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Antimicrobial activity of Drosophyllumlusitanicum, an endemic Mediterraneaninsectivorous plantSandra Gonçalves a , Célia Quintas b , Maria N. Gaspar b , José M.F.Nogueira c & Anabela Romano aa Institute for Biotechnology and Bioengineering , Centre forMolecular and Structural Biomedicine, University of Algarve ,Campus de Gambelas, Faro, Portugalb School of Technology , University of Algarve , Campus da Penha,Faro, Portugalc Faculty of Sciences, Chemistry and Biochemistry Department,Centre of Chemistry and Biology , University of Lisbon , CampoGrande, Lisboa, PortugalPublished online: 29 Oct 2009.

To cite this article: Sandra Gonçalves , Célia Quintas , Maria N. Gaspar , José M.F. Nogueira& Anabela Romano (2009) Antimicrobial activity of Drosophyllum lusitanicum, an endemicMediterranean insectivorous plant, Natural Product Research: Formerly Natural Product Letters,23:3, 219-229, DOI: 10.1080/14786410801972870

To link to this article: http://dx.doi.org/10.1080/14786410801972870

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Natural Product ResearchVol. 23, No. 3, 15 February 2009, 219–229

Antimicrobial activity of Drosophyllum lusitanicum, an endemic

Mediterranean insectivorous plant

Sandra Goncalvesa, Celia Quintasb, Maria N. Gasparb, Jose M.F. Nogueirac andAnabela Romanoa*

aInstitute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine,University of Algarve, Campus de Gambelas, Faro, Portugal; bSchool of Technology, University ofAlgarve, Campus da Penha, Faro, Portugal; cFaculty of Sciences, Chemistry and BiochemistryDepartment, Centre of Chemistry and Biology, University of Lisbon, Campo Grande, Lisboa,Portugal

(Received 26 October 2006; final version received 28 January 2008)

The aim of this study was to evaluate the antimicrobial activity of Drosophyllumlusitanicum leaf extract against various yeasts and bacteria species, including bothstandard and clinically isolated strains. The extract exhibited strongantimicrobial activity against all the tested yeast strains with inhibition zonesranging 23.67–42.23mm and with minimum inhibitory concentration (MIC)values ranging 31–63 mgL�1. All the Gram-positive bacteria studied wereinhibited by the extract, showing inhibition zones ranging 17.67–43.00mm andMIC values comprising between 15.6 and 250mgL�1. In contrast, the growth ofthe tested Gram-negative bacteria was not significantly affected by the extract.Among the microorganisms tested, Staphylococcus epidermidis ATCC 12228was the most sensitive, presenting the lowest MIC value (15.6 mgL�1), whileEnterococcus faecalis ATCC 29212 was the most tolerant (250mgL�1). Theextract of D. lusitanicum was analysed by gas chromatography-massspectrometry and the major constituent found was plumbagin.

Keywords: antimicrobial activity; bacteria; human; pathogens; plant extract;plumbagin; yeasts

1. Introduction

The number of reported cases of human infections has dramatically increased over therecent past, and resistance to antibiotics has become an alarming therapeutic problem(Eloff, 1998). Bioactive compounds from plants may represent alternative sources ofantimicrobial agents, possibly with different mechanisms of action (Motsei, Lindsey, VanStaden, & Jaeger, 2003; Barbour et al., 2004; Melendez & Capriles, 2006). Phytochemicalresearch based on ethnopharmacological information is generally considered a valuableapproach for the identification of unknown anti-infective drugs extracted from plants(Kloucek, Polesny, Svobodova, Vlkova, & Kokoska, 2005). Essential oils and extractsobtained from plants have recently gained popularity and scientific interest, due to their

*Corresponding author. Email: aromano@ualg.pt

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2009 Taylor & Francis

DOI: 10.1080/14786410801972870

http://www.informaworld.com

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potential for controlling pathogens (Tepe, Daferera, Sokmen, Sokmen, & Polissiou, 2005;Kalyoncu, Cetin, & Saglam, 2006; Zhang et al., 2008).

