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Introduction
The infections related to opportunistic yeasts have increased in recent years. The main reason is the high prevalence of diseases related to opportunistic yeasts among the nosocomial infections and immune-compromised patients1 . Candidiasis is containing a broad spectrum of opportunistic diseases that cause superficial skin infections to systemic ones in susceptible patients. Candidiasis is one of important infectious diseases in the world, which compromise high percent of nosocomial infections in intensive care units and total nosocomial infections2 . Candida sp. and especially C. albicans can colonize the vagina and cause vulvovaginal candidiasis. Vulvovaginal candidiasis is the second prevalent infection of female reproductive systems after bacterial vaginitis by Gardnerella vaginalis. Seventy five percent of women are infected by vulvovaginal candidiasis, which can manifest as acute, chronic or recurrent ones3,4. Local treatments with Nizarol, Clotrimazole, Miconazole as the first line treatments are used for management of acute ones. The prolonged local/systematic therapy for at least six month is recommended for chronic, recurrent and resistant vulvovaginal candidiasis5 . 138 million women worldwide annually were affected by recurrent vulvovaginal candidiasis, which 492 million of women are affected during their lifetimes. The patients with recurrent vulvovaginal candidiasis experience repeated episodes of vaginitis6 . The resistant C. albicans strains7 , and the adverse effects related to these treatments8 have encouraged the scientists to search among the essential oils as secondary metabolites of medicinal plants9,10. Ziziphora tenuior, Lavandula angustifolia, Cuminum cyminum are valuable plants for extracting the essential oils. In this investigation, we isolated 80 clinical isolates of C. albicans form women with vulvovaginal candidiasis. Then, the antifungal and anti-biofilm activities of Z. tenuior, L. angustifolia, C. cyminum essential oils were compared on these clinical isolates.
Materials and methods
Essential oils and their specifications
The essential oils were prepared by hydro-distillation method11 in Clevenger type apparatus according to producer’s certificate of analysis. Z. tenuior essential oil with main components of pulegone (37.9%), carvacrol (16%) and thymol (5.9%), C. cyminum essential oil containing cumin aldehyde (25.77%) as the main compounds, and L. angustifolia essential oil with main components of 1,8-cineol (36.6%), limonene (12.43%), linalool (9.24%), and terpinen-4-ol (0.72%) were prepared from TabibDaru Pharmaceutical Company, Kashan, Iran.
Clinical isolates of Candida albicans strains
The fungal strains were isolated from vaginal discharges of 237 women with vulvovaginal candidiasis (22-45 years old). The samples were observed directly by light microscope. The samples with conical cells, mycelium or buds were cultured on Sabauraud dextrose agar with gentamicin and chloramphenicol (Conda Media culture). The plates were incubated at 25 °C for 48-72 h. The plates with creamy colony were screened by biochemical tests (Gram staining, germ tube and chlamydospore formation) 12 and molecular analysis by PCRRFLP with ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers. C. albicans ATCC 10231 was used as control strain13.
Anti-Candidal activity of essential oils against clinical isolates of C. albicans
The antimicrobial evaluations of essential oils against clinical isolates of C. albicans strains were evaluated by disc diffusion and micro-broth dilution assays.
The confirmed clinical isolates of C. albicans were cultured on Sabouraud dextrose at 37 °C for 72 h. One or two colonies of each strain were suspended in sterilized normal saline and their turbidities were adjusted to 0.5 McFarland by spectrophotometric method (1×106 CFU/ml=transmittance 85% at 590 nm). The suspension was cultured on Sabauraud dextrose agar by sterile cotton swab and discs containing different concentrations of essential oils in DMSO as solvent (2.5, 5, 10 μl/disc) were put on cultured media. DMSO and amphotericin B discs were used as controls. The plates were incubated at 35±2 °C for 72 h. After incubations, the inhibition zone diameters (IZ) were measured and the results were reported as means ±SD (Standard deviations). The experiments were performed in triplicates.
The micro-broth dilution assay was performed by diluting the dissolved essential oil in RPMI 1640 at concentrations of 32- 0.125 μl/ml. The Candidal suspension were diluted to 1×105 CFU/ml. 100 μl diluted essential oil and Candidal strain were mixed in wells of 96 microtiter plates. The plates were incubated at above conditions and the first well with inhibitory effect on growth of C. albicans was reported as MIC (minimal Inhibitory Concentration) and the first well with no growth on solid media is defined as MFC (Minimal Fungicidal effect). The means of MIC and MFC values were determined and reported14. Amphotericin B 10 μg/disc (Rosco Diagnostica) and powder (Sigma) was used as positive control.
