Characterization of Candida spp. isolated from vaginal fluid: identification, antifungal susceptibility, and virulence profile

A total of 101 (20.0%) yeast samples were isolated from vaginal fluids of 504 nonhospitalized patients in Maringá, Paraná State, Brazil and Candida albicans was more frequent specie (93.1%) identified by seminested PCR method. All the isolates were susceptible to amphotericin B and nystatin, and 93.1% of them were susceptible to fluconazole. The acid proteinase, hemolytic and phospholipase activities were observed in 99.0, 90.0, and 88.0% of Candida spp., respectively. Around 67.0% of the strains had adherence indexes of 0.5 to 1.5 yeasts by Vero cell, and most of them showed a hydrophilic profile. Correlation studies indicated hydrophilic yeasts presented higher adherence index, proteinase, and phospholipase activities; and a positive correlation between all enzymes was also observed. In addition, the isolates with high hemolytic activity were less susceptible to fluconazole and amphotericin B. These results of Candida prevalence and antifungal susceptibility corroborate with literature’s datas and correlation between virulence factors and MIC values suggest Candida isolates from vaginal fluid less susceptible to antifungal and with higher extracellular enzymes production can be more virulent to cause tissue damage.


Introduction
Vulvovaginal candidiasis (VVC), a Candida infection, is a common disease of the lower genital tract in childbearing women.Despite therapeutic advances, VVC remains a common problem worldwide, occurring in all strata of society (SOBEL, 2007).It is estimated that 70.0-75.0% of all women will experience at least one symptomatic VVC episode during their lifetime.
A significant percentage of these women will experience subsequent episodes, and 5.0% will develop frequent recurrent vulvovaginal candidiasis (RVVC), defined as four or more episodes in one year.Candida spp.can be isolated from the vagina of about 20.0% of asymptomatic healthy women (SOBEL, 2007).Although Candida albicans is the predominant species isolated from vaginal samples (85-95%), Candida non-albicans (CNA) species have been reported with increasing frequency, mainly Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei (BAUTERS et al., 2002;LOPES-CONSOLARO et al., 2004).Interestingly, C. glabrata often is related with RCVV (SOBEL, 2007).
Several factors of the host can be related with VVC, such as genetic susceptibility to colonization by Candida species, pregnancy, diabetes mellitus, use of oral contraceptives with high estrogen content and use of broad spectrum antibiotics.Besides, immunological factors, as low innate and adaptative immunity of the host can be also related with Candida infection (SOBEL, 2007).In addition, many virulence factors of Candida spp.are involved in pathogenic process of VVC.The adherence on host cell and/or medical-devices surfaces is essential for microorganisms to successfully colonize a human host, and is an important step in the infection process.The ability of Candida to persist within the host and to cause infection has also been attributed to its capacity to grow in a range of physiological extremes, to produce hydrolytic enzymes (proteinases such as aspartyl proteinases, phospholipases, and hemolysins), the reversible transition between unicellular yeast and filamentous growth, and phenotypic switching (CALDERONE;FONZI, 2001;SILVA et al., 2011).
A limited number of antifungal agents including azoles (imidazoles and triazoles) and polyenes (amphotericin B and nystatin) are used for the treatment of Candida vaginitis, and strategies and drug combinations may be usefull to VVC therapy (SOBEL, 2007).In addition, oral azoles have a potential side-effect of systemic toxicity, which has dramatically restricted the use of ketoconazole (SOBEL, 2007).Some CNA species, such as C. krusei and C. glabrata, tend to be less susceptible to azoles, and the prolonged and repeated use of these antifungal can contribute to the emergence of resistant Candida strains (COLLIN et al., 1999).Besides the innate or acquired resistance of Candida spp., reduced mucosal immunity of host and inappropriate treatment can contribute to treatment failure (CANUTO; RODERO, 2002).
Diagnosis of VVC is frequently based on the history and physical examination, without corroborative evidence from laboratory tests (SOBEL, 2007) and often the VVC therapy is started without even specie identification, may due to conventional methods for the identification of Candida species are based on micromorphological and physiological traits, but these methods are laborious and time-consuming (KURTZMAN;FELL, 1998).In view of this, the aim of the present study was to identify isolates from vaginal fluid by molecular methods based on the polymerase chain reaction, and to determine their profile of susceptibility to amphotericin B, nystatin, and fluconazole.In addition, virulence factors of the Candida spp.isolates, such as the activity of extracellular enzymes (proteinases, phospholipases, and hemolysins), adherence on mammalian cells (Vero), and cell-surface hydrophobicity were investigated.(Reg. no. 026/2002).The specimens were collected from women who visited the gynecology health service during from September 2002 through March 2003, without previous underlying disease associated with immunodeficiency, including AIDS.These patients were sent to the Clinical Analysis Laboratory of Maringá, where samples of vaginal fluids were obtained by scraping the vaginal walls with a sterile cotton swab.Vaginal swabs were used for microscopy analysis to determine the presence of yeasts through Gram staining and culture on Sabouraud dextrose agar medium (Difco, Becton Dickinson Co., Sparks MD, USA) supplemented with 50 mg mL -1 of chloramphenicol (Sigma Chemical Co., Missouri, USA).The cultures were incubated at 37 o C, for 24-48h, under aerobic conditions.Each isolate was kept in sterile distilled water at 4-8ºC.