The family Droseraceae consists of a large group of carnivorous plants comprisinga large number of species that occur naturally in many parts of the world. Extracts fromDroseraceae contain secondary metabolites, mainly flavonoids, glucosides of the freequinones and naphthoquinones that are used in medicinal preparations and are included inpharmacopoeias. These plants are used in phytotherapeutic preparations and teas forinfections of the respiratory system, particularly bronchitis, whooping cough and generallyfor paroxysmal and hacking coughs. A broad range of biological activities, includingantimicrobial activity, were previously described for several species of this family (Didry,Dubreuil, Trotin, & Pinkas, 1999; Ravikumar, Sudha, & Joseph 2003; Melzig, Pertz,& Krenn 2001; Ferreira et al., 2004).

There is evidence that the naphthoquinone plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), which occurs in plants belonging to Plumbaginaceae andDroseraceae families, is a compound showing a broad spectrum of biological properties.Plumbagin has been shown to have antiplasmodial, antimicrobial (Didry et al., 1999;Wang & Huang, 2005; Lim, Jeon, Jeong, Lee, & Lee, 2007; Krolicka, Szpitter, Gilgenast,Romanik, Kaminski, & Lojkowska, 2006), antiproliferative (Srinivas, Gopinath, Banerji,Dinakar, & Srinivas, 2004) and insecticidal properties (Ganapaty, Thomas, Fotso, &Laatsch, 2004). Moreover, recent studies have shown that plumbagin induces apoptosis inhuman lung cancer cells (Hsu, Cho, Kuo, Huang, & Lin, 2006) and does not exertapoptotic affect on normal cells, and therefore can be developed as an anticancer drug(Hsu et al., 2006; Sandur, Ichikawa, Sethi, Ahn, & Aggarwal, 2006; Kawiak et al., 2007).

Drosophyllum lusitanicum (L.) link is an insectivorous plant of the familyDrosophyllaceae (previously included in the family Droseraceae) (The AngisospermPhylogeny Group, 2003), native to the western Iberian Peninsula and northwest Morocco.According to previous studies, leaves of this species contain flavonoids (luteolin,leucocyanidin, leucodelphinidin), phenolic compounds and naphthoquinones (Nahalka,Nahalkova, Gemeiner, & Blanarik, 1998; Budzianowski, Budzianowska, & Kromer, 2002;Grevenstuk, Goncalves, Nogueira, & Romano, in press). Although these groups ofcompounds have shown a broad spectrum of biological properties, to the best of ourknowledge, there is no information on the biological activity of D. lusitanicum extracts,including their antimicrobial activity. This research describes the first attempt to study theantimicrobial activity of D. lusitanicum extracts against a group of human-pathogenicmicroorganisms (including yeasts and bacteria), using the disc diffusion method and thedetermination of minimum inhibitory concentration (MIC) in liquid media.

2. Materials and methods

2.1. Plant material and extract preparation

Healthy, mature leaves of D. lusitanicum were collected in August 2005 in Monchique(Algarve, Portugal). The plant material was authenticated by Dr A.I. Correia from theBotanical Garden of the University of Lisbon (Lisboa, Portugal), where a voucherspecimen was deposited under the number LISU 206396. Air-dried (at 45�C in a ventilateddrying oven) plant material (20 g) was finely powdered using a blender, extracted twicewith n-hexane (300mL) at room temperature over 2 days and filtered. The combinedfiltrate was concentrated in a rotary evaporator at 40�C. The obtained residue was

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dissolved in n-hexane at a concentration of 50mg of crude extract per mL of n-hexane

(50 mgL�1) and stored at –20�C until further use.

2.2. Gas chromatography-mass spectrometry (GC-MS) analysis

Drosophyllum lusitanicum extract was analysed by GC-MS, using an Agilent 6890 gas

chromatograph interfaced to an Agilent 5973N mass selective detector (Agilent

Technologies, Palo Alto, USA). The injector temperature was set at 250�C and was

operated in the splitless mode. The analysis was performed on a TRB5-MS capillary

column (30m� 0.25mm i.d.; 0.25 mm df; 5% diphenyl and 95% dimethylpolysiloxane;

Teknokroma, Barcelona, Spain) with helium as carrier gas maintained at constant

pressure (35 cm s�1). The oven temperature program was 45�C for 0.20min and then

increased at 5�Cmin�1 to 240�C, followed by an isotherm period of 5min. Samples of

