In vitro antifungal resistance profile of Candida strains isolated from Saudi women suffering from vulvovaginitis

Background Vulvovaginal candidiasis (VVC) represents a universal health hazard that contributes to significant morbidity in women. Resistance of Candida to antifungal therapy has been reported as a public health problem. So, the objective of our current study is to detect resistance profile of different candidal strains. Methods In this study, isolated Candida strains were identified by conventional methods, confirmed by internal transcribed spacer (ITS) sequencing, and phylogenetically analyzed with reference strains in GenBank. Also, sensitivity of different Candida strains to common antifungal agents was evaluated by disc diffusion method. Results Candida albicans was identified as the most frequent strain (63%) followed by non-albicans strains, such as C. glabrata (20%), C. tropicalis (13%), and C. krusei (4%). Sensitivity of Candida strains (C. albicans, C. tropicalis and C. glabrata) to commonly used antifungal agents was evaluated through the disc diffusion method. C. glabrata was the most resistant strain and considered to be a multidrug-resistant pathogen, while both, C. albicans and C. tropicalis showed high susceptibility to terbinafine. In contrast, C. albicans showed resistance to fluconazole, clotrimazole, and nystatin, while C. tropicalis, considered as the most sensitive strain, was susceptible to all the antifungal agents tested except nystatin. Terbinafine was the most effective antifungal agent against both C. tropicalis and C. albicans, and hence its minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) for C. albicans and C. tropicalis were evaluated. MICs of terbinafine against C. albicans and C. tropicalis were 5 μg/ml and 2.5 μg/ml, while their MFCs were 10 μg/ml and 5 μg/ml, respectively. Conclusion The emergence of resistant Candida strains necessitates conduction of the antifungal susceptibility test prior to deciding the medication regime.


Background
Candida albicans, a commensal microorganism, is a part of the normal flora on mucosal surfaces of the human body such as the gastrointestinal, respiratory and genitourinary tracts [1]. Candida species constitute 20-50% of the normal flora colonizing the female genital tract with C. albicans, the causative agent of vaginal candidiasis, being predominant (about 80.5%) followed by Candida glabrata [2][3][4]. Establishment of fungal infection by the Candida sp. is mediated through virulence factors like transition between yeast and hyphal forms, formation of biofilms, secretion of hydrolytic enzymes and expression of invasion and adhesion proteins [5]. Hydrolytic enzymes such as hemolytic enzymes, lipases and phospholipases produced by the Candida sp. contribute to its virulence while the secreted aspartyl proteinases play a role in adherence, penetration and invasion of host

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European Journal of Medical Research tissues, inducing tissue damage, thereby aiding the establishment of infection [6,7]. Vulvovaginal candidiasis is characterized by multiple symptoms such as dyspareunia, pruritis, itching, soreness, and vaginal erythema, and affects majority of the women during their lifetime. The use of antibiotics, diabetes mellitus, pregnancy and immunodeficiency are all risk factors that disturb the vaginal microflora enabling the establishment of vaginal infection [8,9]. In addition, establishment of vulvovaginal candidiasis infection occurs due to many behavioral and host-dependent factors such as the use of oral contraceptives, sexual activity and other hygiene habits [10]. Significant morbidity in women worldwide due to recurrent vulvovaginal candidiasis (RVVC) caused by C. albicans has been reported [11]. Many of these infections may cause high mortality rates due to the development of resistance against antifungal agents [12]. Fluconazole is currently being used as an effective therapy to control RVVC, but the development of antibiotic resistant strains necessitates the discovery of new drugs [13]. Advent of multidrug-resistant C. albicans strains may lead to invasive candidiasis increasing the mortality and morbidity in hospitals [14]. Recently, incidence of vulvovaginal candidiasis in pregnant woman has been recorded to be significantly more than that in non-pregnant woman (28.2% and 7.9%, respectively) [15]. Similar results were confirmed by Bauters et al. [16], who reported vulvovaginal candidiasis incidence of 32% in pregnant and 19.3% in non-pregnant women. Nystatin exhibited a high antifungal activity against all the organisms tested while resistance against azole antifungal drugs varied between species. Only one strain of C. glabrata showed resistance to clotrimazole and fluconazole while others exhibited dose-dependent susceptibility to fluconazole [17]. A study conducted by Salehei et al. [18] showed isolated vaginal Candida strains to be highly susceptible to terbinafine, clotrimazole and miconazole, but resistant to fluconazole and econazole. Antifungal sensitivity test also indicated that clotrimazole was the most effective antifungal agent against 68 of the isolated Candida strains followed by nystatin (51) and fluconazole (29) [19]. The objective of our current study was to isolate Candida strains from vaginitis patients in Riyadh region and identify the most dominant strains. In addition, susceptibility of different strains to antifungal agents commonly used in treatment of vulvovaginal candidiasis was also evaluated.