Yeasts identification
In order to perform a preliminary species identification, all isolates were cultured in CHROMagar Candida medium ® (CHROmagar Company, Paris, France), for 48h, at 37 o C. In addition, PCR-based methods for species identification were performed, using universal and species-specific primers for detection of Candida species as follow below.
DNA extraction: Genomic DNA extraction was performed according Jain et al. (2001) with some modifications.A single colony on SDA was subcultured in the broth Sabouraud dextrose at 37ºC for 48 h.The cells were treated with lysis´s buffer TENTS (10 mM Tris HCl pH 8.0 with 2% v v -1 Triton X-100, 1% SDS, 100 mM NaCl and 1 mM EDTA) and DNA purification was performed by extraction in phenol:chloroform:isoamilic acid (25:24:1, v v -1 ).

Antifungal susceptibility testing
The susceptibility of Candida spp.isolates to amphotericin B, nystatin (Sigma Chemical Co., Missouri, USA), and fluconazole (Pfizer, São Paulo, Brazil) were determined by broth microdilution method as described in document M27-A3 published by Clinical Laboratory Standards Institute (CLSI, 2008) with some modifications.Candida albicans (ATCC 10231) and Candida parapsilosis (ATCC 22019) were included in each experiment as standard strains.Briefly, serial two-fold dilution of the antifungal (amphotericin B, nystatin, and fluconazole) were performed in RPMI 1640 medium (Sigma Chemical Co., Missouri, USA), buffered with MOPS 0.16 M pH 7.0, into 96-well microtitre trays.After, the yeast inoculum was adjusted to 1-5 x 10 6 CFU mL -1 and an aliquot was dispensed into each well to obtain a final concentration of 0.5-2.5 x 10 3 CFU mL -1 .The susceptibility endpoints for polyenes were defined as the lowest concentrations of antifungal which resulted in total inhibition of growth.For fluconazole, minimum inhibitory concentrations of 50% (IC 50 ) and 90% (IC 90 ) were determined by reading the plates in a spectrophotometer (Bio-Tek ® PowerWave XS) at 492 nm (CLSI, 2008).

Cellular surface hydrophobicity (CSH)
Yeasts of Candida spp.grown in Sabouraud dextrose broth for 24-48 h were harvested, washed with phosphate-buffered saline (PBS) at pH 7.4, resuspended in the same solution, and then the turbidity was read at 660 nm.An aliquot of 2.5 mL of this cell suspension was added to a sterile glass tube containing 1 mL of xylene.The tube was vigorously mixed for 2 min., and then left for 20 min at room temperature to allow separation of the two phases.The aqueous phase was collected and the turbidity was read at 660 nm.
The hydrophobicity index (HI) was calculated using the following equation: HI = (A 660nm control -A 660nm test ) x 100/A 660nm control , where A 660nm control = optical density of the samples before xylene treatment, and A 660nm test = optical density of the samples after xylene treatment (TEIXEIRA et al., 1993).
Cellular surface hydrophobicity of Candida isolates was classified as low CSH (HI < 30.0%) and high CSH (HI > 70.0%), which determined them as highly hydrophilic and hydrophobic cells, respectively.

