Distribution of clinical isolates of Candida spp. and antifungal susceptibility of high biofilm-forming Candida isolates

Introduction: The increase in the incidence of fungal infections, especially those caused by Candida albicans and other Candida species, necessitates the understanding and treatment of Candida-associated infections. In this study, we aimed to investigate the identification, distribution, and biofilm formation ability of different clinical Candida isolates and evaluate the distribution and antifungal susceptibilities of high biofilm-forming (HBF) Candida isolates. Methods: For identification, carbohydrate fermentation, carbohydrate assimilation, and ChromAgar tests were used. Biofilm formation was assessed using crystal violet binding assay, while the susceptibility to antifungal agents was determined using ATBTM Fungus 3 test kits. Results: The majority of Candida species were C. parapsilosis (31.3%; 31/99) and C. tropicalis (30.3%; 30/99). C. tropicalis was found to be the most frequently isolated species among all HBF Candida species. HBF Candida isolates were more frequently isolated from vaginal swab (35.7%; 10/28), tracheal aspirate (17.9%; 5/28), and urine (17.9%; 5/28). The majority of tested isolates were resistant to itraconazole and voriconazole, whereas no isolate was deemed resistant to 5-flucytosine. Conclusions: C. tropicalis displays the highest biofilm formation ability among all the Candida species evaluated, and HBF Candida isolates were more frequently seen in vaginal swab, tracheal aspirate, and urine samples. Our findings revealed that 5-flucytosine is the most efficient antifungal agent against HBF Candida isolates.


INTRODUCTION
Candida species are natural colonizers of gastrointestinal and urogenital tracts and known to reside as commensals in the oral and conjunctival flora of the healthy human body 1 .These organisms are known as opportunistic pathogens that may cause various infections ranging from oral candidiasis and esophagitis to hospital-acquired blood stream infections [2][3][4] .Although Candida albicans has been reported as the most predominant Candida species that frequently causes invasive fungal infections, a significant increase in non-C.albicans Candida (NCAC) species such as Candida glabrata, Candida krusei, Candida tropicalis, and Candida parapsilosis in human candidiasis has also been indicated over the last decade 2,4,5 .The increase in the occurrences of all NCAC species as pathogens has led to improvements in diagnostic methods that can sensitively differentiate between NCAC and C. albicans 5 .
On the other hand, the widespread use of a broad range of medical implant devices and an increase in patients that receive immunosuppressive therapy have led to the colonization of different Candida species and various Candida infections 2,6 .Biofilm formation is one of the most important reasons involved in the transformation of Candida species into important human pathogens 6 .Biofilm formation is responsible for many problems, as it avoids penetration and diffusion of various antimicrobial agents, causes generation of biofilm cells that have physiological and metabolic alterations, and provides a suitable environment for horizontal gene transfer mechanisms, which play an important role in antimicrobial resistance 7,8 .As biofilm environments are suitable for the acquisition of new traits via horizontal gene transfer 9 , investigation of the antifungal resistance of Candida isolates with biofilm formation ability and determination of effective antifungal agents against these isolates are necessary to prevent biofilm-associated Candida infections.In this study, we aimed to identify different clinical Candida isolates, determine their biofilm formation ability, and investigate the susceptibility of high biofilm-forming (HBF) Candida isolates to antifungal agents.

Microorganisms
We evaluated 99 clinical Candida isolates that were randomly collected from patients treated at two different hospitals in Ankara, Turkey, between July 2005 and March 2014.Collected isolates were inoculated into the brain heart infusion (BHI) broth (Lab M Ltd, Lancashire, UK) media supplemented with 10% glycerol and stored at -20°C for use in further experiments.

Identification tests
Colony morphologies and microscopic images of collected isolates were examined.By visual inspections, cells and colonies suspected to be Candida were subjected to carbohydrate fermentation, carbohydrate assimilation, and ChromAgar tests.

Carbohydrate fermentation tests
Carbohydrate fermentation tests were performed as per the method described by Bhavan 10 , with some modifications.Briefly, nutrient broth media supplemented with 1% (v/v) bromothymol blue as a pH indicator and carbohydrates such as glucose, galactose, lactose, maltose, and sucrose were separately prepared.A total of 10μL of each Candida isolate suspended in McFarland 0.5 standard in 5mL of saline buffer was added into 96 wells containing 100μL of different carbohydrate media.The plates were incubated at 37°C for 48h.Fermentation of any carbohydrate was considered as positive upon the change in the color of bromothymol blue to yellow.A total of 99 Candida isolates were identified according to their positive/negative carbohydrate fermentation test results, as presented in Table 1 4,[11][12][13][14][15] .