1 mL were injected manually.The transfer line, ion source and quadrupole analyser temperatures were

maintained at 280, 230 and 150�C, respectively, and a solvent delay of 4 min was

selected. In the full-scan mode acquisition, electron ionisation-mass spectra in the

range 40–400 Da were recorded at 70 eV electron energy with an ionisation current of

34.6mA. Data recording and instrument control were performed using Agilent

Technologies MSD ChemStation software (G1701CA; version C.00.00). The identity

of plumbagin was assigned by comparison of its retention time with pure standards,

as well as by comparing the mass spectrum with Wiley’s library spectral data bank

[G1035B; Rev D.02.00]. The quantification of plumbagin in the studied samples was

performed by the external calibration method using standard solutions ranging

1–15mgmL�1. All chemicals were of analytical grade and plumbagin was acquired

from Sigma.

2.3. Microorganisms (bacteria and yeasts)

Drosophyllum lusitanicum extract was tested against nine bacteria (Staphylococcus aureus

ATCC 25923, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC

29212, Streptococcus pneumoniae ATCC 49619, Streptococcus pyogenes ATCC 19615,

Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Enterobacter

sakazakii ATTCC 29544 and Enterobacter sakazakii ATTCC BA-894), and eleven yeasts.

The following strains of yeasts were isolated from hospitalised patients and kindly supplied

by the Faculty of Medicine (Coimbra University, Portugal): Candida albicans YP0048,

C. albicans YP0175 and C. famata YP0011, C. catenulata YP0160, C. guilliermondi

YP0170, Yarrowia lipolytica YP0005, Trichosporon mucoides YP0096, Trichosporon

beigelii YP0005, Cryptococcus neoformans YP0186. Two reference strains of Candida

albicans were also tested: C. albicans ATCC 10231 and C. albicans ATCC 90028. Bacterial

species were grown overnight at 37�C in Plat Count Agar medium, (PCA Scharlau). S.

pneumoniae and S. pyogenes were cultured in Blood Agar Base (BAB, Oxoid) medium

supplemented with 7% defibrinated horse blood. Strains of C. albicans were grown

overnight at 37�C in YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone,

1% glucose and 2% agar), while the other yeasts were cultured in the same medium at

25�C for 48 h.

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2.4. Antimicrobial activity

2.4.1. Disc diffusion method

The antimicrobial activity of a D. lusitanicum extract was evaluated according to theNational Committee for Clinical Laboratory Standards (NCCLS) (1997) using the Agardisc diffusion method. Briefly, a 24 or 48 h-old culture of selected bacteria/yeast wasmixed with sterile physiological saline (0.85%) and the turbidity was adjusted to theMacFarland scale 0.5 [�106 colony forming units (CFU) per millilitre].

Petri plates containing 20mL of Mueller Hinton Agar (MHA Scharlau) were usedfor all the bacteria tested, except for S. pyogenes and S. pneumoniae, which werecultured in BAB supplemented with 7% defibrinated horse blood. Yeasts werecultured in MHA supplemented with 2% glucose and 0.5mgL�1 methlyne blue dye(pH 7.2–7.4). The inoculum was spread on the surface of the solidified media andallowed to dry for 10min. Filter paper discs (6mm in diameter) were placed on thepreviously inoculated plates and impregnated with 20 mL of the extract (1mg extractper disc). Penicillin G (10 units per disc, Oxoid), ampicillin (10mg per disc, Oxoid),erythromycin (15 mg per disc, Oxoid), gentamicin (10mg per disc, Oxoid), ciprofloxacin(10 mg per disc, Oxoid) and chloramphenicol (30mg per disc, Oxoid) were used aspositive controls for bacteria. Amphotericin B (10 mg/disc, Sigma), nystatin (100 unitsper disc, Oxoid) and flucozanole (25 mg per disc) were used for yeasts. Paper discsimpregnated with 20 mL of n-hexane were used as negative control. Plates of bacteriaand C. albicans strains were incubated at 37�C for 24 h and the other yeasts at 25�Cfor 48 h. The inhibition zone diameters were measured in millimeters. All the tests wereperformed in triplicate.

