Braz. J. Biol. vol.66 no.3 So Carlos Aug.

2006
http://dx.doi.org/10.1590/S1519-69842006000500020

BIOLOGY

Phenotypic aspects of oral strains of Candida albicans in children with down's syndrome

Aspectos fenotpicos de cepas de Candida albicans orais em crianas com sndrome de down

Ribeiro, E. L.I; Scroferneker, M. L.II; Cavalhaes, M. S.I; Campos, C. C.III; Nagato, G. M.III; Souza, N. A.III; Ferreira, W. M.I; Cardoso C. G.I; Dias, S. M. S.I; Pimenta, F. C.I; Toledo, O. A.III
I

Institute of Tropical Pathology and Public Health of Universidade Federal de Gois, Goinia, GO, Brazil II Institute of Basic Sciences of the Health of the Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil III School of Dentistry of the Universidade Federal de Gois, Goinia, GO and Universidade de Braslia, Braslia, DF, Brazil Correspondence to

ABSTRACT The aim of this article is to characterize the biological aspects of oral strains of C. albicans in children with Down's syndrome. These yeasts were analyzed as to their macromorphological and enzymatic aspects and were tested as to their in vitro susceptibility to antifungal drugs using broth microdilution to determine the minimum inhibitory concentration (MIC). The morphotyping revealed that all oral C. albicans isolates from children with Down's syndrome promoted the formation of fringes regardless of size, while the control group presented smaller fringes. All oral C. albicans strains produced proteinase, but those with

phospholipolytic activity showed greater enzyme capacity in the test group. In vitro susceptibility showed that all oral C. albicans isolates were sensitive to the drugs used. Keywords: Phenotyping, Candida albicans, Down's syndrome.

RESUMO O objetivo deste artigo foi caracterizar os aspectos biolgicos de cepas de C. albicans orais em crianas com sndrome de Down. Estas leveduras foram analisadas quanto aos seus aspectos macromorfolgicos e enzimticos e foram testadas quanto a sua suscetibilidade in vitro a drogas antifngicas, usando a microdiluio em caldo para a determinao da concentrao inibitria mnima (CIM). A morfotipagem revelou que todos os isolados de C. albicans orais de crianas com sndrome de Down induziram formao de franjas independente do tamanho, enquanto o grupo controle teve franjas menores. Todas as cepas de C. albicans orais produziram proteinase, mas aquelas com atividade fosfolipidoltica mostraram maior capacidade enzimtica no grupo teste. A suscetibilidade in vitro mostrou que todos os isolados de C. albicans orais foram sensveis a drogas empregadas. Palavras-chave: fenotipagem, Candida albicans, sndrome de Down.

INTRODUCTION
The mouth is one of the most diversified sources of microorganisms, where yeasts of the genus Candida play a relevant role due to their presence from the initial development of the oral microbiota to the definitive one (Marcantoni, 1999). In children with Down's syndrome, pathoanatomical disorders predispose them to the proliferation of Candida strains, completely populating the floor of the mouth with this yeast-like fungus, which often causes candidiasis (Campos, 2001). The onset of the infectious process by Candida in the mouth of children with Down's syndrome, in addition to abnormal innate and acquired immunological conditions, is also influenced by the pathogenic capacity of these yeasts, initially acting as colonizers and later on as infectious agents due to the alteration of the oral microbiota produced by microbiological, chemical and physical factors, such as chewing and/or poor mouth cleaning (Campos, 2001; Ribeiro et al., 2002). The methods for typing Candida cultures have been used to better characterize the biological aspects presented by these yeasts. Therefore, the phenotypic characterization of yeast-like fungi enables us to assess macroscopically how these microorganisms behave as a constituent part of the topical microbiota and/or as the triggering mechanism of candidiasis regarding morphological, serological, biological, and antifungal parameters (Prince et al., 1982; Ghannoum & Abu-Elteen, 1990; Odds, 1998; Cndido et al., 2000; Pinto, 2003).