Carbohydrate assimilation tests
Carbohydrate assimilation tests were carried out as per the method described by Marinho et al. 4 , with some modifications.Briefly, 2% (w/v) carbohydrate solutions of glucose, galactose, lactose, maltose, sucrose, xylose, and raffinose were separately prepared and deposited onto sterile blotting paper discs prepared from eight layers of Whatman No. 1 filter paper.Each Candida isolate suspended in McFarland 0.5 standard in 5mL of saline buffer was inoculated onto 1% yeast nitrogen base (YNB) agar medium (Difco™).The sterile carbohydrate discs were placed onto the agar plates and the plates were incubated at 37°C for 48h.Assimilation of any carbohydrate was considered as positive with a presence of a growth zone around the carbohydrate disc.A total of 99 Candida isolates were identified according to their positive/negative carbohydrate assimilation test results, as presented in Table 1 4,[11][12][13][14][15] .

Growths on chromagar media
Each Candida isolate was inoculated into CHROMagar™ Candida medium; (CAC, Becton Dickinson, Heidelberg, Germany), which is designed to identify different Candida species based on their colony colors and morphologies.All plates were incubated at 37°C for 48h and visually observed after incubation.A total of 99 Candida isolates were identified according to their colony morphologies on CHROMAgar™ Candida medium.In CHROMAgar™ Candida medium, smooth colonies that appear light to medium green were considered as C. albicans; while dark blue to metallic blue smooth colonies were considered as C. tropicalis.Pink colonies with a whitish rough border were deemed as C. krusei, whereas pink-lavender smooth colonies were considered as C. glabrata.In addition, pink-salmon smooth colonies were deemed as Candida kefyr, while white-pale pink smooth colonies were considered as C. parapsilosis 4,11,15 .

18S ribosomal RNA gene sequence analysis
Within 99 Candida isolates, Candida isolates that could not be identified by the identification tests used in this study were identified by 18S ribosomal ribonucleic acid (rRNA) gene sequence analysis (RefGen Biotechnology Co. Ltd., Ankara, Turkey).

Biofilm formation on 24-well polystyrene plates
Biofilm formation abilities of 99 different Candida isolates were determined by crystal violet binding assay described by O'Toole 16 , with some modifications.Briefly, single yeast colonies were picked-up from BHI agar plate and inoculated into 10-mL BHI broth medium and incubated at 37°C overnight.The overnight culture was 1:100 diluted into fresh BHI medium and the wells of a polystyrene plate were filled with 1mL of the diluted inoculum.The plates were incubated for 48h at 37°C.
After incubation, the medium was gently removed and the wells were gently washed with sterile distilled water.After allowing wells to dry, each well was stained with 1% (w/v) crystal violet (Merck)/sterile distilled water for 45 min at 25°C.Excess of crystal violet was removed by sterile distilled water and the bound crystal violet in each well was solubilized by adding 1mL of ethanol (96.6%) solution.Solubilized crystal violet from each well was read by a spectrophotometer (Shimadzu UV -1700, Kyoto, Japan) at 560nm wavelength.According to biofilm formations, Candida isolates were classified into four categories as follows: • 0 ≤ OD < 0. The experiment was performed in triplicates.

Antifungal susceptibility tests
Antifungal susceptibilities of HBF Candida isolates [optical density (OD) ≥ 1.60] were determined by ATB TM Fungus 3 test kits (BioMérieux®, France).Antifungal susceptibilities of 25 HBF Candida isolates against 5-flucytosine, fluconazole, itraconazole, and voriconazole were evaluated.Briefly, all isolates were inoculated onto sabouraud dextrose agar (SDA) and incubated at 37°C for 48h.After incubation, each Candida isolate was suspended in saline solution, and yeast cells corresponding to a 2.0 McFarland standard were added into ATB F2 medium (yeast nitrogen base 6.7g; glucose 6.5g; asparagine 1.5g; disodium phosphate 2.5g; trisodium citrate 2.5g; potassium nitrate 5.5g; demineralized water 1,000mL; pH: 6.5-6.8).ATB F2 media with different Candida isolates were transferred into antifungal test strips and all the test strips were incubated at 37°C for 48h.After incubation, minimum inhibitory concentrations (MICs) of antifungal agents were visually determined and all the isolates were classified as resistant (R), intermediate (I), or sensitive (S) according to the MIC standards constituted by the Clinical and Laboratory Standards Institute (CLSI) (M27-A3, 2008) 17

Statistical analysis
Chi-square analysis was applied to estimate differences between the effects of four different antifungal agents.Bonferroni post-hoc test was used to evaluate antifungal susceptibility and agents with more/less significant effects were estimated.The significance level was set at 5% and the difference between the effects of each antifungal agent was considered as significant when p-value < 0.05.Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) 20.0 Software (IBM Corp, New York, USA).

RESULTS
According to all phenotypic identification tests, six different Candida species, namely, C. kefyr (n = 1), C. glabrata (n = 8), C. albicans (n = 13), C. krusei (n = 15), C. tropicalis (n = 30), and C. parapsilosis (n = 31) were identified in this study.One of the Candida isolates could not be clearly identified by phenotypical methods and was thought to be C. tropicalis or C. krusei.The results of 18S ribosomal RNA gene sequence analysis identified this strain as Candida orthopsilosis.