2.4.2. Determination of MIC

Minimum inhibitory concentration of any compound is defined as the lowestconcentration which completely inhibits visible growth (turbidity on liquid media).A broth dilution susceptibility assay was used, as recommended by NCCLS, for thedetermination of the MIC values (NCCLS, 1999). The tests were performed in MuellerHinton Broth (MHB, Scharlau) supplemented with 5% defibrinated horse blood forS. pyogenes and with 2% (w/v) glucose for the yeast species. Two-fold serial dilutions ofthe extract ranging 1.000–0.002 mgL�1 were prepared in the culture medium. Penicillin Gwas used as reference antibacterial agent for Gram-positive bacteria and amphotericin Bfor yeasts. Test tubes with n-hexane, DMSO and ethanol were also included as negativecontrols. Each tube was inoculated with 100 mL of bacterial per yeast suspensioncontaining 104 CFU mL�1. For bacteria and C. albicans strains, tubes were incubated(under normal atmospheric conditions) at 37�C for 24 h and for yeasts at 25�C for 48 h. Allthe tests were performed in triplicate.

3. Results and discussion

In the process of discovering antimicrobial agents in plant extracts or residues, theidentification and quantification of the contained compounds is an indispensable step. TheD. lusitanicum extract used to evaluate the antimicrobial potential was analyzed byGC-MS, in order to study its chemical composition. Confirming previous results with thisspecies (Grevenstuk et al., in press) the major compound found in the extract was

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5-hydroxy-2-methyl-1,4-naphthoquinone, commonly known as plumbagin. The amount of

plumbagin in the extract was 5.5mgmL�1. Figure 1 depicts the total ion chromatogram

obtained and the plumbagin mass spectrum (23.94min). Traces of other compounds,

i.e. neophytadiene and phytol, were also detected.The in vitro antimicrobial activity of D. lusitanicum extract against the employed

microorganisms and their activity potentials were qualitatively and quantitatively assessed

by the presence or absence of inhibition zones, zone diameters and MIC values, and

compared with standard antimicrobial agents. According to the results in Tables 1 and 2,

the extract showed great antimicrobial activity. The extract had significant antimicrobial

Time (min)

Per

cent

age

5.00

100

0

Neophytadiene Phytol

10.00 15.00 20.00 25.00 30.00 35.00 40.00

Plumbagin 188

131 173 120 92

CH3

O

O

OH

160 63

7739 103 51 145 113

30 50 70 90 110 130 150 170 190 210m/z→

Figure 1. Total ion chromatogram and plumbagin mass spectrum of D. lusitanicum extract obtainedby GC-MS.

Table 1. Antimicrobial activity of D. lusitanicum extract against yeasts using the disc diffusionmethod.a

YeastsPlant extract(1mg per disc)

Amphotericin(10mg per disc)

Fluconazole(25mg per disc)

Nystatin(100 units per disc)

C. albicans ATCC 10231 36.67� 0.33 23.33� 1.20 – 24.67� 1.20C. albicans ATCC 90028 31.00� 1.00 19.67� 1.67 15.33� 0.88 25.00� 1.00C. albicans YP0048 33.33� 0.67 24.67� 0.33 22.67� 0.20 26.33� 0.33C. albicans YP0175 23.67� 0.88 19.33� 0.33 – 23.67� 0.88C. famata YP0011 30.00� 1.00 16.00� 0.58 – 26.67� 1.45C. catenulata YP0160 34.33� 1.45 21.00� 0.58 20.00� 1.15 27.67� 0.33C. guilliermondi YP0170 34.33� 0.67 22.67� 1.20 – 32.67� 1.20Y. lipolytica YP0005 29.67� 2.03 25.33� 1.86 27.33� 1.20 34.67� 0.67T. mucoides YP0096 30.67� 0.88 21.67� 0.88 26.33� 0.67 28.67� 0.33T. beigelii YP0005 32.33� 0.67 23.00� 0.58 27.67� 2.33 29.33� 1.20C. neoformans YP0186 42.33� 1.33 20.67� 0.88 – 32.33� 0.33

Notes: aDiameter of inhibition zone (mm) including disk diameter of 6mm; (–) inactive. Valuesrepresent means� SE of three different experiments.