Isolation of microorganisms causing vaginitis infection
Examination of a total of 394 vaginal swabs from pregnant (138) and non-pregnant women (256) suffering from vaginitis, collected from the Regional Laboratory at King Saud Medical City during June 2016 to June 2018, was performed. All the swabs were subjected to wet mount examination by rolling them on glass slides with one drop of saline solution (0.85%) for detection of Trichomonas vaginalis (the causative agent of trichomoniasis). The swabs were also subjected to Gram staining for the detection of bacterial vaginosis infections. The swabs were cultured on Sabouraud dextrose agar (SDA) medium supplemented with chloramphenicol (0.5 g/l), incubated at 37 °C for 48-72 h to isolate Candida vaginitis strains. All vaginal swabs were also cultured on mannitol salt agar, MacConkey agar, and blood agar for the isolation of Gram-negative and Gram-positive infectious bacterial strains.

Identification of isolated Candida strains
The isolated strains were preliminarily identified according to their cultural, microscopic, and chemical characteristics. The Candida albicans and non-albicans strains were differentiated by culturing them in CHROM agar medium as well as performing the germ tube test by inoculating Candida strains in human serum (0.5 ml) followed by incubation at 37 °C for 3 h. The identification of isolated Candida strains was confirmed using the API20C AUX kit and internal transcribed spacer (ITS) sequencing technique.

Molecular identification of concerned Candida strains
The identification of the isolated C. albicans strain and two non-albicans strains was confirmed by bidirectional sequencing technique. Genomic DNA was extracted using GeneJET Genomic DNA Purification Kit K0721 (Thermo Fisher Scientific, US). Polymerase chain reaction (PCR) using universal forward and reverse primers of ITS1 (5ʹ-TCC GTA GGT GAA CCT GCG G-3ʹ) and ITS4 (5ʹ-TCC TCC GCT TAT TGA TAT GC-3ʹ), respectively, was performed to amplify the ITS1-5.8S-ITS2 domain. Bidirectional sequencing of PCR products was performed by Macrogen (Korea). The obtained sequences were blasted to compare with reference strains in Gen-Bank. The sequences were submitted to GenBank (https ://www.ncbi.nlm.nih.gov/genba nk/) and their corresponding accession numbers were obtained. Comparison of sequences of isolated vaginal strains with the reference strains was achieved using MEGA 7 software and phylogenetic tree was built using the neighbor-joining method.

Antifungal susceptibility test
The disc diffusion method was used to check the sensitivity of the isolated vaginal candida strains (Candida albicans, C. tropicalis, C. glabrata) to different antifungal agents. Reference strains, Candida albicans (ATCC 18804), Candida glabrata (ATCC 15545), and Candida tropicalis (ATCC 13803) were used for quality control purposes. Antifungal drugs, namely, fluconazole (Pfizer, UK), terbinafine (Novartis, Switzerland), itraconazole (Janssen, Belgium), clotrimazole, and nystatin (Sigma Aldrich, USA) were dissolved in methanol using an ultrasound sonicator. The three Candida strains were subcultured onto SDA slants and incubated at 37 °C for 48 h. Microbial suspensions were prepared by harvesting the Candida growth in 5 ml of sterile saline water and the absorbance was adjusted to 30% at 560 nm using a spectrophotometer. The yeast cells were enumerated at the corresponding absorbance by the plate count technique, and the viable cell count was 10 7 CFU/ml for each Candida strain. Fifteen milliliter of SDA medium was sterilized, poured into sterile plates (as a basal layer), followed by 10 ml of seeded medium previously inoculated with microbial suspension (1 ml of 10 7 CFU/100 ml of medium) to obtain 10 5 CFU for each ml of the medium. Sterile filter paper discs (8 mm [20].

Determination of minimum inhibitory concentration (MIC) of the most effective antifungal drug (terbinafine) against isolated Candida vaginitis strains
Minimum inhibitory concentration is defined as the lowest concentration of antifungal agents against common Candida strains that inhibits their growth after 48 h of incubation. The most effective antifungal drug (terbinafine), which showed strong antimicrobial activity, was investigated to determine its MIC and to evaluate its efficiency in controlling Candida strains causing vaginitis. A disc diffusion method was used in which 10 ml of SDA medium was poured into sterile petri dishes as a base layer followed by 15 ml of medium seeded with microbial inoculum previously prepared (1 ml of 10 7 CFU of Candida suspension/100 ml of culture medium) to obtain a final concentration of 10 5 CFU/ml of medium. Petri dishes were allowed to solidify and sterile filter paper discs (8 mm

Sample collection and preliminary identification
About 205 vaginal swabs from the 394 clinical samples tested positive for vaginitis infection. Incidence of vaginitis in pregnant and non-pregnant women was 65.9% and 44.5%, respectively, as seen in Table 1. Candida vaginitis was the main cause of vaginal infections (58.5%), followed by bacterial vaginosis (41%) and trichomoniasis infections (0.5%). The prevalence of vulvovaginal candidiasis was higher in both, pregnant (64.8%) and non-pregnant women (53.5%), while bacterial vaginosis was more frequently observed in non-pregnant women than in pregnant women, as seen in Table 2. The isolated Candida vaginitis strains were preliminarily identified in order to determine the most predominant disease-causing strains. About 166 isolates of Candida vaginitis strains and 87 bacterial vaginosis strains were isolated, as shown in Table 3. High incidence of vaginal infections among pregnant (57.1%) and non-pregnant (34.2%) women in the age group of 26-35 years was observed (refer Table 4).