The aim of this article is to establish the phenotypic profile of oral Candida yeasts in children with Down's syndrome given the morphotyping of fringes and the fungal topography of colonies, production of exoenzymes (proteinase and phospholipase) and antifungaltyping.

MATERIAL AND METHODS

Strains of C. albicans The 35 strains of C. albicans used in this work were due to a case-control study involving oral yeasts of Candidaisolated from children with and without trisomy of the 21st chromosome. There were twenty five (25/30) (83.3%) oral strains of C. albicans from children with Down's syndrome and ten (10/60) (16.7%) from the control group (children without the syndrome) treated at the Pediatric Dental Clinic of the School of Dentistry at the Universidade Federal de Gois (CO/FO/UFG) in Goinia, state of Gois, Brazil. The yeasts used in this study were isolated and identified according to Kreegen-van Rij (1984). These strains of C. albicans were obtained from individuals between 0 and 10 years old with an intact oral mucosa and who were not on any type of drug therapy. The present study was previously accepted by the Committee of Ethics in the Human Medical Investigation and Animal of the Clinics Hospital at the Universidade Federal de Gois (HC/UFG) and the parents or guardians of the children gave their consent to the investigators. Phenotyping Morphological, enzyme and antifungal phenotyping used in the present study aims at establishing an epidemiological correlation between the Candida strains analyzed herein. Morphotyping A phenotypic marker was detected in malt extract agar with the seeding of two strains of Candida (inoculum with turbidity adjusted in 3 on Mc Farland scale) on each Petri dish with sterilized swabs. After the incubation of the dishes at 25 C for 10 days, the reading was based on the macromorphological aspects of the fringes and on the topography of the colony by way of the model proposed by Hunter et al. (1989) (Table 1). Enzymetyping The production of extracellular enzymes by Candida species was detected for proteinase in a basic medium enriched with albumin and vitamins (Rchel et al., 1982) and for phospholipase in Sabouraud dextrose agar (SDA) and egg yolk (Prince et al., 1982). After incubation in a greenhouse at 37 C for 48 h for proteinase and for 96 h for phospholipase, the reading of test isolates and of the standard strain ( Candida albicans CBS 562) granted by the mycological collection of the Institute of Biomedical Sciences at the Universidade de So Paulo (ICB/USP), Brazil was performed based on the production of the precipitation zone around the inoculation point of Candidaisolates. The enzyme activity (Pz) resulted from the relation between the diameter of the colony (dc) and the diameter of the colony and precipitation zone (dcp). The results were classified as negative (Pz = 1 cm), positive (0.64 cm > Pz < 1 cm) and strongly positive (Pz < 0.64 cm). Antifungaltyping

The in vitro susceptibility test was carried out by broth microdilution, standardized by the National Committee for Clinical Laboratory Standards (NCCLS) (Atlanta, USA) to determine the minimum inhibitory concentration (MIC). Amphotericin B and 5 fluorocytosine were obtained from Sigma Chemical Company. Fluconazole and itraconazole were used in their commercial formulation available in Brazil: 150 mg and 100 mg respectively. To prepare the standard solution, 5 mg of each drug were weighed, yielding concentrations of 640 g/mL and 1280 g/mL for amphotericin B and itraconazole, respectively, which were solubilized in dimethyl sulfoxide (DMSO) at the concentration of 1% of the volume of the standard solution. Both water-soluble fluconazole and 5 fluorocytosine yielded concentrations of 1.250 g/mL and 1.000 g/mL, respectively. From these solutions, serial dilutions of the drugs were prepared in MOPS (morpholinepropanesulfonic acid)-buffered RPMI-1640 (pH 7.0) in order to obtain the desired concentrations of antifungals. The following concentrations were used: 0.02 at 16.0 g/mL for amphotericin B, 0.012 at 12.5 g/mL for 5-fluorocytosine, 0.04 at 32.0 g/mL for itraconazole and 0.08 at 64.0 g/mL for fluconazole. The inoculum of Candida strains was grown in Sabouraud dextrose agar (SDA) for 48 h at 35 C. This solution was prepared in 5 mL of deionized and sterilized water and the cellular density was adjusted on a spectrophotometer for an 85% transmittance reading in the 530 nm wavelength, resulting in a solution with one inoculum between 0.5 x 10 2 and 2.5 x 103 CFU/mL (colony forming units per milliliter). Covered acrylic dishes were used for microdilution, with 96 wells (Difco, Detroit, USA). Each well received 100 g/mL of the antifungal drug concentration and 100 g/mL of the test suspension of Candida strain. The dishes were incubated at 37 C for 48 h. The MIC for azole drugs was defined as the first well with a 50% reduction in yeast cell growth, comparatively to the positive control (drug-free medium). For amphotericin B and 5fluorocytosine, the MIC was defined as the lowest concentration able to inhibit any visible growth.