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Table

2.AntibacterialactivityofD.lusitanicum

extract

usingthediscdiffusionmethod.a

Bacteria

Plantextract

(1mgper

disc)

PenicillinG

(10unitsper

disc)

Ampicillin

(10mg

per

disc)

Erythromycin

(15mg

per

disc)

Gentamicin

(10mg

per

disc)

Ciprofloxacin

(10mg

per

disc)

Chloramphenicol

(30mg

per

disc)

S.epidermidisATCC

12228

43.00�0.58

18.00�1.15

18.00�1.15

27.33�0.33

27.67�0.33

28.00�0.00

23.67�0.33

S.aureusATCC

25923

35.67�0.33

29.33�0.33

28.33�0.67

23.67�0.33

23.33�0.67

25.33�0.67

20.33�0.33

S.pyogenes

ATCC

19615

24.67�0.67

34.33�0.33

33.00�0.00

28.67�0.33

10.33�0.33

20.67�0.67

23.00�0.58

S.pneumoniaeATCC

49619

23.33�0.88

31.67�0.88

33.67�0.33

34.33�0.33

16.67�0.33

27.00�0.58

27.00�0.58

E.faecalisATCC

29212

17.67�0.33

19.00�0.58

19.00�0.58

20.00�0.58

12.00�0.00

21.33�0.33

16.00�0.58

P.aeruginosa

ATCC

27853

––

––

21.00�0.00

29.67�0.33

–E.coliATCC

25922

14.67�0.33

–14.00�1.53

28.67�0.33

19.67�0.33

32.00�0.58

24.00�0.58

E.sakazakiiATCC

29544

13.67�0.33

–16.33�0.88

12.00�0.58

23.67�0.88

34.33�0.33

23.00�1.00

E.sakazakiiATCC

BA-894

13.67�0.33

–18.33�0.33

13.33�0.33

26.33�3.84

33.33�1.67

24.00�0.58

Values

representmeans�

SE

ofthreedifferentexperim

ents.

Notes:

aDiameter

ofinhibitionzone(m

m)includingdiskdiameter

of6mm.(–)Inactive.

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activity (inhibition zones 423.67mm) against all yeasts tested, including several

flucozanole-resistant strains (Table 1). Moreover, in the majority of yeasts the plant

extract induced larger inhibition zones than the standards.The clinically isolated and flucozanole-resistant strain C. albicans YP0175 was the most

tolerant yeast, while the human pathogen C. neoformans YP0186 was the most sensitive

one, showing the largest inhibition zone (48.33� 0.88mm). These results are of the utmost

relevance since finding new potential antifungal agents may allow the replacement of the

antifungal drugs that induce many types of toxicity in patients (Somchit, Reezal, Nur, &

Mutalib, 2003) and induce resistant microbial strains.Staphylococcus epidermidis ATCC 12228 and S. aureus ATCC 25923 were the most

sensitive bacteria with inhibition zones of 43.00� 0.58mm and 35.67� 0.33mm,

respectively (Table 2). In the case of S. aureus, the extract was significantly more

active, showing much wider inhibition zones than the six antibiotics tested. E. faecalis

ATCC 29212 was found to be the most tolerant (17.67� 0.33mm) Gram-positive

bacteria tested. The Gram-negative bacteria P. aeruginosa ATCC 27853 was not

inhibited by the extract and Escherichia coli ATCC 25922, Enterobacter sakazakii

ATTCC 29544 and E. sakazakii ATTCC BA-894 were tolerant with small inhibition

zones. The tolerance of Gram-negative bacteria to plant extracts has been previously

documented (Palombo & Semple, 2001; Daud, Gallo, & Riera, 2005). Moreover,

Gram-negative bacteria are frequently reported to have developed multi drug

resistance to many of the antibiotics currently available on the market (Sader,

Jones, & Silva, 2002). Dissimilar sensitivity patterns observed in Gram-positive

bacteria compared with the Gram-negative may be related to the different cell wall

structures reported in these groups and to the target compounds in the bacterial cell.