Hydrolytic enzymes production
All analyses were carried out in minimum medium agar (6 g NaNO 3 , 1.5 g KH 2 PO 4 , 0.5 g KCl, 0.5 g MgSO 4 .7H 2 O, 0.01 g FeSO 4 , 0.01 g ZnSO 4 , 10 g glucose, and 20 g agar-agar per 1,000 mL), supplemented with: 0.6% skim milk, pH 4.0 for acid proteinase activity; 4.0% egg yolk, 350 mM NaCl and 6.5 mM CaCl 2 , pH 4.5 for phospholipase activity; and 7.0% sheep blood for hemolytic activity.Candida spp.cultures 24-48 h old in Sabouraud dextrose broth were used to determine the enzymatic activities.Yeasts were centrifuged and washed with PBS, counted in an improved Neubauer chamber to obtain a yeast suspension of 1 x 10 6 CFU mL -1 .An aliquot of 10 μL of this fungal suspension was placed on the surface of the minimum medium and the cultures were incubated at 37 o C for 96h for acid proteinase and phospholipase activities.To determine hemolytic activity, the plates were incubated in atmosphere at 5% CO 2 , at 37 o C, for 96h (LUO et al., 2001;RUCHEL et al., 1982;SAMARANAYAKE et al., 1984).
Candida isolates that showed degradation or a precipitation halo around the colony were considered positive for enzyme production.The enzymes activities were expressed as Pz values (a/b), where meaning the ratio of the colony diameter (a) to the degradation halo diameter around the colony (b).According this definition, low Pz values mean high enzyme production and, inversely, high Pz values indicate low enzymatic production.The Pz value was scored into four categories: 0.00 -0.39: high activity, 0.4 -0.69: moderate activity, 0.70 -0.99: low activity and 1.00: without activity (PRICE et al., 1982).
Candida albicans (ATCC 10231) was used as a positive control for the determination of acid proteinase and phospholipase activity, and the bacteria Aeromonas hydrophila (ATCC 7966) for hemolytic activity.

Statistical analysis
Statistical analyses were performed with the program Statistic for Windows, version 6.0 (Statsoft, Inc., Oklahoma City, Oklahoma, USA) and a p-value less than 0.05 was regarded as significant.The Pearson's test was performed to correlate enzymes activities and MIC values.

Species distribution
One hundred and one yeasts (20.0%) were isolated from vaginal fluids of the 504 women between 13 and 80 years old.In microscopic analyses by Gram's stain, the presence of yeasts was detected in only 81 (16.0%) samples of the vaginal fluid.There was an agreement of 78.6% in detecting yeasts by culturing on Sabouraud dextrose agar and by microscopic analysis.
In the identification by the snPCR method, C. albicans occurred in 93.1% of samples (94/101), and CNA in 5.7% (6/101), consisting of one C. parapsilosis, one C. tropicalis, and 4 Candida sp.(non-albicans, non-parapsilosis, non-tropicalis, and non-glabrata).One isolate was not identified as a member of the Candida genus.

Extracellular Enzymes Production
After 72h of Candida growth on supplemented minimum medium at 37ºC, we observed almost all the isolates (99.0%) produced acid protease with Pz values below 0.3 characterizing high enzymatic activity.The hemolysis was observed in 90.0% of isolates being 38.0% with high production and 39.0% with moderate activity, whilst the phospholipase production occurred in 88.0% of Candida spp., where the most of them (74.0%)presented moderate activity (0.4 < Pz < 0.69) (Table 2).Among enzymes production, the acid protease presented the best enzymatic activity in comparison with hemolysin and phospholipase (p < 0.0001) followed by hemolytic activity when compared with phospholipase (p < 0.0001) (Figure 1).