Determining the capability of C. albicans strains in biofilm formation
100 μl of suspension containing 106 CFU/ml C. albicans in RPMI were poured in the wells of 96 micro titer plates. The wells containing culture media with fungal strain and C. albicans ATCC 10231 were used as negative and positive wells, respectively. The plates were incubated at 25 °C for 48 h. After incubation, the plates were washed by 200 μl PBS, and dried. The plates were dyed by Crystal violet and washed by acetic acid 30% and read at 550 nm15. The percent of biofilm formation was determined in comparison with C. albicans ATCC 10231 and negative wells.
Anti-biofilm activity of essential oils against the strains with high potency in biofilm formation
After determining the best strains in producing the biofilm, the biofilms were established by inoculating the fungal suspension (106 CFU/mL) into the wells and incubating at 37 ºC for 24 h as above. After that, the culture media were removed and the wells were washed with distilled water to remove the planktonic cells. The diluted essential oils (16-0.125 µl/ mL) were added to wells and they were incubated at 37 ºC for 24 h, again. The biofilm staining with crystal violet and estimating the biofilm killing effects was performed and the biofilm killing effects of each compounds were estimated by determining the OD550 of each well in comparison with control wells (fungal wells without essential oil) (16.
Statistical analysis
The difference between the antimicrobial activities of essential oils by disc diffusion and micro-broth dilution assays were determined by SPSS software (version 21.0). One-way analysis of variance (ANOVA) is used to determine the statistically significant differences between the means of independent (unrelated) groups at level of 0.05 (pvalue<0.05).
Results
237 vaginal discharges were gathered from the infected women, which 109 samples showed conical yeast cells with buds in direct microscopically evaluations. After culturing these samples on Sabouraud dextrose agars and identification tests, 80 samples showed the presence of C. albicans. C. albicans is the most prevalent etiological agent of acute vulvovaginal infections17. As our results showed 73.3% of isolated Candida strains was belonged to C. albicans strains. Rather than vulvovaginal candidiasis, C. albicans as opportunistic yeast is the major cause of oral candidiasis in immunocompromised individuals, or superficial skin infections or systemic candidiasis18. Therefore, the essential oils with antifungal activity against C. albicans strains can be a suitable alternative or complementary treatments in these patients. Evaluating the anti-Candidal effects of essential oils against 80 clinical isolates of C. albicans showed the dose response for anti-Candidal activity of each evaluated essential oil. Regardless the essential oil concentration, the inhibition zone diameters of C. cyminum essential oil (21.9 mm) statistically was higher than that of Z. tenuior essential oil (21.05 mm), while L. angustifolia oil (8.48 mm) had the lowest inhibition zone diameter against clinical isolates of C. albicans. 10 μl/ disc Z. tenuior essential oil had the higher inhibition zone diameter (29.5±5.74 mm) than that of C. cyminum essential oil (27.5±7.7 mm) at the same concentration (Table 1). The inhibition zone diameter for 10 µg amphotericin B was 22.0±3.3 mm.