Gram-negative bacteria hold an outer phospholipidic membrane carrying the

structural lipopolysaccharide components, which forms a barrier to the entry of

antimicrobial substances into the cell (Madignan & Martinko, 2006). Gram-positive

bacteria are more susceptible, having only an outer peptidoglycan layer which is

not an effective permeability barrier (Tadeg, Mohammed, Asres, & Gebre-Mariam,

2005).Although the pharmacological mechanism underlying the antimicrobial actions

of the compounds present in the extract is currently unknown, our results suggest

that these compounds may act by damaging the cell wall function, since Gram-

negative bacteria were not inhibited by the extract. This assumption is reinforced

by the fact that both antibiotics tested which prevent cell wall synthesis (Penicillin

G and Ampicillin) have not been effective against the Gram-negative bacteria

tested.Subsequent experiments were conducted to determine MIC of the extract against the

microorganisms (Table 3). The MIC values ranged 31–63 mgL�1 for yeasts and from

15.6 to 250 mgL�1 for bacteria. S. epidermidis ATCC 12228 was the most sensitive

microorganism with the lowest MIC value (15.6 mgL�1), while E. faecalis ATCC 29212

was the most tolerant (250mgL�1). It would also be worth pointing out that the various

C. albicans strains tested, although with different diameters of inhibition zone, showed the

same MIC value (31mgL�1). Moreover, the strains that presented the largest inhibition

zones (diffusion method) are not always the most sensitive (lower MIC values). As

emphasised elsewhere, this can be explained by the fact that the antimicrobial effectiveness

of a compound is affected by factors such as the solubility of the extract, the diffusion rate

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in the agar and the evaporation (that can affect the dose) (Cimanga et al., 2002;Hernandez et al., 2005).

The antimicrobial activity of the D. lusitanicum extract was apparently related to thepresence of plumbagin, which is the main compound present in the extract, and known tohave antimicrobial activity (Didry et al., 1999; Wang et al., 2005; Lim et al., 2007; Krolickaet al., 2006). However, several studies suggest that minor components present in theextract may be critical to its activity, since they may promote a synergistic effect withplumbagin (Skocibusic, Bezic, & Dunkic, 2006). Therefore, further phytochemical researchis required to determine the types of compounds that may be responsible for the extractbioactivity.

In conclusion, the results of the disc diffusion method, complemented with theevaluation of the MIC values, demonstrate the antimicrobial activity of D. lusitanicumextract. By inhibiting the growth of all yeasts and Gram-positive bacteria tested,D. lusitanicum extract showed a broad antimicrobial spectrum. Moreover, the clinicallyisolated yeasts, which are resistant to flucozanole, were found to be sensitive to the extract.This result may suggest that the extract can be used as an antimicrobial agent in new drugsfor therapy of infectious diseases in humans. However, further research should beconducted to identify the specific bioactive compounds that are responsible for theobserved antimicrobial activity. Moreover, the toxicity of the extract against animalor human cells, its mechanisms of action and its effects in vivo must be investigated(Rıos & Recio, 2005).

Table 3. Minimum inhibitory concentration (MIC) of D. lusitanicum extracts incomparison with standard antimicrobial agents.

MicroorganismPlant extract(mgmL�1)

Controla

(mgmL�1)

YeastsC. albicans ATCC 10231 31 0.039C. albicans ATCC 90028 31 0.078C. albicans YP0048 31 0.020C. albicans YP0175 31 0.039C. famata YP0011 63 0.078C. catenulata YP0160 31 0.078C. guilliermondi YP0170 63 0.078Y. lipolytica YP0005 63 0.039T. mucoides YP0096 63 0.039T. beigelii YP0005 31 0.039C. neoformans YP0186 63 0.078

BacteriaS. aureus ATCC 25923 31 0.039S. epidermidis ATCC 12228 15.6 0.625E. faecalis ATCC 29212 250 1.250S. pyogenes ATCC 19615 125 0.010

Note: aAmphotericin B was used as control for yeasts, and penicillin Gfor bacteria.

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Acknowledgements

We kindly acknowledge the supply of yeast strains by Prof. T. Goncalves from the Faculty ofMedicine, University of Coimbra, Portugal. Sandra Goncalves acknowledges a grant fromFundacao para a Ciencia e a Tecnologia (Grant SFRH/BD/19850/2004).

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