RESULTS
Morphotyping The morphotyping of 35 C. albicans strains from the oral mucosa of children with and without Down's syndrome enabled us to detect nine different morphotypes; the test group (children with Down's syndrome) showed a predominance of morphotype 5530 (colony with continuous fanlike fringes, equal to or greater than 6 mm, with an intermediate texture and smooth topographical aspect). In the group of children without Down's syndrome, a higher incidence of morphotype 5240 (colony with continuous fanlike fringes, equal to or greater than 2 mm, fine texture and smooth topographical aspect) was observed (Table 2). Enzymetyping Proteinase production was detected in all oral strains of C. albicans obtained from the mouth of children with and without Down's syndrome, whereas the test group (children with Down's syndrome) was highly proteolytic. As for phospholipase, 88.0% (22/25) of oral C. albicans strains in the group of children with Down's syndrome produced this exoenzyme in

remarkable amounts, while in the group of children without the syndrome, only 80.0% (8/10) of the oral strains showed enzymatic capacity (Table 3). Antifungaltyping All oral strains of C. albicans in the test and control groups (children with and without Down's syndrome) revealedin vitro sensitivity to the antifungal drugs used in broth microdilution (Table 4).

DISCUSSION
A deeper understanding concerning the phenotypic behavior of Candida cultures analysing each sample, either as colonizer and/or pathogen, enables us to evaluate the biological diversity of existing strains and how such characteristics express themselves in the sequencing of the fungal process. Yeast-like aspects of Candidaobserved in genotypic characteristics through molecular biology by way of one-dimensional electrophoretic karyotyping and pulsed field electrophoresis with DNA probes, polymerase chain reaction (PCR), or also the combination of these techniques enables us to differentiate phenotypically undistinguishable strains, finding the genetic mapping sequence of these yeasts and showing the phenotypic aspect of each gene of the fungus (Fukazawa et al., 1997; Radford et al., 1997; Lpez-Ribot et al., 2000; Melo et al., 2003). The morphological aspect of C. albicans colonies showed that the predominance of a smooth surface does not depend on the analyzed group (children with and without Down's syndrome). As for the disposition of the colonies in fringes, 84.0% (21/25) of the strains isolated from the oral mucosa of children with Down's syndrome revealed fringes throughout the periphery of the fungal colony, whose length was greater than or equal to 6 mm in 48.0% (12/25) of the strains, and rough structure in 68.0% (17/25). Concerning the control group (children without the syndrome), fringes were present in 80.0% (8/10) of C. albicans strains on the entire colony surface, but with a length shorter than 5 mm in all strains and rough texture in approximately 60.0% (6/10) of the fringes. This phenotypic aspect thus showed that the adherence capacity of oral strains of C. albicans in the test group (children with Down's syndrome) is more exacerbated in vitro than in the control group, although all these yeasts are of colonizing type and originate from the oral mucosa of children with or without this chromosome disorder. Therefore, the presence of fringes in yeasts of Candida obtained from the mouth is possibly attributed to the virulence of this fungus, thus showing a higher tendency towards the development of the mycelial form by the yeast and favoring the adaptation to the oral cavity (Melhem, 1997; Radford et al., 1997; Melo et al., 2003). Patients with neoplasms or with AIDS have shown oral morphotypes of C. albicans with fringes more often than those obtained from the mouth of healthy individuals, which reinforces its association with yeast virulence (Melhem, 1997). The production of extracellular enzymes by Candida yeasts is another capacity that this fungus has to establish itself as a colonizing and/or infectious microorganism in man. Proteinase favors the adherence of Candida isolates to the surface of infected mucosa due to the penetration of germ tubes into the infected tissue (Odds, 1998). However, phospholipase induces the control of fungal growth by its presence in the extremities and destruction of lipids that constitute the cell membrane (Ribeiro et al., 2002). Approximately 96.0% (24/25) of oral strains of C. albicans from children with Down's syndrome were highly proteolytic, produced phospholipase and were highly phospholipidolytic, while 80.0%

