Distribution of secreted aspartyl protease ( SAP ) virulence genes and antifungal resistance genes at vulvovaginal candidiasis isolates

Virulence factors and antifungal resistance features of Candida albicans considering a growing health problem worldwide. This study made to show the expression of both virulence and azole resistance genes at 100 clinical isolates of Candida we used a model of infection of human vaginal epithelial cells with C. albicans strains isolated from Egyptian women with vulvovaginal candidiasis (VVC). The detection and expression of virulence genes and azole resistance genes were performed using PCR technic. All isolates were susceptible to ketoconazole (KTC), 3 isolates (3%) only were resist to both nystatin (NY) and amphotericin B (AMB) ,all isolates found resist to griseofulvin (AGF) 10 μg, eighty five isolates (85%)were resist to flucytosine (AFY), four isolates (4%)were resist to miconazole (MCL), seventy one isolates (71%) were resist to voriconazole (VO), thirty three isolates (33 %) were resist to itraconazole (ITC), forty one isolates and twenty seven isolates (27%)were resist to 100 μg fluconazole (flu). All isolated strains expressed SAP4SAP6 (100%) and almost all expressed SAP1-SAP3 (91%) In this study, fluconazole resistance was identified in 27% of the strains, whereas (27%) had positive ERG11 gene, (27%) were positive MDR1 gene and (14%) were positive CDR1 gene. The results indicate that the strains that infect Egyptian patients suffering from VVC are highly virulent and virtually all are insensitive to fluconazole.


Introduction
Candida albicans is a domain fungal species of the human microbiota and asymptomatically colonizes healthy people.Also, it is an opportunistic microorganism which can cause fatal bloodstream infections [1].C. albicans is the prevalent cause of invasion the fungal infections [2] and represents a serious challenge with increasing the medical and the economic importance due to the high mortality rates and increased costs of care and duration of hospitalization [3], [4].Clinical strains of C. albicans have many virulence genes that directly influence the pathogenesis of vulvovaginal candidiasis (VVC), including genes responsible for phenotypic switching [5], HWP1 (hyphal wall protein 1) [6] ALS(agglutinin-like sequence) [7], SAP (secreted aspartyl proteases), PL (phospholipases) [8], and LIP (lipases) [9] The aspartyl proteases, phospholipases, and lipases are involved in the breakdown of cell membranes in host epithelia, which promotes colonization and invasion [10].
The raising of C. albicans resistant to antifungal agents, spicially to azoles, is a serious health problem and hampers the treatment of VVC.Many mechanisms responsible for azoles resistance include overexpression of CDR1 and CDR2 from the ABC transporter family and MDR1 which encodes a multidrug efflux pump.Mutations in ERG5 and ERG11, which cause alterations in C22-desaturase and sterol 14α-demethylase, respectively, are also associated with azole resistance [11].
The expression of ALS and SAP gene families in C. albicans of vaginal origin has recently been studied [12][13].Secreted aspartyl proteinases (Saps), encoded by the SAP gene family with 10 members, (SAP1-SAP10) have a major role in C. albicans virulence during fungal infections [14][15].Development of molecular techniques for targeted gene disruption in diploid eukaryotic organisms recently gives a powerful genetic tool to address virulence studies in C. albicans.Destruction of SAP1-SAP3 genes affected the vaginopathic potential of the strain was previously reported in vitro studies [16].Using Sap-deficient mutants, different researchers showed that Sap1-Sap 3 have major role in experimental mucosal infections.On the other hand, Sap4-Sap6 appear to be critical for systemic infections [17][18].
The aim of our study was to investigate the Sap distribution among different C. albicans isolates using SAP specific primers in polymerase chain reaction (PCR) assay then relationship with resistance gens distribution among our isolates.