DISCUSSION
In many studies, C. albicans has been regarded as the most prevalent Candida species 18,19 .However, the results of the present study show that C. parapsilosis and C. tropicalis were observed in high frequencies.In addition, the frequency of C. krusei was higher than that of C. albicans, confirming that the occurrence of NCAC species such as C. tropicalis, C. parapsilosis, and C. krusei is increasing 11,18,20 as observed in a recent study.C. tropicalis was indicated as the most prevalent Candida species among all Candida species isolated and is regarded as an important emerging fungal pathogen associated with high mortality rate 11 .In line with the results of the present study, NCAC species are shown to be more prevalent than C. albicans in pediatric (< 3 year) and older (> 60 year) patients than in patients from other age groups (4-18, 19-60 years) and intensive care unit (ICU) patients 21 .According to other studies carried out with neonates, the prevalence of C. parapsilosis was  higher than that of C. albicans, and C. parapsilosis has been indicated as a predominant pathogen of invasive candidiasis in neonates 22,23 .
Urine, vaginal swab, blood, indwelling biomaterial, and respiratory tract samples are found to be the most prevalent specimens for Candida isolation 11,21,24 .In parallel with these findings, the majority of Candida isolates were isolated from vaginal swab specimen, followed by specimens of tracheal aspirate, blood, sputum, and urine.However, C. tropicalis was found as the most frequent Candida species in tracheal aspirate and sputum specimens (Figure 1), contradicting the results of previous studies on the predominance of C. albicans in lower respiratory tract specimens 12,25 .The most prevalent Candida species isolated from blood was C. albicans, confirming that C. albicans remains the most frequent fungal species in blood specimen 18 .
The investigation of the distribution of Candida species in different biofilm groups showed that C. tropicalis, which was more frequently isolated in this study, was also found as the most prevalent HBF Candida species, whereas all other C. albicans isolates were NBFs (Table 2).Therefore, the predominance of C. tropicalis instead of C. albicans was thought to be related to its enhanced biofilm formation ability 24 .
Applications of temporary or permanent biomaterials and medical devices in medicine have particularly led to an increase in the incidence of biofilm-associated infections 26,27 .One of the clinical specimens positive for HBF Candida isolates was catheter (Figure 2).Furthermore, HBF Candida isolates were more frequently isolated from the clinical specimens (vaginal swab, tracheal aspirate, and urine) that were related to the body parts that may be exposed to biomaterials such as intrauterine devices, endotracheal tubes, and urinary catheters.
The treatment of invasive fungal infections is usually carried out with five major groups of antifungal agents, including azoles, polyenes, allylamines, echinocandins, and pyrimidine analogues 28 .Fluconazole, voriconazole, and itraconazole belong to azole class, while 5-flucytosine is a pyrimidine analogue (Figure 3).Of these, azoles that target ergosterol biosynthesis via blockage of the enzyme lanosterol 14a-demethylase 28 are the most widely used group of antifungal agents 29 .A recent study evaluating the susceptibilities of different Candida species to fluconazole, voriconazole, itraconazole, ketoconazole, and 5-flucytosine showed that the majority of Candida isolates were sensitive to fluconazole and 5-flucytosine 30 .However, 5-flucytosine known to inhibit both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) synthesis after being converted to 5-fluorouracil 31 was the most effective antifungal agent against all HBF Candida species tested in the present study (Figure 3).
The high resistance to fluconazole may be mainly related to the high biofilm formation ability of the tested Candida isolates.Biofilms are known as suitable environments for horizontal gene transfer mechanisms 32 .Therefore, high biofilm formation ability may play an important role in the acquisition of new antifungal resistance traits in various Candida species.
Candida tropicalis isolates that demonstrated high biofilm formation capacity were shown to display higher rate of resistance to fluconazole in one of the recent studies 33 .
In conclusion, the present study shows that C. tropicalis displays the highest biofilm formation ability among the Candida species evaluated.Our findings indicate high frequency of HBF Candida isolation from clinical samples of vaginal swab, tracheal aspirate, and urine.We also found that 5-flucytosine is the most efficient antifungal agent against HBF Candida isolates.

FIGURE 1 :
FIGURE 1: Distribution of different Candida species in various clinical materials (n = 99 isolates).

FIGURE 3 :
FIGURE 3: Antifungal susceptibilities of HBF Candida isolates (biofilm formation OD ≥ 1.60) (n = 25 isolates.).All antifungal agents had significantly different effects (Chi-square test, p-value < 0.05).HBF: high biofilm-forming; OD: optical density.*Indicates that the tested HBF Candida isolates were significantly more sensitive to 5-flucytosine than to other antifungal agents used in this study (Bonferroni post-hoc test, p-value < 0.00).**Indicates that the tested HBF Candida isolates were significantly less sensitive to voriconazole than to other antifungal agents used in this study (Bonferroni post-hoc test, p-value < 0.00).

TABLE 1 :
Carbohydrate fermentation and carbohydrate assimilation test results of different Candida species.