(8/10) of the yeasts in the control group (children with Down's syndrome) showed to produce both enzymes. Nevertheless, the individual analysis of the productive enzyme capacity of each strain of C. albicans revealed that strains from the mouth of children with Down's syndrome showed enhanced production. Cndido et al. (2000) studied 79 Candida strains isolated from the mouth of patients with lesions characteristic of candidiasis and from individuals with clinically normal oral cavity treated at the School of Dentistry in Ribeiro Preto, Universidade de So Paulo (FO/RP/USP), Brazil, and found out that 83.3% and 66.7% of C. albicans strains from oral lesions produced phospholipase and proteinase, respectively. However, the strains of this yeast obtained from the mouth of individuals with no lesions produced phospholipase in 71.9% and proteinase in 68.7% of the cases. Among the strains of C. albicans in both groups, enzymetype 22 (positive phospholipase and weakly positive proteinase) was the most prevalent. This showed that the phospholipidolytic capacity of oral strains of C. albicans is a preponderating factor for candidiasis (Cndido et al., 2000; Ribeiro et al., 2002). Ghannoum & Abu-Elteen (1990) and Cndido et al. (2000) affirm that these hydrolytic enzymes produced by Candida yeasts are virulence factors for this yeast-like fungus. In vitro tests for sensitivity of oral yeasts of C. albicans in children with or without Down's syndrome regarding the antifungal agents used (amphotericin B, 5-fluorocytosine, fluconazole and itraconazole) showed to be susceptible to all antifungals. The strains of C. albicans obtained from the mouth of children with this chromosome disorder revealed a CI50 (concentration index able to inhibit 50% of the strains) equal to 0.25 g/mL for amphotericin B, CI50 = 0.39 g/mL for 5-flourocytosine, IC50 = 1.00 g/mL for fluconazole and IC50 = 0.50 g/mL for itraconazole. The control group (children without Down's syndrome) showed a CI50 = 0.25 g/mL for amphotericin B, CI50 = 0.39 g/mL for 5fluorocy-tosine, CI50 = 2.00 g/mL for fluconazole and CI50 = 0.50 g/mL for itraconazole. Batista et al. (1999) observed the susceptibility of 19 strains of C. albicans, isolated from patients with protein-induced stomatitis, and found that polyene antibiotics had a lower minimal inhibitory concentration than azole derivatives, although their in vitro antifungal action fell within the serum blood levels reached by these drugs in man. Pinto (2003) observed that the C. albicans strains obtained from patients with HIV, cancer, diabetes and other diseases, also showed in vitro susceptibility to 5-fluorocytosine.

