grown for 2411 at 37[degrees]C in the presence of phytochemical used at MIC or 1/2 MIC. Then, the biofilm formed in each well was assessed using a L1VE/DEAD[R] BacLightTm kit (L7007, Molecular Probes. USA), as recommended by the manufacturer. Results are presented as the percentage of the amount of the formed biofilm, calculated from mean fluorescence values (measured at 485/535 nm for green Syto9 and at 485/620 nm for red PI)[+ or -] S.D. of the control (100%) and the tested wells. A second set of plates was designed to investigate the influence of the compound on the already preformed (24-h-old) biofilm of S. aureus ATCC 29213. Given the known very low sensitivity of biofilm to antibiotics/antiseptics, we used for this purpose higher concentrations (supraM1C) of 7-(2-oxohexyl)-taxodione, such as 50, 100 and 200 [micro],g [m1.sup.-1]. After subsequent 24-h co-incubation, the degree of biofilm survival (%) was assessed as described above. The results obtained by staining in a LIVE/DEAD Viability Assay were confirmed by CFU counting of bacteria recovered from mechanically dispersed biofilms.

Results and discussion

Two diterpenoids extracted from hairy roots of S. austriaca, a newly isolated and identified as 7-(2-oxohexyl)-taxodione and, for comparison, well known taxodione, were screened for their in vitro antimicrobial activity. Unfractionated n-hexane extract was also examined. 7-(2-oxohexyl)-taxodione exhibited potent antagonistic effect against the reference Gram-positive S. aureus strain, at a much higher level compared to that of taxodione. Its activity against E. faecalis was similar to taxodione (Table 1). Antimicrobial action of the unfractionated n-hexane extract against these bacteria was also significant, ranged from 20.0 vg [m1.sup.-1] to 40.0 [micro]g [m1.sup.-1]. The growth of Gram-negative bacteria (E. coli, P. aeruginosa) and pathogenic fungi (C. albicans) was not significantly inhibited by any of the investigated compounds with MIC > 160.0 [micro]g [m1.sup.-1] (data not shown). These results are consistent with data reported by other authors testing the activity of synthetic taxodione and its derivatives (Yang et al. 2001; Tada et al. 2010).

Table 1
Minimum inhibitory concentrations (MICs) of 7-(2-oxohexyl)-taxodione
(oxohexyl-TAX) and taxodione (TAX), compared to MIC of the reference
antibiotics-oxacillin (OXA) and vancomycin (VAN).

Bacterial strain     MIC([mu]g
                   Oxohexyl-TAX  TAX         OXA

S. aureus
ATCC 29213 (MSSA)           2.5  10.0        0.25
A3(MRSA)                   1.25   5.0       128.0
A7(MRSA)                   1.25   5.0           >
D5(MRSA)                   1.25   5.0      >128.0

Bacterial strain     MIC([mu]g
                   Oxohexyl-TAX  TAX       VAN

E. faecalis/E.

ATCC 29212 (VSE)           10.0  10.0         2.0
2003/06 (VSE)
                           10.0  10.0         2.0
138/09 (VRE)               10.0  10.0        64.0
988/09 (VRE)               20.0  20.0      >256.0

This has encouraged us to undertake further studies on 7-(2-oxohexyl )-taxodione activity against multidrug-resistant strains. For the experiment we selected some clinical isolates-members of an important group of "alert" human pathogens-Staphylococcus and Enterococcus (Willems et al. 2011; Arias and Murray 2012). Potent antibacterial activity against S. aureus strains: A3, A7, D5 and against enterococci: strains 138/09, 988/09, 203/06 was observed (Table 1). Following to MIC

evaluation, the CFU counting test revealed > 99.9% reduction in the original inoculum by MIC of taxodione, which proved its bactericidal effect (MBC). 7-(2-oxohexyl)-taxodione was found to have bactericidal activity only when used in a higher concentration (MBC = 40.0 [micro]g [m1.sup.-1]).