Identification of isolated Candida strains
Primary identification of the isolated Candida strains was achieved by germ tube test, CHROM agar and API20C AUX which revealed Candida albicans to be the most predominant strain (63%) followed by C. glabrata (20%), C. tropicalis (13%) and C. krusei (4%).

Antifungal susceptibility test
Disc diffusion method was used to evaluate resistance of C. albicans and non-albicans strains (C. tropicalis, C. glabrata) to common antifungal drugs. Antifungal sensitivity test revealed that C. albicans was highly sensitive to terbinafine and itraconazole drugs exhibiting inhibition zone diameters of 27.2 and 17.9 mm, respectively, while its resistance to clotrimazole, nystatin and fluconazole was interpreted according to CLSI guidelines (refer Table 5). Candida tropicalis showed resistance to nystatin, but was highly sensitive to terbinafine, fluconazole and clotrimazole, exhibiting inhibition zone diameters of 24.4, 24.1 and 21.2 mm, respectively, as seen in Table 6. Its sensitivity to itraconazole was dose dependent. Candida glabrata showed resistance to all antifungal drugs used in the current study.

Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)
Terbinafine was identified to be the most effective antifungal drug against C. albicans and C. tropicalis with MIC values of 5 and 2.5 μg/ml and inhibition zone diameters of 9.2 and 11.2 mm, respectively (refer Table 7). Candida tropicalis was more sensitive to terbinafine compared to C. albicans as shown in Fig. 4. MFC of   terbinafine against C. albicans was 10 μg/ml while it was 5 μg/ml for C. tropicalis. MFC results confirmed that C. tropicalis was more susceptible to terbinafine than C. albicans.

Discussion
Approximately, 58.5% of the women examined in this study suffered from vulvovaginal candidiasis episodes. This result is in accordance with that of Kamath et al. [21] who recorded 47.7% of pregnant women to be infected with vulvovaginal candidiasis and the disease incidence in non-pregnant woman to be 20.3%. The high infection rate among pregnant woman may be attributed to higher secretion of sex hormones during pregnancy, especially during the last trimester [16,21,22]. The present study identified C. albicans to be the predominant causative agent of vulvovaginal candidiasis disease (63%) followed by non-albicans strains C. glabrata (20%) and C. tropicalis (13%). Our study results are in agreement with that of Amouri et al. [23], who ascertained that C. albicans represented the predominant strain (76.3%) followed by C. glabrata (19.3%) and C. tropicalis (1.4%) among the strains causing vulvovaginal candidiasis. Preliminary identification of vaginal yeast strains from vulvovaginal candidiasis patients showed that C. glabrata represented the second most dominant strain and this coincides with the results obtained by Richter et al. [12], Mahmoudabadi et al. [24] and Hedayati et al. [25]. Internal transcribed spacer (ITS) sequencing is considered as a rapid and accurate tool for identification of fungal pathogens [26]. Genetic variation within ITS region is considered to be sufficient for identification and typing of different fungal strains [27][28][29]. Azoles and allylamines (terbinafine) act as antifungal agents by inhibition of ergosterol biosynthesis while polyenes (nystatin) disrupt cell functions by binding to ergosterol in plasma membrane [30,31]. Moreover, allylamines disrupt synthesis of ergosterol synthesis through inhibition of squalene epoxidase enzyme [32]. Resistance of C. albicans to fluconazole antifungal drug was recorded in current study as seen in Fig. 4 and this may be due to its use for longterm treatment [33,34]. Similar result was obtained by Scocozza et al. [35] who reported the resistance of C. albicans strains to fluconazole. C. glabrata showed high resistance to azole drugs similar to that observed in previous studies recorded by Oxman et al. [36] and Pfaller et al. [37]. Resistance of C. albicans and C. glabrata to azole antifungal drugs may be due to several factors including the induction of drug efflux pumps and sequestration of antifungal agents [38][39][40]. C.   albicans and C. tropicalis resistance to nystatin has also been previously demonstrated in studies conducted by Khan et al. [19]. C. tropicalis isolated from infected patients show high sensitivity to terbinafine and clotrimazole and these results were in accordance with the results of Salehi et al. [18]. Terbinafine was highly effective against C. albicans and C. tropicalis strains with MIC values 2.5 and 5 µg/ml, respectively, as shown in Table 7. Similar results have been reported by several researchers and hence terbinafine is the first drug of choice in treatment of infections caused by dermatophytes [41,42]. Antifungal resistance of C. glabrata to all antifungal agents tested in the current study may be attributed to its ability to form biofilms [43].

Conclusions
Terbinafine was the most effective therapeutic agent against isolated C. albicans and C. tropicalis strains. Also, we can conclude that performing antifungal sensitivity test in hospitals prior to start of medical therapy is essential owing to the high emergence of multidrug-resistant Candida strains.