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BATISTA, J. M., BIRMAN, E. G. & CURI, A. E., 1999, Suscetibilidade a antifngicos de C. albicans isoladas de pacientes com estomatite protica. Rev. Odontol. Univ. So Paulo, 4(13): 234-239. [ Links ] CAMPOS, C. C., 2001, Contagem e identificao de estreptococos do grupo mutans em crianas com sndrome de Down. Dissertao de Mestrado em Medicina Tropical Instituto de Patologia Tropical e Sade Pblica da Universidade Federal de Gois, 56p. [ Links ] CNDIDO, R. C., AZEVEDO, R. V. P. & KOMESU, M. C., 2000, Enzimotipagem de espcies de Candida isoladas da cavidade bucal. Rev. Bras. Med. Trop., 5(33): 456463. [ Links ] FUKAZAWA, Y. & KAGAYA, K., 1997, Molecular bases of adhesion of C. albicans. J. Med. & Veter. Mycol., 35: 87-99. [ Links ]

GHANNOUM, M. A. & ABU-ELTEEN, K. H., 1990, Pathogenicity determinants of Candida. Mycoses, 33: 265-82. [ Links ] HUNTER, P. R. & FRASER, C. A. M., 1989, Application of a numerical index of discriminatory power to a comparison of four physiochemical typing methods for C. albicans. J. Clin. Microb., 27: 215-218. [ Links ] KREEGER-VAN RIJ, N. J. W., 1984, The yeast: a taxonomic study, Elsevier, Amsterdam, 1080p. [ Links ] LPEZ-RIBOT, J. L., MCATEE, R.K. & KIRKPATRICK, W. R., 2000, Comparison of DNAbasead typing methods to assess genetic diversity and relatedness among Candida albicans clinical isolates. Rev. Ibero. Micol., 17: 49-54. [ Links ] MARCANTONI, M., 1999, Ecologa de la cavidad bucal, pp. 473-497. In: Negroni, Marta (ed), Microbiologa estomatolgica. 630p. Mdica Pan-Americana, Mxico. [ Links ] MELHEM, M. S. C., 1997, Bitipos, caritipos e perfis de suscetibilidade a antifngicos de amostras seriadas de C. albicans de pacientes com candidase bucal associada sndrome de imunodeficincia adquirida. Tese de Doutorado em Cincias Faculdade de Medicina da Universidade de So Paulo, 167p. [ Links ] MELO, A. S. A., SERAFIM, R. C. S. & BRIONES, M. R. S., 2003, Identification of genes differentially expressed in hyphae of Candida albicans. Braz. J. Microb., 34(1): 135137. [ Links ] ODDS, F. C., 1998, Candida and Candidosis. 2nd ed. Bavillire Tindall, London, 280p. [ Links ] PRINCE, M. F., WILKINSON, I. D. & GENTRY, L. O., 1982, Plate Methods for detection of phospholipase activity inCandida albicans. Sabouraudia, 20: 15-20. [ Links ] PINTO, P. M., 2003, Caracterizao fenotpica e anlise da variabilidade gentica de espcies do gnero Candida isoladas de pacientes portadores e no portadores de doenas de base. Tese de doutorado em Cincias Biolgicas Instituto de Cincias Biolgicas da Universidade Federal de Minas Gerais, 148p. [ Links ] RADFORD, D. R., CHALLACOMBE, S. J. & WALTER, J. D., 1997, Scanning electron microscopy of the development of structured aerial mycelia and satellite colonies of phenotypically switched C. albicans. J Med Microbiol., 46: 326-332 [ Links ] RIBEIRO, E. L., CAMPOS. C. C., CRESPO, A. M. C., CASTRO, J. S., ROCHA, F. C., ALVES, M. P., GOULART, M. S., CARDOSO, C. G., FERREIRA, W. M., NAVES, P. L. F., SOARES, A. J., MIRANDA, S. R. & PIMENTA, F. C., 2002, Deteco de C. albicans fosfolipidolticas isoladas da saliva de crianas com sndrome de Down. Acta Md. Portug., 14: 33-42. [ Links ] RCHEL, R., TESELLER, R. & TROST, M. A., 1982, A comparison of secretory proteinases from different strains ofC. albicans Sabouraudia, 20: 233-244. [ Links ]