Patients analyzed and sampling
One hundred and fifty-seven (157) vaginal swabs were obtained from women aged from (15 to 45 years old) which clinically diagnosed of genitourinary tract infections attending to AL-Sabaa Banat health care unit and obstetrics and gynecology private clinic.The lower vagina (vaginal introitus) was swabbed.One vaginal swab was collected from each patient with an informed consent.
The vaginal swabs were placed into Sabouraud dextrose broth (SDB) (Difico, USA) transporting media.The transporting media maintained Candida viability for up to 4 days at room temperature or under refrigeration [19].SDB (Difico, USA) Transporting media was prepared according to manufacturer's instruction: 30 grams were suspended in 1000 ml of cold distilled water heated to dissolve, sterilized by autoclaving then distributed into 15ml capped plastic tubes and stored in a cool place.Sterile cotton-tipped swabs were used to collect the specimen.Each vaginal swab then was pushed down the medium depth.Swabs were kept cool, in an insulated box containing sufficient ice packs, during the transport period [19].
Amplification procedure.The standard PCR mixture contained 0.5 U of Taq DNA polymerase, 200 mol/L of deoxynucleoside triphosphates, 10 pmol of each primer, 25 mmol/L of MgCl2, 10× of buffer in 25 L of PCR mixture.PCR was performed in a thermal cycler (Techne, UK) was done as follows: 5 min at 95 °C, 30 cycles of 30 s at 95 °C, 45 s at 58 °C, 1 min at 72 °C, then terminal step (5 min, 72 °C), and the mixture was held at 4 °C.The PCR products were separated by 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized with UV light [17], [25][26].SAP1-SAP3 has band at 253 bp, SAP4 give band at 106 bp, while SAP5 band observed at 267 bp and SAP6 give positive product at 314 bp [24].

Identification of C. albicans and antifungal resistance phenotypes
The present study was conducted with a total of 157 women clinically diagnosed of genitourinary tract infections and by using the conventional culture technique only 100 isolates were identified as Candida with a prevalence rate (64%).
Table 5 Percentage of resistance for antifungal agents

Discussion
In this study, we isolated and identified both resistance and SAP virulence gene of C. albicans strains in vaginal samples.VVC is a condition that affects a quarter of young women [27].The chronicity of VVC or recurrent episodes by C. albicans is due to prolonged use of antifungal agents, which select for resistant strains, and due to the many virulence genotypes that enhance the adhesion to epithelial cells, production of hydrolytic enzymes, colonization, and invasion [10].The observation in this study that C. albicans had the high incidence rate (63.69%) among the yeast isolates studied agrees with the reports of other workers [28], [29].
This study used highly sensitive method for the detection of C. albicans SAP-expression directly in yeast DNA samples.
To investigate each Sap isoenzyme, we individually used SAP specific primers in PCR screening.We used 4 different specific primer pairs for SAP1-SAP3, SAP4, SAP5 and SAP6 [24].Likewise, all isolated strains expressed SAP4-SAP6 (100 %) and almost all expressed SAP1-SAP3 (91%).These results are similar to those previously described for SAP expression in VVC in humans [30].The expression of SAP4-SAP6 has a relevant role in pathogenesis by promoting hyphae formation [31] and by the inhibition of phagocytosis [32].
The use of azoles for the treatment of VVC for prolonged time has resulted in the promote of strains resistant to these agents [28].In this study, fluconazole resistance was identified in 27% of the strains (Table 5), whereas (27%) had positive ERG11 gene, (27%) were positive MDR1 gene and (14%) were positive CDR1 gene.The high frequencies of strains resistant to fluconazole can be explained by many such as incomplete therapy, over growth of resistant strains, and induction of drug resistance in the particular species, colonization and subsequent infection with a resistant organism [33], [34].The reason in our study is due to self-treatment and drug abuse of the patient.Therefore, in vitro testing of the susceptibility of yeasts to antifungal agents will likely play an ever-increasing role in the appropriate selection of antifungal agents for the treatment of fungal infections.Nonetheless, the high susceptibility rate of Candida species to ketoconazole drugs so can use as azole antifungal agent for treatment genitourinary tract infections among women in Egypt.

Conclusion
The results show that the strains that infect Egyptian women suffering from VVC are highly virulent and virtually all are insensitive to fluconazole.These results help in identification of resistance and virulence patterns of strains causing VVC in Egypt.Further researches on different agents are required to solve this issue.

Figure 1
Figure 1 Identification by multiplex PCR

Table 1
Primer sequences and PCR conditions for multiplex PCR

Table 3
Primer sequences used in PCR