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

Rev Soc Bras Med Trop 51(5):644-650, Sep-Oct, 2018 doi: 10.1590/0037-8682-0136-2018 Major Article Distribution of clinical isolates of Candida spp. and antifungal susceptibility of high biofilm-forming Candida isolates Gulcan Sahal[1] and Isil Seyis Bilkay[1] [1]. Biotechnology Section, Department of Biology, Hacettepe University, Ankara, Turkey. Abstract 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. Keywords: Antifungal resistance. Biofilm formation. Candida albicans. Non-Candida albicans. Candida Species. 5-Flucytosine 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 body1. These organisms are known as opportunistic pathogens that may cause various infections ranging from oral candidiasis and esophagitis to hospital-acquired blood stream infections2-4. 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 decade2,4,5. 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. albicans5. Corresponding author: Dr. Gulcan Sahal. e-mail: Received 22 April 2018 Accepted 7 August 2018 644 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 infections2,6. Biofilm formation is one of the most important reasons involved in the transformation of Candida species into important human pathogens6. 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 resistance7,8. As biofilm environments are suitable for the acquisition of new traits via horizontal gene transfer9, 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. Sahal G and Bilkay IS - Candida distribution and susceptibility 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 14,11-15. METHODS 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. 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. parapsilosis4,11,15. 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 Bhavan10, 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 considere (...truncated)