Antimicrobial potential of some plant extracts against Candida species

Potencial antimicrobiano de extratos de plantas na inibio de leveduras do gnero Candida

Hfling, JF.I, *; Anibal, PC.I,II; Obando-Pereda, GA.I; Peixoto, IAT.I,III; Furletti, VF.I,III; Foglio, MA.II; Gonalves, RB.I
I

rea de Microbiologia e Imunologia, Departamento de Diagnstico Oral, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas Unicamp, Av. Limeira, 901, CP 052, CEP 13414-900, Piracicaba, SP, Brazil II Centro Pluridisciplinar de Pesquisas Qumicas, Biolgicas e Agrcolas, Departamento de Fitoqumica, Universidade Estadual de Campinas Unicamp, Campinas, SP, Brazil III Centro Pluridisciplinar de Pesquisas Qumicas, Biolgicas e Agrcolas, Departamento de Microbiologia, Universidade Estadual de Campinas Unicamp, Campinas, SP, Brazil

ABSTRACT The increase in the resistance to antimicrobial drugs in use has attracted the attention of the scientific community, and medicinal plants have been extensively studied as alternative agents for the prevention of infections. The Candida genus yeast can become an opportunistic pathogen causing disease in immunosuppressive hosts. The purpose of this study was to evaluate dichloromethane and methanol extracts from Mentha piperita, Rosmarinus officinalis, Arrabidaea chica, Tabebuia avellanedae, Punica granatum andSyzygium cumini against Candida species through the analysis of Minimum Inhibitory Concentration (MIC). Results presented activity of these extracts against Candida species, especially the methanol extract. Keywords: Candida, plant extracts activity, antifungal activity, antimicrobial agents.

RESUMO Devido ao aumento da resistncia aos antimicrobianos em uso, as plantas medicinais tm sido intensamente estudadas como agentes alternativos para a preveno de doenas e

infeces. A levedura do gnero Candida,por ser um patgeno oportunista, tem sua virulncia aumentada ao adquirir resistncia aos antifngicos, desencadeando doenas, principalmente em hospedeiros imunossuprimidos. O propsito deste trabalho foi avaliar os extratos diclorometano e metanol das plantas Mentha piperita, Rosmarinus officinalis, Arrabidaea chica, Tabebuia avellanedae, Punica granatum e Syzygium cumini contra linhagens do gnero Candida atravs dos testes de Concentrao Inibitria Mnima (CIM). Os resultados demonstraram atividade dos extratos sobre as espcies de Candida, particularmente o extrato metanol. Palavras-chave: Candida, atividade de extratos de plantas, atividade antifngica, agentes antimicrobianos.

1. Introduction
Medicinal plants and corresponding preparations have been used for a wide range of purposes and for many centuries people have been trying to treat diseases as well as alleviate symptoms by using different plant extracts and formulations (Cowan, 1999). Plants such as Mentha piperita (Iscan et al., 2002), Rosmarinus officinalis (Nascimento et al., 2000), Arrabidaea chica (Taylor, 1998), Tabebuia avellanedae (Machado et al., 2003), Punica granatum (Nascimento et al., 2000; Duraipandiyan et al., 2006) and Syzygium cumini(Chandrasekaram and Venkatesalu, 2004)have been used due to their antimicrobial properties. In the 2000-2006 period, approximately 50% of new chemical molecules extracted from natural products demonstrated their importance for the development of drugs in the treatment of infectious diseases (Newman and Cragg, 2007). The choice of an appropriate antifungal treatment is important, though limited to a few licensed agents (Provine and Hadley, 2000). The increasing resistance of microorganisms to antimicrobial drugs in use has attracted the attention of the scientific community regarding the search for new costeffective drugs of natural or synthetic origin (Pai et al., 2004). Members of the Candida genus are commensal aerobic microorganisms, which are part of the indigenous microbial flora in humans and can be found in the oral cavity and the digestive as well as vaginal tracts (Odds, 1988). They may become opportunistic pathogens causing a number of diseases in immunosuppressive hosts (De Repentigny et al., 2004), mainly HIV-positive patients (Erkse and Erturan, 2007). A particular characteristic of Candida is its ability to invade oral mucosa tissues by the development of hyphae, which adhere to tissue surfaces and lead to inflammation (Ellepola and Samaranayake, 2001). Although the colonisation by Candida albicans is common and causes severe injuries in immunocompromised patients, other Candida species have been isolated from such patients, healthy patients and children with Down's syndrome, such as C. glabrata, C. krusei, C. tropicalis, C. lusitaniae, C. parapsilosis, C. guilliermondii (Erkse and Erturan, 2007; Hfling et al., 2001; Ribeiro et al., 2006; Rodrigues et al., 2004) and C. dubliniensis. These yeasts were recognised as the source of mucosal infection in HIV-positive patients