Table 1 The anti Candidal activity of essential oils against clinical strains of C. albicans (n=80) by disc diffusion method (diameter in millimeter)
The significance at the level 0.05 (pvalue<0.05)
As table 2 is shown, determining the means of MIC and MFC values for clinical isolates of C. albicans in micro-broth dilution assay, showed that three samples of essential oils were belonged to two subsets. C. cyminum and Z. tenuior essential oils showed non-significant anti-Candidal effects against 80 clinical isolates of C. albicans (p>0.05). The MIC values for C. cyminum and Z. tenuior essential oils were 6.0±3.58 and 6.3±2.75 μl/ml, respectively. The corresponding MFC values were 9.58±6.51 and 10.0±3.48 μl/ml, respectively. L. angustifolia essential oil showed less anti-Candidal activity with MIC and MFC values of 10.3±3.64, 18.4±5.8 μl/ml, respectively. The MIC and MFC values for amphotericin B were 4.29±3.27 and 4.94±3.01 µg/ml, respectively. The higher anti-Candidal activity of C. cyminum and Z. tenuior essential oils were related to their main components. C. cyminum essential oil with main components of cumin aldehyde and Z. tenuior essential oil with main components of pulegone (37.9%), carvacrol (16%) and thymol (5.9%) are belonged to the essential oils with high antimicrobial activities19. The antimicrobial activity of C. cyminum essential oils was evaluated in different studies20-24. The anti-Candidal effects of C. cyminum essential oils with α-pinene (30%), limonene (21%), and 1,8-cineole (18.5%) against C. albicans ATCC 10231 with MIC value of 289 μg/ml25 was confirmed, also C. cyminum essential oil with cuminic alcohol (30.3%), γ-terpinene (25.3%) and cuminic aldehyde (11.2%) showed the less activity against the Candida albicans ATCC 1023125. There is one report on anti-Candidal effect of C. cyminum essential oil with γ-terpinene (21.1%) against clinical isolates of C. albicans from recurrent vulvovaginal candidiasis with MIC value of 8.0±1.89 μg/ml20. The difference between our results and the last investigation is about the main components of C. cyminum essential oil and the MIC values (5.4 mg/ml vs. 8.0 μg/ml). According to the density of C. cyminum essential oil (0.904 g/mL), the means of MIC and MFC values of our essential oils were 5.4±3.2 and 8.6±5.8 mg/ml against clinical isolates of C. albicans from vulvovaginal Candidiasis. Z. tenuior essential oil with main components of pulegone (46.8%), p-menth-3-en-8-ol (12.5%), isomenthone (6.6%) had 80% germ tube inhibitory effects against C. albicans at concentration of 0.16 μl/ml of Z. tenuior essential oil26.
Discussion
Although, the anti-Candidal effect of Z. tenuior essential oil against C. albicans has been compared with clotrimazole27, our investigation is the first study which compared the antiCandidal effects of three essential oils with main components belonged to different groups of chemical structures against clinical isolates of C. albicans.
C. albicans can cause life threatening biofilm-based-infections in many areas of the body, particularly in gastrointestinal and genitourinary organs of human body. These biofilms form on implanted medical devices and intrinsically are resistant to treatment more than planktonic ones28.
In this regard, evaluating the potency of strain in biofilm formation in comparison with C. albicans ATCC 10231 showed the strains had the less potency in formation of biofilms (Table 3 ). Therefore, 12 strains of C. albicans with higher potency in biofilm formation were chosen for further studies.
Table 3 The capability of C. albicans strains in biofilm formation in comparison with C. albicans ATCC 10231
Evaluating the biofilm killing effects of essential oils against clinical isolates of C. albicans showed the biofilm killing effects of essential oil were dose dependent. The higher concentrations of essential oils showed the higher biofilm killing effects. Among the screened essential oil, C. cyminum and L. angustifolia essential oils had the highest biofilm killing effects, followed by Z. tenuior essential oils (Table 4). Although, L. angustifolia showed the less antifungal activity than that of C. cyminum and Z. tenuior essential oil, but screening the biofilm killing effects of L. angustifolia essential oil was a little higher than C. cyminum essential oil, followed by Z. tenuior essential oil. The anti-biofilm effect of L. angustifolia essential oil with main components of linalool was confirmed against Staphylococcus aureus and Escherichia coli biofilms29. Understanding the precise mechanism related to anti-Candidal effects of essential oils on biofilms need more investigations and one hypothesis related to it, can be the nature of main component(s) or interactions of components and biofilm.
Table 4 Biofilm killing effects of essential oils (%) against clinical isolates of C. albicans (n=12)
In conclusion, our report is the first in vitro study, which compared the anti-Candidal effects of C. cyminum, Z. tenuior and L. angustifolia essential oils against 80 clinical isolates of C. albicans from women with vulvovaginal candidiasis. 73% of Candida isolated from vaginal discharge were belonged to C. albicans. 22.5% of these isolates produced biofilm between 10-22.5% in comparison with C. albicans ATCC 10231. The C. cyminum, Z. tenuior essential oils had the higher anti-candidal effects than that of L. angustifolia essential oil, while the biofilm killing effects of L. angustifolia essential oils was a less higher than C. cyminum essential oil, followed by Z. tenuior essential oil.