Correlations
The hydrophobicity index showed a negative correlation with adherence on Vero cells (r = -0.3)and positive correlation with proteinase and phospholipase Pz values (r = 0.3 and r = 0.4, respectively).The hemolytic activity had positive correlations with acid proteinase and phospholipase (r = 0.4 and r = 0.3, respectively), and proteinase and phospholipase also presented positive correlation between them (r = 0.3).In addition, the hemolytic activity correlated negatively with fluconazole and amphotericin B MIC values (r = -0.2 for both antifungal), suggesting that isolates with hemolysin high production were less susceptible to antifungal agents.

Discussion
In this study, 101 yeasts were isolated from 504 fluid vaginal samples (20.0%) of symptomatic and asymptomatic patients and this frequency was consistent with previously published data (ECKERT et al., 1998;LOPES-CONSOLARO et al., 2004).C. albicans was more frequent specie in vaginal fluid samples; it was identified in 93.1% of isolates by snPCR method.Although research had shown C. albicans is responsible for around 90.0% of symptomatic VVC (SOBEL, 2007), CNA species isolation had increased (RUHNKE, 2006).Different morphological and physiological properties of C. albicans strains have been reported by other investigators (KENNEDY et al., 1988;LAN et al., 2002) and these different properties can make species identification difficult.Yeasts have traditionally been classified on the basis of their morphological, physiological and biochemical properties (KURTZMAN; FELL, 1998), however, this is complex and time consuming, and can lead to incorrect classification.Phenotypic identification methods are broadly supplemented by new genotypic methods, which allow a more refined and detailed differentiation of related microorganisms (BUSCH; NITSCHKO, 1999).The gene amplification technology by snPCR used in this study, can detect members of the genus Candida by means of a specific and conserved nucleotide sequence, in region 5.8S and 28S rDNA.This methodology has shown specificity and sensitivity for Candida species (AHMAD et al., 2002).
In this study, all vaginal isolates were susceptible to amphotericin B and nystatin, in agreement with previously reported data (GUALCO et al., 2007).Even though polyenes were widely used for more than 40 years in many fungal infections, the incidence of resistance in Candida spp. is rare (ELLIS, 2002).On the other hand, resistance of Candida spp. to azoles continues to increase in response to the widespread application of triazole therapeutics, although few fluconazole-resistant strains have been isolated from VVC cases (SOBEL, 2007).In agreement with previous studies (GROSS et al., 2007;SOBEL, 2007), we demonstrated resistance to fluconazole in 5.9% of the isolates from vaginal fluid.
For some human pathogenic fungi the virulence factors to have been selected for environmental survival and function in mammalian virulence more by accident or happenstance (CASADEVALL, 2007).These events may be regulated by different pathways, but MAP kinases are key elements that control of essential virulence factors such as invasion and oxidative stress in C. albicans.MAP kinases sense different activating signals, which enable them to integrate, amplify and modulate the appropriate protective and adaptive response (ROMÁN et al., 2007).Several virulence factors of Candida species can be related to candidiasis pathogenesis, such as CSH, adhesion, hydrolytic enzymes production, and morphogenesis (CALDERONE;FONZI, 2001;SILVA et al., 2011).
The adhesion is an extremely important step for establishing of this infection, colonization and tissue invasion by digesting and destroying cell membranes and/or extracellular matrix components; and the extent of adhesion is dependent on microbial, host and abiotic surfaces properties, such as CSH and fungal cell wall composition (SILVA et al., 2011).In this work, 68% of vaginal isolates showed hydrophilic characteristics and adherence capacity occurred of 0.04 to 3.86 yeasts/Vero cells.Several reports have shown that CSH can be involved in factors related to pathogenesis, such as the cell wall, germ-tube formation, adhesion to cells and matrix proteins, and phagocytosis (HAZEN, 1989).Reports have indicated that CSH is an important factor for adhesion mechanisms of yeasts in cells (ENER; DOUGLAS, 1992), showing hydrophobic yeasts present more adherence than hydrophilic yeasts.However, we show a negative correlation between CSH