The experiment designed to test the time-dependent growth inhibition showed that both, 7-(2-oxohexyl)-taxodione and taxo-dione (each at their MIC or 1/2 MIC), efficiently reduced the number of growing staphylococci, starting just from 41-1 of incubation. After 8 h-growth inhibition caused by both compounds was calculated to be approximately 90%. This effect was comparable to the action of a standard beta-lactam antibiotic, oxacillin (Fig. 1A). A certain proportion of bacteria in the samples taken from 24-h co-culture with taxodione derivative and taxodione, stained by a LIVE/DEAD[R] BacLightTm kit, showed red fluorescence under the microscope, which indicated the cell wall and membrane leaking. The percentage of P1-positive cells was higher with an increasing concentration of the compounds and length of co-incubation time (data not shown).

The inhibition of biofilm formation in the constant presence of 7-(2-oxohexyl)-taxodione at a concentration equal to MIC, 1/2 or 1/4 MIC, calculated at approximately 25-30%, was considered as a promising observation (Fig. 1B). Moreover, this compound applied to already formed biofilm at the concentration range 50-200 [micro]g [m1.sup.-1] was able to reduce biofilm viability in a dose dependent manner (46.8 [+ or -] 2.74% to 65.6 [+ or -] 2.0%), assessed after a further 24 h of incubation (Fig. 1C). Given the known extremely high resistance of biofilm population, even partial destruction of the biofilm by 7-(2-oxohexyl)-taxodione should be regarded as encouraging. Many studies in vitro and clinical observations indicate that complete eradication of biofilm-embedded bacteria by chemotherapy is hard to achieve (Hoiby etal. 2010). Therefore, it is important to search for natural or synthetic compounds that could increase the susceptibility of biofilms to conventional therapeutic strategies. It is hoped that 7-(2-oxohexyl)-taxoclione could be such forward-looking compound. In summary, the present study has shown for the first time the potent antibacterial activity of a novel taxodione derivative, isolated from S. austriaca hairy roots. It is accepted that natural, semi-synthetic or synthetic compounds capable of inhibiting bacterial growth at a concentration equal to or lower than 10.0 lig [m1.sup.-1] are worth further study. These requirements are met by 7-(2-oxohexy1)-taxoclione, showing MIC ranging from 1.25 [m1.sup.-1] to 10.0 [m1.sup.-1] against MRSA and VRE, respec- tively. Thus, the demonstrated level of activity of the compound allows it to be placed at the top of the list of natural diter-penoicis exhibiting antimicrobial activity, including anti-biofilm potential.


We would like to thank to U. Krajewska for technical assistance. This study was supported by a grant from University of Lodz (807), and from Medical University of LOc12, Poland (grants 503/3-01502/503-01 and 503/3-012-01/503-01).

* Corresponding author. Tel.: +48 42 635 41 85; fax: +48 42 665 58 18. E-mail address: (B. Rozalska).

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L. Kuzma (a), H. Wysokinska (a), M. Roza1ski (b), A. Budzynska (c), M. Wieckowska-Szakiel (c), B. Sadowska (c), M. Paszkiewicz (c), W. Kisiel (d), B. Rozalska (c), (*)

(a.) Department of Biology and Pharmaceutical Botany, Medical University of Lodz Muszynskiego 1, Pl-90-151 Lodz, Poland

(b.) Department of Pharmaceutical Biochemistry, Medical University of Lodz. Muszynskiego 1, Pl-90-151 Oa Poland

(c.) Department of Infectious Biology. University of1.6di. Banacha 12/16. Pl-90-237 Lodz, Poland

(d.) Department of Phytochemisny. Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, Pl-31-343 Krakow, Poland
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Title Annotation:Short communication
Author:Kuzma L., Wysokinska H., Rozalski M., Budzynska A., Wieckowska-Szakiel M., Sadowska B., Paszkiewicz
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Nov 15, 2012
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