and regarded as a significant cause of infections in humans, such as abdominal infections and fungemia (Erkse and Erturan, 2007). The purpose of this study was to evaluate the potential activity of extracts from six selected plants against tenCandida species.

2. Material and Methods

Selected plants: the species Mentha piperita L. (leaves, voucher no. UEC 1253), Arrabidaea chica (Bonpl.) B. Verl. (leaves, voucher no. UEC 1254), Rosmarinus officinalis L. (leaves, voucher no. UEC 1264), Syzygium cuminiL.(seeds, voucher no. UEC 143724), Punica granatum L. (fruit, voucher no. UEC 143723) and Tabebuia avellanedae Lor. ex Griseb (bark, voucher no. UEC 1256) were collected from the experimental field of the Research Centre for Chemistry, Biology and Agriculture, State University of Campinas (CPQBA/UNICAMP), Brazil. Voucher specimens were deposited at the Herbarium of the State University of Campinas (UEC) and were identified by Professor Jorge Yoshio Tamashiro, Ph.D. Preparation of extracts: Each selected plant (5 g) was extracted with 600 mL of dichloromethane (Labsynth PA) with a Dispersive Extratur (Quimis Q-252-28 model) and filtered thereafter. The vegetal residue was reextracted with methanol (Labsynth PA). Solvents were evaporated under reduced pressure and dried using a rotary evaporator (Buchi R-200 model). Crude extracts were monitored by chromatography in Thin Layer in silicagel chromatoplaques (60 F254 Merck 1.05554). The dried plant extracts were dissolved in Tween20 (Labsynth) and sterile distilled water, filtered through a 0.22-m membrane filter (TPP) and stored at 4 C until further use. The extracts were diluted at the moment of use with the concentration ranging from 1 to 0.001 mg/mL. Microorganisms: The test organisms used were Candida albicans CBS-562, C. dubliniensis CBS-7987, C. parapsilosis CBS-604, C. tropicalis CBS-94, C. guilliermondii CBS566, C. utilis CBS-5609, C. krusei CBS-573, C. lusitaniae B-06, C. glabrata B-07, C. rugosa B-12, proceeding from the Microbiology and Immunology Laboratory, at the School of Dentistry of Piracicaba (FOP/UNICAMP). Screening for antimicrobial activities: Preparation of inoculum for susceptibility tests was carried out by microdilution as set forth by the CLSI's M27-A2 recommendation protocol (CLSI, 2002). The yeasts were grown overnight at 37 C in Sabouraud Dextrose Agar (Merck) plates, and inocula for the assays were prepared by diluting scrape cell mass in 0.85% NaCl solution, adjusted to 0.5 Mc Farland scale and confirmed by spectrophotometric reading at 625 nm. Cell suspensions were finally diluted to 5.0 10 3 CFU/mL. 50 L of diluted extract were added in 50 L of RPMI-1640 in microplates (96 wells) + 100 L of microorganisms. After that, the samples were incubated at 37 C for 24-48 hours, in duplicate. Fluconazol was used as control standard in concentrations ranging from 64-0.125 g/mL. The microplates were incubated at 37 C for 48 hours.