and adherence on Vero cells.Kennedy et al. (1988) had similar results, which demonstrated hydrophobic cells presented fewer adherences in buccal epithelial cells.This may suggest many factors can be involved in the adhesion process of Candida spp. on cells or tissues, in that CSH may not be predominant, and that effect of CSH on adherence depends on the yeast isolated (HAZEN, 1989;KENNEDY et al., 1988).On the other hand, other authors did not find a correlation between CSH and adhesion of Candida spp.isolates on urinary catheters, demonstrating that CSH alone was not a predictor of adhesion (CAMACHO et al., 2007).An important factor that has correlated with adhesion ability of Candida spp.and biofilm formation is the presence of specific cell-wall proteins, often referred to as adhesins, being agglutinin-like sequence (ALS) a major Candida adhesins group (SILVA et al., 2011).
Many microorganisms produce hydrolytic enzymes to obtain nutrients and assure their survival.However, the hydrolytic enzymes activities of several bacteria and fungi, including proteases, phospholipases and hemolysins are associated with adhesion, cell damage, tissue invasion and blood dissemination.Most of the strains from vaginal fluid isolated in this work presented proteinase, phospholipase, and hemolysis production.Interestingly, we observed positive correlations between all enzymes analyzed; meaning Candida isolates that present high acid proteinase production also are characterized by be hemolysins and phospholipases good producers.
The secretion of aspartyl proteinases (Sap1-Sap10) by C. albicans is recognized as an important virulence determinant for this specie.Saps facilitate colonization and invasion of host tissue through the disruption of host mucosal membranes and by degrading important immunological and structural defenses proteins (PICHOVÁ et al., 2001;SILVA et al., 2011).Beyond Saps, phospholipases had been described for Candida species and are enzymes that hydrolyze phospholipids to fatty acids contributing to host cell-membrane damage, which promote cell damage and/or expose receptors to facilitate adherence of Candida (GHANNOUM, 2000).Several studies have suggested that Candida isolates that present increased production of proteinases and phospholipases, may have more ability to cause infection, invasion, and tissue damage (GHANNOUM, 2000;SUGITA et al., 2002).
Pathogenic microorganisms can grow in the host by using hemin or hemoglobin as a source of iron by hemolysins action.These enzymes are likely to be key virulence factors as might promote pathogen survival, persistence in the host and can facilitate the microorganism's dissemination (BONASSOLI et al., 2005;MANNS et al., 1994;SILVA et al., 2011;TAKADA et al., 2003).Few studies on the hemolytic activity of yeasts of the Candida genus have been reported.Luo et al. (2001) andBonassoli et al. (2005) showed that some Candida species produced hemolysin in vitro.In the isolates examined in this study, high hemolytic activity was correlated with lower susceptibility to amphtericin B and fluconazole.Previous study demonstrated  2006;SAMARANAYAKE et al., 1984).These enzymes are also correlated with increase of pathogenesis and overexpression of a gene encoding a multidrug resistance (MDR1) efflux pump (WU et al., 2000).

Conclusion
We observed 20.0% of the vaginal fluid samples in symptomatic and asymptomatic patients showed a positive culture on Sabouraud dextrose agar, and around 90.0% of the isolates were identified as C. albicans by snPCR method.All isolates were susceptible to polyene agents and resistance to fluconazol was rare (5.9%).Moreover, we showed virulence factors of Candida isolates (such as CSH, adherence and hydrolytic enzymes activity) had significant correlations between them, which may be related with Candida pathogenicity and lower susceptibility to amphotericin B and fluconazole.
This study was carried out at the Laboratory of Applied Microbiology at Natural and Synthetic Products of the State University of Maringá, Paraná State, Brazil.The study was approved by the local Ethics Committee on Research involving Humans

Table 1 .
Minimum inhibitory concentrations (MICs) of antifungal agents and susceptibility profiles of yeasts isolated from vaginal fluid.

Table 2 .
Virulence profiles of Candida spp.isolated from vaginal fluid: adherence on mammalian cells, cellular surface hidrophobicity, and hydrolytic enzymes activity (acid proteinase, phospholipase and hemolytic activity).