3. Results

All Candida species in the in vitro test presented sensitivity to the plant extracts in use, though not for all extracts (Table 1).

4. Discussion
Because of the increasing development of drug resistance to human pathogens and the appearance of undesirable effects of certain antifungal agents, the search for new antimicrobial agents is of great concern today (Phongpaichit et al., 2005). A multidisciplinary approach to drug discovery, involving the generation of truly novel molecular diversity from natural product sources combined with total and combinatorial synthetic methodologies, and including the manipulation of biosynthetic pathways, provides the best solution to the current productivity crisis facing the scientific community engaged in drug discovery and development (Newman and Cragg, 2007). Our results revealed a strong activity of Punica granatum, Syzygium cumini and Rosmarinus officinalis(dichloromethane and methanol extracts), Arrabidaea chica (dichloromethane extract), Mentha piperita andTabebuia avellanedae (methanol extract), with MIC varying from 0.06 to 0.001 mg/mL; and no or less activity ofArrabidaea chica (methanol extract), Mentha piperita and Tabebuia avellanedae (dichloromethane extract). The methanol extract of A. chica showed activity against yeasts within 24 hours (data not shown). However, these yeasts showed resistance to this extract after 48 hours. Among the 10 yeasts used in this study, the most resistant were C. glabrata and C. utilis, and C. krusei and C. guilliermondii were the most sensitive strains to the tested extracts. Data obtained from other studies demonstrated positive results for these plants as well. Duraipandiyan et al. (2006) observed inhibitory effects of P. granatum, while Nascimento et al. (2000) showed activity of extracts ofP. granatum, S. cumini and R. officinalis against Candida albicans. Chandrasekaram and Venkatesalu (2004) reported effectiveness of methanol extract of S. cumini and Portillo et al. (2001) found positive results for the dichloromethane extract of T. avellanedae against yeast C. albicans, thus validating our results. On the other hand, Duarte et al. (2005) obtained moderate activity testing M. piperita oil against C. albicans while Duraipandiyan et al. (2006), Portillo et al. (2001) and Dulger and Gonuz (2004) observed resistance of the extracts of S. cumini, T. avellanedae (methanol extract) and R. officinalis (ethanolic extract), respectively. Although these results are not in accordance with ours, there may be many other contributing factors, such as the seasonal period when plants are collected. However, some compounds like tannins (P. granatum, A. chica, S. cumini and R. officinalis), anthocyanins (A. chica), flavonoids (A. chica and R. officinalis), naphtoquinones (T. avellanedae), menthol and menthone (M. piperita) are known to have antimicrobial properties against microorganisms (Alcerito et al., 2002; Cordeiro et al., 2006; Gershon, 1975; Hussein et al., 1997; Iscan et al., 2002; Wagner et al., 1989). Components like tannins found in plants may act on the cell membrane and precipitin proteins (Nawwar et al., 1994), which turns out to be a target for studies. The findings presented in this paper indicate that the extracts obtained from the selected plants had anti-Candida activity. Punica granatum and Syzygium cumini extracts exerted strong antifungal activity and can be a source for the development of new therapeutic agents, as they inhibited the growth of Candida. Subsequently, bioguided fractionation shall

be conducted on Puninca granatum to identify the active compounds against Candida genus species. Acknowledgements This study was supported by the Research Foundation for the State of So Paulo (Fundao de Apoio Pesquisa do Estado de So Paulo FAPESP) and the Fund for Education, Research and Extension (Fundo de Apoio ao Ensino, Pesquisa e Extenso FAEPEX).

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Received April 16, 2009 Accepted January 12, 2010 Distributed November 30, 2010